Precision Medicine Resource Guide

2016

The Precision Medicine Resource Guide highlights resources that provide awareness and understanding

VERSION 2.0 | ISSUE 1 NO. of this technology. Opinions expressed in this document are the authors’ own and do not necessarily reflect an endorsement by the CAP of any organization, equipment, reagents, materials, or services used by participating laboratories.

© 2016 College of American Pathologists. All rights reserved. None of the contents of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means (electronic, mechanical, photocopying, recording, or otherwise) without the prior written permission of the publisher. Goal of this Resource Guide and How to Use It

What is the Resource Guide?

The Precision Medicine Resource Guide is one of four CAP Resource Guides that brings a collected set of resources together in one place that are focused on a specific hot-topic technology important to pathologists. Each comprehensive guide highlights current resources such as a curated set of journal articles, and a collected set of CAP resources that includes learning opportunities, proficiency testing, and accreditation related to this technology. Also, each Resource Guide includes an “Insights From Adopters” section to gain perspective from pathology leaders in the field. In sum, each Resource Guide provides a one-stop resource that will assist busy pathologists to find valuable information about a dynamic and important emerging technology.

How to Use This Precision Medicine Resource Guide

This Resource Guide is designed in a modular manner to facilitate its use in several different ways. For example, the guide may be used in its entirety as a comprehensive guide to the rapidly evolving field of genomic and molecular pathology. Conversely, it may be used by a pathologist to focus on and gain a current understanding of the application of genomic and molecular pathology to a very specific organ system or disease process. The tables are designed not only to organize and summarize the contents of a section, but also to serve as stand- alone, quick reference guide to a topic. To some, these tables may hold the greatest value and become a frequently used reference. The Adopters sections will undoubtedly prove to be of great value to those contemplating taking or actually taking the plunge into the enhanced application of genomic and molecular pathology approaches to their practice.

Special Features of the Precision Medicine Resource Guide

Be sure and see the Quick Reference Table: Selected Tests by Tumor Type in Solid Tumors in Section 2.1.2. There are other valuable tables such as Mutated Genes of Potential Clinical Significance (Section 2.12.1), Commonly Tested Genes for Hereditary Disease (Section 3.1), Inherited Cardiovascular Disorders and Associated Genes (Section 3.2), Hereditary Cancers and Associated Genes (Section 3.12), High Multiplexed IVD Assays to Detect Important Human Pathogens (Section 4.6) and Pharmacogenomics: Commonly Tested Gene-Drug Pairs (Section 5). Be sure and see Section 9.1 on the Short Presentations on Emerging Concepts (SPECs) which provides a valuable tool for tumor boards or in discussion with local clinicians about emerging molecular tests that are actionable for patient care today. Also, the CAP webinar series on genomic and molecular topics listed in Section 9.2.

Editors of the Precision Medicine Resource Guide Allison M Cushman-Vokoun, MD, PhD, FCAP – The University of Nebraska Medical Center Matthew W Anderson, MD, PhD, FCAP – BloodCenter of Wisconsin

Tarek Bismar, MD – University of Calgary, Cumming School of Medicine Pranil K Chandra, DO, FCAP – PathGroup Joshua F Coleman, MD, FCAP – The Ohio State University Regina Gandour-Edwards, MD, FCAP – University of California Davis, Department of Pathology and Laboratory Medicine Aaron M. Gruver, MD, PhD, FCAP – Eli Lilly and Company Jill H. Kaufman, PhD – College of American Pathologists Frederick L Kiechle, MD, PhD, FCAP – Memorial Healthcare System Jordan S Laser, MD, FCAP – Northwell Health Franklin Moore, MD, PhD, FCAP – Baystate Health Ann M Moyer, MD, PhD, FCAP – Mayo Clinic Randall J Olsen, MD, PhD, FCAP – Houston Methodist Hospital

The Precision Medicine Resource Guide is a product of the CAP’s Personalized Health Care Committee. The CAP has four Pathology Resource Guides: Pathology Resource Guide: Precision Medicine Pathology Resource Guide: Digital Pathology Pathology Resource Guide: In Vivo Microscopy Pathology Resource Guide: Clinical Informatics

The National Institute of Health says, “Precision medicine is an emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle for each person. While significant advances in precision medicine have been made for select cancers, the practice is not currently in use for most diseases. Many efforts are underway to help make precision medicine the norm rather than the exception.”

President Obama’s Precision Medicine Initiative says, “Most medical treatments have been designed for the “average patient.” As a result of this “one-size-fits-all- approach,” treatments can be very successful for some patients but not for others. This is changing with the emergence of precision medicine, an innovative approach to disease prevention and treatment that takes into account individual differences in peoples’ genes, environments, and lifestyles. Precision medicine gives clinicians the tools to better understand the complex mechanisms underlying a patient’s health, disease, or condition and to better predict which treatments will be most effective.”

Table of Contents

Section 1 Emerging Concepts and Technologies || Page 1 1.1 Emerging Diagnostic Technologies || Page 1 1.2 Immunology as an Emerging Area for Precision Medicine || Page 30 1.3 The Microbiome || Page 37 1.4 Regulatory Framework and Precision Medicine || Page 52 1.5 Reimbursement || Page 55 1.6 Evolving Standards for Precision Medicine || Page 60 1.7 Precision Medicine Approaches in Health Care Systems || Page 78 1.8 Pathologists Education in Molecular Pathology and Genomics || Page 84

Section 2 Molecular and Genomic Pathology of Cancer || Page 87 2.1 The Basics || Page 87 2.2 Breast Cancer || Page 115 2.3 Central Nervous System Tumors || Page 123 2.4 Gastrointestinal Cancer || Page 128 2.5 Lung Cancer || Page 139 2.6 Melanoma || Page 143 2.7 Thyroid Cancer || Page 146 2.8 Bone and Soft Tissue || Page 153 2.9 Head and Neck || Page 155 2.10 Genitourinary Cancer || Page 160 2.11 Cancer of Unknown Primary Origin || Page 168 2.12 Hematopoietic Neoplasms || Page 171

Section 3 Molecular and Genomic Testing for Hereditary Disease || Page 197 3.1 The Basics - Quick Reference Table: Commonly Tested Genes for Hereditary Disease || Page 197 3.2 Cardiovascular Disorders – Quick Reference Table: Inherited Cardiovascular Disorders and Associated Genes || Page 212 3.3 Cystic Fibrosis || Page 214 3.4 Overgrowth Syndromes || Page 216 3.5 Inflammatory Bowel Disorders || Page 218 3.6 Eye Disorders || Page 220 3.7 Renal Disorders || Page 221 3.8 Neurologic, Neuromuscular and Neuropsychiatric Disorders || Page 223 3.9 Ethical Issues || Page 225 3.10 Prenatal || Page 231 3.11 Mitochondrial Disorders || Page 235 3.12 Hereditary Cancer Syndrome – Quick Reference Table: Hereditary Cancers and Associated Genes || Page 237 3.13 Post Mortem || Page 241

Section 4 Molecular and Genomic Infectious Disease || Page 243 4.1 Type of Testing for Infectious Disease || Page 243 4.2 Viruses || Page 249 4.3 Bacteria || Page 269 4.4 Fungi || Page 282 4.5 Parasites || Page 291 4.6 Pan-Infection Detection || Page 302 4.7 Clinical Application of Genomics in Infectious Disease || Page 316

Section 5 Pharmacogenomics || Page 335 5.1 Overview of Pharmacogenomics and Its Clinical Utility and Implementation || Page 340 5.2 Cardiovascular Pharmacogenomics || Page 344 5.3 Pharmacogenomics in Cancer || Page 350 5.4 Pharmacogenomics in Anesthesia || Page 354 5.5 Pharmacogenomics of Infectious Diseases and Interactions with the Immune System || Page 355 5.6 Psychiatric Pharmacogenomics || Page 358 5.7 Other Pharmacogenomics || Page 359

Section 6 Databases and Bioinformatics Primer || Page 361 6.1 Analysis Pipeline and Software || Page 361 6.2 Clinical Informatics and Reporting || Page 365 6.3 Databases || Page 369 6.4 Considerations of Depth of Coverage || Page 372 6.5 Bioinformatics for Pathogens || Page 373

Section 7 Issues for the Practicing Pathologists || Page 377 7.1 Insights from Adopters || Page 378 7.2 Pathology Reports in the Era of Genomics || Page 392 7.3 Patient Consent Forms, Privacy and Ethics || Page 395

Section 8 CAP Resources: Guidelines, Accreditation and Proficiency Testing || Page 403 8.1 CAP Guidelines || Page 403 8.2 CAP Cancer Biomarker Reporting Templates || Page 406 8.3 CAP Electronic Cancer Checklists (eCC) || Page 407 8.4 CAP Proficiency Testing (PT) || Page 410 8.5 CAP LAP: Accreditation || Page 413

Section 9 CAP Genomics and Molecular Pathology Education Resources || Page 415 9.1 CAP Short Presentations on Emerging Concepts (SPECs) || Page 415 9.2 CAP Webinars || Page 416 9.3 CAP Learning || Page 418 9.4 Molecular Oncology Tumor Board Series || Page 422

Section 10 Feedback || Page 423

Contributor Acknowledgment || Page 425

Bibliography || Page 427

Hot Topic Spotlight These sections are editorials on various emerging topics within genomics and molecular. The author of this section also includes a list of references that will be helpful for further reading on the subject.

Topic Author Section

New Frontiers in Clinical Next- Sophia Yohe, MD, FCAP 1.1.1 Generation Sequencing Next-generation Sequencing for HLA Matthew W. Anderson, 1.2.1.1 Genotyping MD, PhD, FCAP

Insights from Adopter These sections are short lists of tips from experts in the field on what works best for adoption of genomics and molecular. In addition, each adopter recommends a few articles that are helpful in preparing for the technology

Featured Adopter Section Lynn Bry, MD, PhD, FCAP Section 1.3.1 Samuel K. Caughron, MD, FCAP Section 7.1.3 Pranil K. Chandra, DO, FCAP Section 7.1.1 Eric J. Duncavage, MD, FCAP Section 2.1.1.3 David G. Hicks, MD, FCAP Section 2.1.1.1 Jordan S. Laser, MD, FCAP Section 7.1.2 Shannon J. McCall, MD, FCAP Section 1.6.1 Abhijit A. Patel, MD, PhD Section 1.1.2 Colin Pritchard, MD, PhD, FCAP Section 2.1.1.2

Quick Reference Table Index

The information presented in these tables is for quick reference only. Clinical decision making should not be based solely on this information.

Table Name Section Location

Selected Tests by Tumor Type in Section 2.1.2 Solid Tumors

Hematopoietic Neoplasms: Mutated Genes of Section 2.12.1 Prognostic and Diagnostic Significance

Commonly Tested Genes for Hereditary Section 3.1 Disease

Inherited Cardiovascular Disorders and Section 3.2 Associated Genes

Hereditary Cancers and Associated Genes Section 3.12

High Multiplexed IVD Assays to Detect Section 4.6 Important Human Pathogens

Pharmacogenomics: Commonly Tested Gene- Section 5 Drug Pairs

Section 1 Emerging Concepts and Technologies

This section includes the following topics:

• Emerging Diagnostic Technologies (Section 1.1); • Immunology as an Emerging Area for Precision Medicine (Section 1.2); • The Microbiome (Section 1.3); • Regulatory Framework for Precision Medicine (Section 1.4); • Reimbursement (Section 1.5); • Evolving Standards for Precision Medicine (Section 1.6); • Precision Medicine Approaches in Health Care Systems (Section 1.7); and • Pathologists Education in Molecular Pathology and Genomics (Section 1.8).

1.1 Emerging Diagnostic Technologies

This section includes the following topics:

• New Frontiers in Clinical Next-Generation Sequencing (Section 1.1.1); • Insights from Adopter - Abhijit A. Patel (Section 1.1.2); and • Mass Spectrometry Application for Infectious Disease (Section 1.1.3).

A) Emerging Technologies for Diagnostic Pathology Bucar O. Emerging Technologies for Diagnostic Pathology. DISCOVERIES. 2015; 3(2): e46.

Summary: Many technological advances have been made in the recent years, several of them with a great potential of significantly improving the diagnostic pathology field. This article discusses three of the most promising technologies, which emerged in the last one year. Fluorescent in situ sequencing can lead to the simultaneous identification of the transcriptome- wide RNA in individual cells across a tissue sections. 3D microscopy together with advanced image analysis can be used in diagnostic pathology and will especially be useful in hard to diagnose cases where the spatial

relationship of the tissue components is important. Expansion microscopy physically expands the biological specimen, and is of great interest for diagnostic pathology since the cheap conventional microscopes can be used to image a symmetrically expanded tissue. In addition, digital analysis and computational pathology are an integral part of each of these three emerging technologies, which underline their importance for the future developments in diagnostic pathology.

Free full text available from Discoveries

1.1.1 Hot Topic Spotlight - New Frontiers in Clinical Next-Generation Sequencing Hot Topic written by Sophia L. Yohe, MD, University of Minnesota Medical Center, Spotlight Minneapolis, MN

Introduction Next generation sequencing (NGS) or massively parallel sequencing is a method of simultaneous sequencing of thousands to millions of fragments of DNA or cDNA that has moved into the clinical laboratory. As with any new technology, its use in the clinical laboratory has evolved and will continue to evolve over time. This article is an overview of recent trends in clinical NGS as well as evolution that will likely occur in the near future. There have also been many changes or proposed changes to the regulation and reimbursement of NGS but these topics are beyond the scope of this article.

The Status Quo (Current state and methods of clinical NGS) Next generation sequencing is an established test method for inherited and somatic genetic mutations in many clinical laboratories. For inherited disease, testing may include targeted panels or whole exome and the genomic DNA or mitochondrial DNA may be tested. Targeted panels for genes associated with a clinical phenotype are usually the first-line of testing; while whole exome is reserved for cases where targeted testing has been unrevealing. Whole exome testing often involves testing the child and both parents (trio testing) to assist in the interpretation of variants. In addition, NGS technologies are used in analyzing cell free DNA in the prenatal setting. Somatic testing in the clinical setting consists of targeted panels, the composition of which varies from laboratory to laboratory.[1]

The vast majority of clinical sequencing is performed on one of two main types of instruments: Illumina sequencers (San Diego, CA) including the HiSeq, MiSeq, and NexSeq and the Ion Torrent series of machines including the IonPGM, IonProton, and IonS5 (ThermoFisher, Waltham, MA). These two types of

machines differ in their chemistry and detection methods and have different advantages and disadvantages (Table 1).

Table 1 Platform Local clonal Detection Pros Cons amplification Illumina Flow cell Fluorescent Paired end Errors in GC reads rich regions Ion torrent Bead and Ion (pH) Short run Homopolymer emulsion time error (>5) = 3.5%

See appendix A for more technical details about library preparation, DNA selection, and sequencing. The raw data reads from either type of instrument undergo a series of bioinformatics steps to ultimately deliver a variant call file (VCF). These steps include de-multiplexing, quality analysis, mapping of the reads to a reference genome (re-sequencing), identification of variants compared to the reference, and annotation of the variants. See appendix B for more details about mapping and identification of variants.

Pushing the Limits Although there is a desire to use NGS as a single method to detect all clinically relevant genetic changes, significant limitations currently exist. These limitations include background noise affecting the analytical sensitivity of mutation detection, areas of the genome that are difficult to sequence or analyze, and limitations in the knowledge of how to interpret novel or rare mutations. Furthermore, structural variations and copy number variation are not the primary output (ie. sequence data) and require different methods of analysis.

Analytic sensitivity Due to background noise, the sensitivity of NGS for single nucleotide variant (SNV) detection is approximately 5-10%. Although this sensitivity is acceptable for inherited disease testing, it limits testing in oncology for minimal residual disease, when a low tumor percentage is present, or to detect low level mutation due to tumor heterogeneity. There are several causes, including PCR noise compounded by C to T transversions with formalin fixed paraffin embedded tissue and systematic errors. Studies have shown that systematic errors lead to a 4-6% error rate, counterintuitively the rate is higher with increasing coverage.[2] Systematic errors may be sequence specific errors, errors at a particular location of the read (for example the ends for Illumina sequencers), and the base pair content (GC rich for Illumina).[2, 3] As neither PCR nor fixation causes insertions/deletions (indels) outside of repeat regions, there is better sensitivity for detecting small indels compared to SNVs.

There are two main methods for improving sensitivity; however, both of these methods decrease the number of useable reads and therefore will increase the sequencing cost to obtain a comparable coverage.[4] The first method is the use of overlapping paired end reads. This method works only for areas where the paired ends overlap, therefore the DNA insert size must be the same size or smaller than the number of reads. This technique is well-suited to amplicon based sequencing where the DNA insert size/amplicon size can be strictly controlled. In this situation, the DNA insert would be completely sequenced by both paired end reads (ie. the forward read and the reverse read). The sequence of these two read should match and any base pair that does not match in both reads is discarded.

The second technique is to use random nucleotide tags, referred to as unique identifiers (UID) or primer IDs, as they are often incorporated into the PCR primer.[4, 5] This method works with both sequence capture and amplicon based DNA selection techniques. In this method, random nucleotide tags are added to DNA fragments, assigned if DNA was sheared, or incorporated during the first round or two of PCR for amplicon based methods. Importantly, these steps occur prior to other amplification and result in a DNA fragment with a random and unique nucleotide sequence at one or both ends. After amplification, multiple identical template molecules will be present and will be sequenced (Figure 1), therefore duplicate reads must be retained during analysis. All reads that map to the same location and have an identical UID are considered part of a UID family and will be analyzed as a group. Targeted areas should be covered by many different UID families. If a mutation is present in a majority (for example >95%) of that UID family the mutation is considered present (and is considered one read). This process is repeated for all other UID families.

Figure 1. A. Prior to amplification, random tags (short bars) are added to DNA fragments (black) some of which have a mutation (orange). B. The tags randomly attach to DNA fragments. C and D. During amplification some copies will develop an error (red). All fragments will be sequenced. Only mutations that are detected within a majority (for example 95%) of all the sequencing reads with the same ID tag will be identified as true mutations (C). Mutations present in a minority of reads with the same ID tag are considered errors (D).

Difficult to sequence areas Homologous regions, repetitive regions, and GC rich regions are not reliably interpretable by the current NGS platforms and standard bioinformatics algorithms. Homologous regions, including pseudogenes, are areas of the genome with high homology that may differ from the gene of interest by only a few base pairs. Fragments of DNA that are sequenced from the target gene and homologous regions may be as similar in sequence as to be indistinguishable and the shorter the length of sequence the more likely this is to occur. This is not a problem unique to NGS as Sanger sequencing is also susceptible to inadvertent sequencing of homologous regions and test design is important to mitigate the problem. In NGS analysis, fragments of DNA from a target gene and homologous regions will have poor mapping quality and reads belonging to the homolog can be mismapped to the real gene and vice versa.[6] (Figure 2) Mismapping may lead to both false positive and false negative calls (e.g. mutations being missed and mutations being erroneously called). Many clinically relevant genes have pseudogenes and are challenging to interpret by NGS. This problem may be solved by newer instruments that have much longer sequencing reads (See section “New Instrumentation”).

Figure 2. The right side shows the CYP21A2 gene with baits designed for sequence capture (green bars). The left side shows the CYP21A2 pseudogene without baits. A similar number of sequencing reads are being mapped to the pseudogene as to the real gene, since the reads are so similar the actual source cannot be determined. These reads would have a low mapping quality score as the reads are mapping to more than 1 location, as indicated by the faded colors. Black arrow: coverage (gray peaks), green circle: location of baits (if any) (cropped IGV screenshot)

For repeat areas, unique sequence flanking the repeat is required to reliably map a sequencing read and determine the size of the repeat. Repetitive regions larger than the size of the DNA insert will not have flanking sequence and therefore will not be accurately mapped. Smaller repeats sizes will have unique flanking sequence on at least a proportion of the DNA fragments and therefore will map, although at lower coverage since some reads will not be informative. Even so, identification of the repeat size requires specialized bioinformatics algorithms and errors still occur which require interpretation. Source of errors include stutter (polymerase slippage leading to small shifts in repeat size) and PCR sequencing mistakes.[7] Homopolymers (ie. poly A or poly T) are difficult for the Ion Torrent sequencers, as the degree of change in voltage loses resolution above 5 base pairs.

GC rich regions appear to have higher background noise and lower quality of sequencing. In particular Illumina sequencers give substitution errors in areas of high GC content and long G/C homopolymers.[3] GC rich regions are known to form secondary structures which may represent part, but perhaps not all, of the problem. There may also be accumulation of G or C fluorophores after washing or out of phase sequencing.[3]

Validation of an NGS assay should include assessment for areas that cannot reliably be genotyped by NGS methods and at a minimum these areas should be documented. Alternative testing strategies such as Sanger sequencing or long range PCR may possible for some regions. [6]

Limitation in databases and knowledge Although the technical ability exists to perform whole genome analysis at a reasonable cost, especially for inherited disease, the ability to interpret all of that data lags behind. Sources to help with interpretation include databases (both publicly available, private, or laboratory specific databases), genetic and medical knowledge, medical literature, patient information, clinical experience, and team discussion. There are different types of databases with different amounts of data within them. A layer 1 database or Clinical Genomic Variant Repository contain only sequence/variant information, a layer 2 database or Genomic Medical Data Repository contains sequence/variant information with clinical/phenotype data, and a layer 3 database or Genomic Medical Evidence Database contains the medical evidence of classification or association with the sequence/variant information.[8] Most databases contain data for either inherited disease or somatic mutations but not both.

Although databases are extremely useful in the interpretation of variants, there are limitations of the current databases and no database is comprehensive or error free. Many databases lack assurance of the quality of sequence data or other data within the database. Databases may not be up to date or may include conflicting data. Both the medical literature and databases must be used with caution as some variants have been described as pathogenic by outdated criteria (ie. absent in 100 controls). Furthermore, there is limited knowledge about digenic or multigenic effects.

The significance of variants in introns or untranslated regions (UTRs) is often unknown and rare or novel exon variants may also be difficult to interpret. Novel or rare mutations that cause a frameshift or change an amino acid to a stop codon (stop loss or nonsense mutations) will generally be pathogenic if that mechanism has been described for the gene in question, but even then there may be exceptions. Missense mutations are more difficult to interpret. Many factors are taken into consideration when interpreting these cases including details about the specific mutation, details about mutations known to cause disease, similarity to known mutations, whether the mutation is in cis/trans with another known mutation or is de novo, presence/absence in other individuals (such as populations, normal controls, or affected and unaffected family members), or predicted protein effect (using in-silico models).[9]

Another challenging area is determining what genes to test in a given clinical scenario, especially in oncology. Although guidelines are present that define the common mutations or genes of interest (which are usually reimbursed), the literature and/or clinician interest may suggest other genes (which usually are not reimbursed) that may be useful. Commercially and locally available panels often

differ to some degree in the genes that are tested or the portions of the genes that are tested. Knowing the pros and cons associated with the different panels is challenging and databases or tools to assist with this do not exist. Furthermore, in oncology testing multiple gene mutations that may indicate differing prognosis or therapeutic response may be difficult to resolve. Lastly, oncology testing may identify possible germline mutations. Although simultaneous testing of matched tumor/normal is performed in large research studies, in clinical laboratories this practice is difficult as it doubles the cost of testing but is not reimbursed. This is usually addressed by a disclaimer or sometimes by follow up testing of a germline sample in select cases.

Structural variation and copy number variation NGS performs well at detection of single nucleotide variants (SNVs) and small insertion/deletions (indels), but does not perform as well at detecting structural rearrangements or copy number variations (CNVs) especially when using sequence enrichment to perform targeted NGS. Additionally, detecting structural variation and CNVs requires different bioinformatics algorithms than SNV detection. Despite these limitations, several clinical laboratories are using NGS data to detect CNVs.[10-12]

The techniques that have been used to detect CNVs including depth of coverage (read depth), read pair, split pair, assembly based, or a combination of these techniques. Clinical CNV analysis usually uses some combination of two or more of these techniques. [10-12] All the methods detect deletions better than duplications, cannot detect CNVs in repeat regions or difficult to map areas, and are limited by coverage (although the depth of coverage technique is more affected by coverage that the other techniques).[13] False positives are a problem, especially when applying CNV analysis over large areas of the exome, and have been reported to occur 10-89% of the time.[14] However, some areas of the genome are more prone to false positives than others.

Using depth of coverage or read depth to detect CNVs works well with uniform sequencing which is assumed for standard bioinformatics tools. These tools analyze for increased or decreased coverage to detect duplication/amplification or deletion respectively. However, coverage varies between run, within run, and between patients especially when sequence enrichment is used and spurious calls will be detected when sequencing is not uniform. With sequence enrichment, the pattern of coverage tends to be similar but the absolute coverage varies, requiring some sort of normalization. This may require comparison to a control as well as to control genes within the sample to normalize for the performance of the individual sample. The advantage of the read depth technique is the ability to detect large CNVs and to predict the actual copy

number; however, this method cannot detect the breakpoint or detect rearrangements.[13]

Read pair (or mate pair) analysis compares the distance of two ends of a read pair to the average insert size. Read pair analysis requires paired end reads, is limited by the insert size, and will only detect smaller CNVs.[13] One advantage of read pair analysis is that it detects both CNVs and rearrangements (translocations and inversions). However, it will only detect duplications/amplifications smaller than the average insert size and deletions less than 1 Kb and it cannot accurately estimate the number of copies.

Split pair (or split read) analysis looks specifically at paired reads where one of the paired reads fails to map or only maps partially. Split pair analysis also requires paired end reads, will only detect smaller CNVs and does not perform well in regions of low complexity. However, it can pinpoint the breakpoint and detect rearrangements.[13]

Finally, assembly based analysis uses de novo alignment of the reads. De-novo alignment matches the individual reads to each other instead of to a reference genome. Because it is computationally intensive, this technique works better for small genomes, such as bacteria.

Expanding uses of NGS Several new applications of NGS are being used clinically or are being actively researched for clinical use, including cell free DNA testing, HLA typing, microbiome analysis, RNA sequencing and expression, and methylation. Some of these new uses of NGS may be helped by the unique advantages of new instruments that are now available. The use of NGS for HLA typing had some challenges to overcome: differentiating low frequency alleles from high frequency artifacts and distinguishing two similar alleles as two distinct alleles.[15]

However, newer data analysis techniques such as step wise threshold clustering have allowed NGS to be explored as a clinical option for HLA typing. The use of NGS for identity testing using short tandem repeats (STRs) runs into the same problems as other repeat regions (see above); however, again newer data analysis techniques are making headway against this problem and may be applicable to other repeat regions.[7] Other uses for clinical NGS include pharmacogenetics and advanced blood group typing (for example type A1 versus type A2).

NGS of cell free DNA (cfDNA) has been used for some time for prenatal testing; however, NGS of circulating tumor DNA (ctDNA), a component of cfDNA, is a more recent development that is now clinically available. This testing is often

referred to as a liquid biopsy. Research studies have evaluated the ability of cfDNA sequencing to detect somatic mutations and the ability to monitor disease.[16, 17] Monitoring a known mutation by sequencing of cfDNA has been shown in several studies to correlate with relapse/progression of disease.[18] Using this technique to diagnose cancer is more problematic. Although some studies have shown promise, most studies are small and do not contain normal controls. Furthermore, studies with normal controls show some degree of mutation detection (false positives), albeit usually at a low level, in these controls. Lastly, as cfDNA is hypothesized to be released due to apoptosis and necrosis some tumor types are less likely to have significant ctDNA and would be falsely negative.[19]

New instrumentation Two sequencing instruments are currently available for research use that provide longer sequencing reads and are able to read the sequence of a single molecule: the PacBio SMRT (single molecule real time) (Menlo Park, CA) and the Oxford Nanopore (Oxford, UK), sometimes referred to as third generation sequencers. These instruments use different chemistry. The PacBio SMRT uses multiple wells each of which has a DNA polymerase affixed to the bottom with one long DNA fragment. Each fluorescently tagged nucleotide (A, C, G, T) gives a different fluorescent signal when incorporated. Illumination and detection occur from the bottom of the well and detection is sensitive enough to detect the single fluorescent signal that is released when a base pair is added to the DNA strand. The Oxford Nanopore uses a protein pore inserted into a membrane. A current is applied and flows through the pore between the two sides of the membrane. As the structure (DNA or RNA strand) passes through the pore the current changes and the degree of change correlates with the individual base (A, C, G, or T) and also correlates with the methylation status of C therefore methylation and hydroxymethylation can be detected.[20] The PacBio SMRT can also infer methylation status by analyzing the change in DNA polymerase kinetics (the time to incorporate a base and the time between incorporation of two bases). Neither instrument requires amplification steps which should reduce the background noise. Both instruments can perform long reads (14,000-40,000 for the PacBio SMRT and 8,000-100,000 for the Nanopore) which can overcome issues with pseudogenes and repeat regions and may help with identifying RNA isoforms. Both have high error rates. The errors on the PacBio are random and therefore can be overcome by replicate sequencing of the molecule and using a consensus result. The errors on the Nanopore are biased (meaning they occur in the same areas) and therefore cannot be overcome by replicate sequencing. These instruments show promise and may address problems in many clinically relevant regions such as trinucleotide repeat regions, HLA, and homologous regions. However, these sequencers have not yet moved into the clinical realm,

possibly due to their higher price and lower throughput and possibly due to the challenges of clinically validating an instrument with high intrinsic error rates.

Summary Next generation sequencing is being implemented in clinical laboratories and the use will only increase as the technology, bioinformatics, and resources evolve to address the limitations, improve quality of results, and increase the number of clinically useful applications. Clinical NGS has expanded to detect single nucleotide variants as well as structural rearrangements and CNVs, to monitor circulating tumor DNA, and analyze areas of the genome that previously were challenging for standard bioinformatics algorithms to manage. Further improvements will continue to occur; however, the challenge for clinical laboratories is to ensure testing is clinically relevant, cost effective, and can be integrated into clinical care.

Definitions:  Alignment: To compare a sequence read to another sequence and determine where it belongs. There are two types of alignment: de-novo assembly or resequencing.  De-novo assembly: a sequence read is compared to all the other sequence reads of that sample to determine a consensus sequence  Resequencing: a sequence read is compared to a reference sequence (for example the reference human genome). Also, referred to as mapping.  Bait: an artificial construct that is able to target the sequence of interest (for example a complementary DNA or RNA sequence) and can be used to isolate that target sequence. Used for sequence capture target enrichment.  Map/mapping: To compare a sequence read to a reference and determine where it belongs. See also Alignment-resequencing  Read: may refer to either the sequence result of a single base pair position or to the sequence result of a sequential length of base pair reads from a single clonally amplified DNA cluster.

Appendix A Samples undergo DNA extraction, DNA library preparation, target enrichment, and sequencing. The raw data from a sequencer then needs to be de- multiplexed and go through various bioinformatics algorithms to analyze and annotate the data for final interpretation. Library preparation refers to the process of preparing DNA for use on a sequencer. Although many methods are available, they all result in breaking DNA into fragments or amplifying target DNA regions and adding adaptors to the ends. Adaptors may include molecular barcodes (to allow pooling of patient samples), universal PCR primers, hybridization

sequences to bind the DNA fragment to a surface, and sequences required for the sequencing itself. The size of the DNA fragment between the adaptors is referred to as the insert size. The insert size may vary and there are different advantages to short insert sizes and long insert sizes. Shorter fragments are more likely to have both ends fall within an exon, which is often the area of interest, while longer fragments are more likely to have one end fall in an intron which will increase detection of structural rearrangements. (Figure 3)

Exon

Figure 3. Fragments with a short DNA insert size (top) are more likely to have both paired end reads (red bars) fall within the exon. Fragments with a long insert size are more likely to span the breakpoint of a rearrangement which often occurs in the intron.

The resulting library (fragments of DNA that have been prepared for sequencing) undergoes enrichment for whole exome analysis and targeted testing or is sequenced directly for whole genome analysis. Enrichment may be performed by hybridization to complementary sequences (sequence capture) or by PCR. Enrichment by PCR is generally combined with the library preparation step. The choice of enrichment strategy is often dictated by the clinical use, sequence capture is preferred for large genomic regions and PCR for smaller regions where high enrichment is desired.

The first sequencing step for both the Illumina and Ion Torrent platforms is to immobilize each DNA fragment and clonally amplify it. Clonal amplification is needed to generate a large enough signal for detection. The Ion Torrent uses a bead emulsion for immobilization and clonal amplification, whereas the Illumina sequencers use a flow cell. The flow cell or bead contains sequences that hybridize to part of the adaptor on the DNA fragments. The input DNA concentration is critical to ensure only one DNA fragment binds per bead and that the DNA fragments are well spaced out on the flow cell. The clonal amplification step creates a bead or cluster with approximately 1000 identical copies of a unique parent DNA molecule that are physically isolated from other molecules. For the Ion Torrent the beads are then placed in a well (one bead per well).

Next the actual sequencing begins. Illumina sequencers use sequencing by synthesis with fluorescent detection. (Figure 4) All four fluorescently tagged nucleotides are added and compete for the next space. The complementary tagged nucleotide will bind but a blocker prevents addition of more than 1

nucleotide per round. The remaining non-bound nucleotides are washed away. Laser excitation leads to a fluorescent emission which is recorded (simultaneously for each DNA fragment cluster). The fluorescent tag and blocker are removed. The next round begins. Each round “reads” one base pair from each DNA cluster. This process can be repeated on the opposite end of the DNA fragment referred to a paired end reads.

Figure 4. Schematic of sequencing by synthesis. A. Fluorescently tagged nucleotides (black circles with colored circles) compete for the next complementary space on the DNA strand (gray circles). B. Once a fluorescently tagged nucleotide is incorporated, it blocks further addition of nucleotides. C. The flow cell is washed removing additional fluorescently tagged nucleotides and a laser signal leads to fluorescent emission. D. The fluorescent tag and blocker are removed and washed away allowing incorporation of the next base during the next cycle. This occurs simultaneously for all DNA strands in a cluster and all clusters on the flow cell.

Ion Torrent sequencing is different, only a single base is added each round (for example: A in round 1, T in round 2, etc). When an added base is incorporated a hydrogen ion is released accompanied by a pH change which is detected for each bead within a well, if a base is not incorporated there is no voltage generated. Incorporation of more than one of the same base leads to a proportionately higher voltage signal up to about 5 bases. (Figure 5) If more than 5 bases are incorporated the signal in no longer proportional and the exact amount cannot be determined.

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Figure 5. Schematic of Ion Torrent sequencing. Each cycle, a single base is added in a set pattern. For this example, the order of base additions is A, T, C, G which then repeats. If a base is incorporated an ion is released leading to a pH (voltage) change which is proportional to the number of bases added in a row.

Appendix B The use of barcodes to tag a specimen’s DNA fragments, allows multiple samples to be pooled and sequenced together decreasing the sequencing cost. However, this process requires a de-multiplexing step where all reads are sorted by barcode/sample before further analysis. The de-multiplexed file with raw reads is referred to a FASTQ file. Following de-multiplexing, the individual reads for a sample are mapped to a reference genome and any difference between the reference and the sequencing read is noted (BAM file). For whole genome sequencing or sequence capture, duplicate reads are discarded. If multiple unique reads show the same difference a variant is called (the threshold for the number or percentage of reads required is set and should be validated). For example, a heterozygous SNV should be present in 50% of reads; however, in actual practice the range has been shown to vary as much as 23-74%.[21] The quality of signal for an individual base read and the mapping quality are also factors considered when calling a variant. The output file that defines all the variants for a sample and their allelic fractions is referred to as a variant call file (vcf). This list of variants undergoes interpretation. The vcf will contain all variants including common variants, although additional bioinformatics tools can be used to filter out variants meeting certain criteria (minor allele frequency above a threshold or variants previously identified as benign by a lab for example).

Spotlight References: 1. Zutter MM, Bloom KJ, Cheng L, et al. The Cancer Genomics Resource List 2014. Arch Pathol Lab Med. 2015;139:989-1008. 2. Wall JD, Tang LF, Zerbe B, et al. Estimating genotype error rates from high-coverage next-generation sequence data. Genome Res. 2014; 24:1734-1739. 3. Shin S, Park J. Characterization of sequence-specific errors in various next-generation sequencing systems. Mol Biosyst. 2016;12:914-922.

4. Zhang TH, Wu NC, Sun R. A benchmark study on error-correction by read-pairing and tag-clustering in amplicon-based deep sequencing. BMC Genomics. 2016; 17:108. 5. Kinde I, Wu J, Papadopoulos N, et al. Detection and quantification of rare mutations with massively parallel sequencing. Proc Natl Acad Sci U S A. 2011; 108:9530-9535. 6. Mandelker D, Amr SS, Pugh T, et al. Comprehensive diagnostic testing for stereocilin: an approach for analyzing medically important genes with high homology. J Mol Diagn. 2014; 16:639-647. 7. Van Neste C, Vandewoestyne M, Van Criekinge W, et al. My-Forensic- Loci-queries (MyFLq) framework for analysis of forensic STR data generated by massive parallel sequencing. Forensic Sci Int Genet. 2014; 9:1-8. 8. Yohe SL, Carter AB, Pfeifer JD, et al. Standards for Clinical Grade Genomic Databases. Arch Pathol Lab Med. 2015; 139:1400-1412. 9. Richards CS, Bale S, Bellissimo DB, et al. ACMG recommendations for standards for interpretation and reporting of sequence variations: Revisions 2007. Genet Med. 2008;10:294-300. 10. Pugh TJ, Amr SS, Bowser MJ, et al. VisCap: inference and visualization of germ-line copy-number variants from targeted clinical sequencing data. Genet Med. 2015. 11. Yang R, Nelson AC, Henzler C, et al. ScanIndel: a hybrid framework for indel detection via gapped alignment, split reads and de novo assembly. Genome Med. 2015; 7:127. 12. Nord AS, Lee M, King MC, et al. Accurate and exact CNV identification from targeted high-throughput sequence data. BMC Genomics. 2011; 12:184. 13. Zhao M, Wang Q, Jia P, et al. Computational tools for copy number variation (CNV) detection using next-generation sequencing data: features and perspectives. BMC Bioinformatics. 2013; 14 Suppl 11:S1. 14. Pinto D, Darvishi K, Shi X, et al. Comprehensive assessment of array- based platforms and calling algorithms for detection of copy number variants. Nat Biotechnol. 2011; 29:512-520. 15. Stutz WE, Bolnick DI. Stepwise threshold clustering: a new method for genotyping MHC loci using next-generation sequencing technology. PLoS One. 2014; 9:e100587. 16. Leary RJ, Sausen M, Kinde I, et al. Detection of chromosomal alterations in the circulation of cancer patients with whole-genome sequencing. Sci Transl Med. 2012; 4:162ra154. 17. Forshew T, Murtaza M, Parkinson C, et al. Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA. Sci Transl Med. 2012; 4:136ra168.

18. Yamada T, Iwai T, Takahashi G, et al. Utility of KRAS mutation detection using circulating cell-free DNA from patients with colorectal cancer. Cancer Sci. 2016. 19. Jahr S, Hentze H, Englisch S, et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res. 2001; 61:1659-1665. 20. Schreiber J, Wescoe ZL, Abu-Shumays R, et al. Error rates for nanopore discrimination among cytosine, methylcytosine, and hydroxymethylcytosine along individual DNA strands. Proc Natl Acad Sci U S A. 2013; 110:18910-18915.

Additional Resources:

A) Nanopore-Based Fourth-Generation DNA Sequencing Technology Feng Y, Zhang Y, Ying C, Wang D, Du C. Nanopore-Based Fourth- Generation DNA Sequencing Technology. Genomics Proteomics Bioinformatics. 2015; 13(1): 4-16.

Summary: Nanopore-based sequencers, as the fourth-generation DNA sequencing technology, have the potential to quickly and reliably sequence the entire human genome for less than $1000, and possibly for even less than $100. The single-molecule techniques used by this technology allow us to further study the interaction between DNA and protein, as well as between protein and protein. Nanopore analysis opens a new door to molecular biology investigation at the single-molecule scale. In this article, we have reviewed academic achievements in nanopore technology from the past as well as the latest advances, including both biological and solid-state nanopores, and discussed their recent and potential applications.

Free full text available from PubMed PMID: 25743089

B) The Evolution of Nanopore Sequencing Wang Y, Yang Q, Wang Z. The Evolution of Nanopore Sequencing. Front Genet. 2014; 5: 449.

Summary: The "$1000 Genome" project has been drawing increasing attention since its launch a decade ago. Nanopore sequencing, the third- generation, is believed to be one of the most promising sequencing technologies to reach four gold standards set for the "$1000 Genome" while the second-generation sequencing technologies are bringing about a revolution in life sciences, particularly in genome sequencing-based

personalized medicine. Both of protein and solid-state nanopores have been extensively investigated for a series of issues, from detection of ionic current blockage to field-effect-transistor (FET) sensors. A newly released protein nanopore sequencer has shown encouraging potential that nanopore sequencing will ultimately fulfill the gold standards. In this review, we address advances, challenges, and possible solutions of nanopore sequencing according to these standards.

Free full text available from PubMed PMID: 25610451

1.1.2 Insights from Adopter - Abhijit A. Patel, MD, PhD

Abhijit A. Patel, MD, PhD, is a board-certified radiation oncologist at Yale University School of Medicine. He is an Assistant Professor in the Department of Therapeutic Radiology in the Yale Cancer Center. He is a physician-scientist, and maintains an active clinical practice and leads translational research efforts in his laboratory. Dr. Patel’s clinical interests include thoracic, genitourinary, and gastrointestinal malignancies. His laboratory has been developing NGS-based methods for ultrasensitive measurement of circulating tumor DNA (ctDNA). He

Abhijit A. Patel, MD, PhD works collaboratively with pathologists and medical oncologists to develop and validate technologies to address a variety of clinically important questions with regard to ctDNA.

ctDNA is likely to have clinical applications in both non-invasive mutation profiling as well as therapeutic response monitoring. With the rapid pace of developments in the field, Dr. Patel predicts that it will be used in a more widespread fashion very soon. He urges pathologists and other physicians to become familiar with the various unique biological features of ctDNA and technological options for identifying and measuring circulating tumor DNA in a clinical setting.

Dr. Patel’s insights for the next wave of adopters (June 2016):

1 The rate of utilization of It is important to familiarize oneself with the circulating tumor DNA, possibilities and also the limitation of this especially for non- approach relative to standard tissue-based invasive mutation mutation profiling. profiling, could rapidly increase in the upcoming years.

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2 ctDNA is shed from In patients who have low tumor burden or dying cancer cells into whose cancer has been effectively treated, the blood. ctDNA can be present at very low abundance in the blood. It is found amidst a background of normal DNA in blood, at a low allelic fraction. This presents significant technical challenges for the identification and measurement of ctDNA. 3 ctDNA is cleared ctDNA is highly fragmented and gets cleared quickly from blood. quickly from the bloodstream with a half-life of approximately two hours, and therefore its levels provide a real time estimate of active cell death. It is important to recognize that after effective cancer therapy, circulating tumor DNA levels can change rapidly. 4 ctDNA is expected to Circulating tumor DNA is identified based on have higher specificity the presence of tumor-specific mutations than most serum which are unlikely to be found in the blood of protein biomarkers healthy individuals. Thus, false-positive test results should be rare if the technical error rate of the assay is low. 5 Understand pros and Digital PCR can very sensitively detect and cons of digital PCR quantify ctDNA containing one particular technologies for ctDNA mutation or a small number of mutations in a detection given blood sample. Thus, one would need to define a priori which mutations to evaluate. This approach is more focused than NGS, but it's also cheaper and has a faster turnaround time. It can also be simpler to implement.

6 Understand pros and Next-generation sequencing based methods cons of next-generation for circulating tumor DNA can evaluate sequencing (NGS) thousands of mutations simultaneously from a based methods for given sample. Thus, prior knowledge of the ctDNA detection tumor’s mutation profile is not required. However, this approach can be more expensive and can have a slower turn-around time than PCR-based methods.

Because of the technical complexity and bioinformatic challenges of low-abundance mutation detection via NGS, it can be difficult to develop such an assay in-house. Some companies are beginning to offer kits that may facilitate development of in-house workflows. But several reference labs are already offering such tests. If you are aiming to offer next- generation sequencing based ctDNA testing, sending out may be a better option in the near term. 7 ctDNA for monitoring Incorporation of circulating tumor DNA into treatment response is corollary studies or clinical trials will help to an emerging accelerate this process and would help us to application; more rapidly understand how circulating tumor pathologists should DNA behaves in this context. Pathologists are participate in related encouraged to participate in such trials. research 8 Cancer screening using A promising future application of circulating ctDNA is an exciting tumor DNA is cancer screening. This would potential future take advantage of ctDNA’s exquisite cancer application specificity. Both commercial and academic groups are working in this direction but more research is needed. This is exciting because it could have a tremendous impact on cancer outcomes.

Dr. Patel’s suggested articles:

A) Liquid Biopsies: Genotyping Circulating Tumor DNA Diaz LA, Jr.,Bardelli A. Liquid Biopsies: Genotyping Circulating Tumor DNA. J Clin Oncol. 2014; 32(6): 579-586.

Summary: Genotyping tumor tissue in search of somatic genetic alterations for actionable information has become routine practice in clinical oncology. Although these sequence alterations are highly informative, sampling tumor tissue has significant inherent limitations; tumor tissue is a single snapshot in time, is subject to selection bias resulting from tumor heterogeneity, and can be difficult to obtain. Cell-free fragments of DNA are shed into the bloodstream by cells undergoing apoptosis or necrosis, and the load of circulating cell-free DNA (cfDNA) correlates with tumor staging and prognosis. Moreover, recent advances in the sensitivity and accuracy of DNA analysis have allowed for genotyping of cfDNA for somatic genomic alterations found in tumors. The ability to detect and quantify tumor mutations has proven effective in tracking tumor dynamics in real time as well as serving as a liquid biopsy that can be used for a variety of clinical and investigational applications not previously possible.

Free full text available from PubMed PMID: 24449238

B) Going with the Flow: From Circulating Tumor Cells to DNA Bidard FC, Weigelt B,Reis-Filho JS. Going with the Flow: From Circulating Tumor Cells to DNA. Sci Transl Med. 2013; 5(207): 207ps214.

Summary: Molecular analyses of circulating tumor DNA (ctDNA) in plasma from cancer patients have the potential to deliver minimally invasive diagnostic and disease-monitoring biomarkers. Drawing from experience gained through the translation of circulating tumor cell detection to clinical tests, we discuss ctDNA as a source of tumor material for biomarker development.

Full text available from Science Translational Medicine (USD 30.00) PMID: 24132635

C) Cell-Free Nucleic Acids as Biomarkers in Cancer Patients Schwarzenbach H, Hoon DS,Pantel K. Cell-Free Nucleic Acids as Biomarkers in Cancer Patients. Nat Rev Cancer. 2011; 11(6): 426-437.

Summary: DNA, mRNA and microRNA are released and circulate in the blood of cancer patients. Changes in the levels of circulating nucleic acids have been associated with tumour burden and malignant progression. In the past decade a wealth of information indicating the potential use of circulating nucleic acids for cancer screening, prognosis and monitoring of the efficacy of anticancer therapies has emerged. In this Review, we discuss these findings with a specific focus on the clinical utility of cell-free nucleic acids as blood biomarkers.

Full text available from Nature Reviews (subscription required) PMID: 21562580

1.1.3 Mass Spectrometry Applications for Infectious Disease

A) MALDI-TOF MS: An Upcoming Tool for Rapid Detection of Antibiotic Resistance in Microorganisms Kostrzewa M, Sparbier K, Maier T, Schubert S. MALDI-TOF MS: An Upcoming Tool for Rapid Detection of Antibiotic Resistance in Microorganisms. Proteomics Clin Appl. 2013; 7(11-12): 767-778.

Summary: MALDI-TOF MS profiling for microorganism detection has already been demonstrated in the 1990s, but has evolved to the first-line identification method in many laboratories just during the past five years. While this application of MALDI-TOF MS has proven its broad applicability, accuracy, robustness, and cost-effectiveness it is of particular interest to expand the capabilities of the mass spectrometric platform. Resistance detection is the most desirable further application of MALDI-TOF MS in microbiology, but maybe also the most challenging. Different approaches have been published regarding diverse antibiotic drugs and distinct microorganism classes. The current review shall give an overview about the developments of the recent years and their potential to get transformed in clinical useful assays in the future.

Free full text available from Proteomics – Clinical Applications PMID: 24123965

B) Proteome-Based Bacterial Identification Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS): A Revolutionary Shift in Clinical Diagnostic Microbiology Nomura F. Proteome-Based Bacterial Identification Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS): A Revolutionary Shift in Clinical Diagnostic Microbiology. Biochim Biophys Acta. 2015; 1854(6): 528-537.

Summary: Rapid and accurate identification of microorganisms, a prerequisite for appropriate patient care and infection control, is a critical function of any clinical microbiology laboratory. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a quick and reliable method for identification of microorganisms, including bacteria, yeast, molds, and mycobacteria. Indeed, there has been a revolutionary shift in clinical diagnostic microbiology. In the present review, the state of the art and advantages of MALDI-TOF MS-based bacterial identification are described. The potential of this innovative technology for use in strain typing and detection of antibiotic resistance is also discussed. This article is part of a Special Issue entitled: Medical Proteomics.

Full text available from Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics (USD 35.95) PMID: 25448014

C) MALDI-TOF Mass Spectrometry for Microorganism Identification Dingle TC, Butler-Wu SM. MALDI-TOF mass spectrometry for microorganism identification. Clin Lab Med. 2013; 33(3): 589-609.

Summary: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid, reliable, and high-throughput diagnostic tool for the identification of microorganisms. The technology is unique in clinical microbiology, allowing laboratories to definitively identify

bacterial and fungal isolates within minutes. The rapid turnaround time and minimal cost for consumables per specimen compared with conventional identification methods have resulted in MALDI-TOF MS being increasingly used in clinical laboratories worldwide. This article summarizes the current literature on MALDI-TOF MS for microbial identification and provides a preview of the method's potential future applications in clinical microbiology.

Full text available from Clinics in Laboratory Medicine (USD 31.50) PMID: 23931840

D) Integrating Rapid Pathogen Identification and Antimicrobial Stewardship Significantly Decreases Hospital Costs Perez KK, Olsen RJ, Musick WL, et al. Integrating Rapid Pathogen Identification and Antimicrobial Stewardship Significantly Decreases Hospital Costs. Arch Pathol Lab Med. 2013; 137(9): 1247-1254.

Summary: CONTEXT: Early diagnosis of gram-negative bloodstream infections, prompt identification of the infecting organism, and appropriate antibiotic therapy improve patient care outcomes and decrease health care expenditures. In an era of increasing antimicrobial resistance, methods to acquire and rapidly translate critical results into timely therapies for gram- negative bloodstream infections are needed. OBJECTIVE: To determine whether mass spectrometry technology coupled with antimicrobial stewardship provides a substantially improved alternative to conventional laboratory methods. DESIGN: An evidence-based intervention that integrated matrix-assisted laser desorption and ionization time-of-flight mass spectrometry, rapid antimicrobial susceptibility testing, and near-real-time antimicrobial stewardship practices was implemented. Outcomes in patients hospitalized prior to initiation of the study intervention were compared to those in patients treated after implementation. Differences in length of hospitalization and hospital costs were assessed in survivors. RESULTS: The mean hospital length of stay in the preintervention group survivors (n = 100) was 11.9 versus 9.3 days in the intervention group (n = 101; P = .01). After multivariate analysis, factors independently associated with decreased length of hospitalization included the intervention (hazard ratio, 1.38; 95% confidence interval, 1.01-1.88) and active therapy at 48 hours (hazard ratio, 2.9; confidence interval, 1.15-7.33). Mean hospital costs per patient were $45 709 in the preintervention group and $26 162 in the intervention group (P = .009). CONCLUSIONS: Integration of rapid identification and susceptibility techniques with antimicrobial stewardship significantly improved time to optimal therapy, and it decreased hospital length of stay and total costs. This innovative strategy has ramifications for other areas of patient care.

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PMID: 23216247

E) Comparative Evaluation of Matrix-Assisted Laser Desorption Ionisation- Time of Flight Mass Spectrometry and Conventional Phenotypic-Based Methods for Identification of Clinically Important Yeasts in a UK-Based Medical Microbiology Laboratory Fatania N, Fraser M, Savage M, Hart J, Abdolrasouli A. Comparative Evaluation of Matrix-Assisted Laser Desorption Ionisation-Time of Flight Mass Spectrometry and Conventional Phenotypic-Based Methods for Identification of Clinically Important Yeasts in a UK-Based Medical Microbiology Laboratory. J Clin Pathol. 2015; 68(12): 1040-1042.

Summary: AIMS: Performance of matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was compared in a side-by side-analysis with conventional phenotypic methods currently in use in our laboratory for identification of yeasts in a routine diagnostic setting. METHODS: A diverse collection of 200 clinically important yeasts (19 species, five genera) were identified by both methods using standard protocols. Discordant or unreliable identifications were resolved by sequencing of the internal transcribed spacer region of the rRNA gene. RESULTS: MALDI-TOF and conventional methods were in agreement for 182 isolates (91%) with correct identification to species level. Eighteen discordant results (9%) were due to rarely encountered species, hence the difficulty in their identification using traditional phenotypic methods. CONCLUSIONS: MALDI-TOF MS enabled rapid, reliable and accurate identification of clinically important yeasts in a routine diagnostic microbiology laboratory. Isolates with rare, unusual or low probability identifications should be confirmed using robust molecular methods.

Full text available from Journal of Clinical Pathology (USD 37.00) PMID: 26307074

F) Impact of Rapid Organism Identification Via Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Combined with Antimicrobial Stewardship Team Intervention in Adult Patients with Bacteremia and Candidemia Huang AM, Newton D, Kunapuli A, et al. Impact of Rapid Organism Identification Via Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Combined with Antimicrobial Stewardship Team Intervention in Adult Patients with Bacteremia and Candidemia. Clin Infect Dis. 2013; 57(9): 1237- 1245.

Summary: BACKGROUND: Integration of rapid diagnostic testing via matrix- assisted laser desorption/ionization time-of-flight (MALDI-TOF) with antimicrobial stewardship team (AST) intervention has the potential for early

organism identification, customization of antibiotic therapy, and improvement in patient outcomes. The objective of this study was to assess the impact of this combined approach on clinical and antimicrobial therapy-related outcomes in patients with bloodstream infections. METHODS: A pre-post quasi-experimental study was conducted to analyze the impact of MALDI- TOF with AST intervention in patients with bloodstream infections. The AST provided evidence-based antibiotic recommendations after receiving real- time notification following blood culture Gram stain, organism identification, and antimicrobial susceptibilities. Outcomes were compared to a historic control group. RESULTS: A total of 501 patients with bacteremia or candidemia were included in the final analysis: 245 patients in the intervention group and 256 patients in the preintervention group. MALDI-TOF with AST intervention decreased time to organism identification (84.0 vs 55.9 hours, P < .001), and improved time to effective antibiotic therapy (30.1 vs 20.4 hours, P = .021) and optimal antibiotic therapy (90.3 vs 47.3 hours, P < .001). Mortality (20.3% vs 14.5%), length of intensive care unit stay (14.9 vs 8.3 days) and recurrent bacteremia (5.9% vs 2.0%) were lower in the intervention group on univariate analysis, and acceptance of an AST intervention was associated with a trend toward reduced mortality on multivariable analysis (odds ratio, 0.55, P = .075). CONCLUSION: MALDI- TOF with AST intervention decreased time to organism identification and time to effective and optimal antibiotic therapy.

Free full text available from Clinical Infectious Diseases PMID: 23899684

G) Utility of the MALDI-TOF MS Method to Identify Nontuberculous Mycobacteria Kodana M, Tarumoto N, Kawamura T, et al. Utility of the MALDI-TOF MS Method to Identify Nontuberculous Mycobacteria. J Infect Chemother. 2016; 22(1): 32-35.

Summary: In comparison to the conventional real-time polymerase chain reaction method (PCR method) or the DNA-DNA hybridization method (DDH method), the utility of NTM identification by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method has seldom been reported. In this study, 75 clinical NTM isolates from our hospital between April 2013 and July 2014 were identified and analyzed using PCR, DDH, and MALDI-TOF MS methods, and the results for the MALDI-TOF MS method were compared with the others. Identification at the species level was in agreement for 71 (94.5%) of the 75 isolates. For further details, identification was possible for 23 (95.8%) of 24 Mycobacterium avium, 11 (100%) of 11 Mycobacterium intracellulare, and 1 (50%) of 2 isolates mixed with M. avium and M. intracellulare. Mycobacterium ksansasii, Mycobacterium abscessus, Mycobacterium

fortuitum, Mycobacterium gordonae, and Mycobacterium chelonae identified by DDH method were same result by MALDI-TOF MS. Additionally, Mycobacterium mucogenicum, which could not be identified by the DDH method, was identified by the MALDI-TOF MS method. However, two isolates identified as Mycobacterium terrae by DDH method could not be identified by the MALDI-TOF MS method and were determined to be Mycobacterium arupense by 16S ribosomal RNA (rRNA) sequence analysis. The present findings show that, for rare bacterial species, identification is sometimes not possible, but, in most cases, the results of identification by the MALDI-TOF MS method have a high concordance rate with the results of the PCR and DDH methods.

Free full text available from Journal of Infection and Chemotherapy PMID: 26603481

H) Rapid and Reliable Discrimination between Shigella Species and Escherichia Coli Using MALDI-TOF Mass Spectrometry Paauw A, Jonker D, Roeselers G, et al. Rapid and Reliable Discrimination between Shigella Species and Escherichia Coli Using MALDI-TOF Mass Spectrometry. Int J Med Microbiol. 2015; 305(4-5): 446-452.

Summary: E. coli-Shigella species are a cryptic group of bacteria in which the Shigella species are distributed within the phylogenetic tree of E. coli. The nomenclature is historically based and the discrimination of these genera developed as a result of the epidemiological need to identify the cause of shigellosis, a severe disease caused by Shigella species. For these reasons, this incorrect classification of shigellae persists to date, and the ability to rapidly characterize E. coli and Shigella species remains highly desirable. Until recently, existing matrix-assisted laser desorption/ionization- time of flight mass spectrometry (MALDI-TOF MS) assays used to identify bacteria could not discriminate between E. coli and Shigella species. Here we present a rapid classification method for the E. coli-Shigella phylogroup based on MALDI-TOF MS which is supported by genetic analysis. E. coli and Shigella isolates were collected and genetically characterized by MLVA. A custom reference library for MALDI-TOF MS that represents the genetic diversity of E. coli and Shigella strains was developed. Characterization of E. coli and Shigella species is based on an approach with Biotyper software. Using this reference library it was possible to distinguish between Shigella species and E. coli. Of the 180 isolates tested, 94.4% were correctly classified as E. coli or shigellae. The results of four (2.2%) isolates could not be interpreted and six (3.3%) isolates were classified incorrectly. The custom library extends the existing MALDI-TOF MS method for species determination by enabling rapid and accurate discrimination between Shigella species and E. coli.

Full text available from International Journal of Medical Microbiology (USD 35.95) PMID: 25912807

I) Enhancing Molecular Approaches for Diagnosis of Fungal Infections Zhang SX. Enhancing Molecular Approaches for Diagnosis of Fungal Infections. Future Microbiol. 2013; 8(12): 1599-1611.

Summary: Molecular tests can improve the diagnosis of fungal infections. Despite the increasing application for fungal detection, molecular tests are still not accepted as a diagnostic criterion to define invasive fungal diseases. This limitation is largely due to a lack of a standardized method. Method standardization can be achieved by following a consensus protocol developed by a working group, by performing a molecular test in a centralized laboratory or by using a commercial assay that provides a standardized method and quality-controlled reagents. Forming a consortium or a working group consisting of large-scale diagnostic mycology laboratories can accelerate the process of validating and implementing a commercial molecular assay for clinical use through a joint effort between industry partners and clinicians. Development of molecular tests not only for the detection of fungi but also for the identification of antifungal drug resistance directly in blood, bronchoalveolar lavage fluid, cerebrospinal fluid, and formalin-fixed and paraffin-embedded tissues greatly enhances fungal diagnostic capacities. Advances of developing quantitative assays and RNA detection platforms may provide another avenue to further improve fungal diagnostics.

Free full text available from Future Microbiology PMID: 24266359

J) Strategy for Rapid Identification and Antibiotic Susceptibility Testing of Gram-Negative Bacteria Directly Recovered from Positive Blood Cultures Using the Bruker MALDI Biotyper and the BD Phoenix System Wimmer JL, Long SW, Cernoch P, et al. Strategy for Rapid Identification and Antibiotic Susceptibility Testing of Gram-Negative Bacteria Directly Recovered from Positive Blood Cultures Using the Bruker MALDI Biotyper and the BD Phoenix System. J Clin Microbiol. 2012; 50(7): 2452-2454.

Summary: Decreasing the time to species identification and antibiotic susceptibility determination of strains recovered from patients with bacteremia significantly decreases morbidity and mortality. Herein, we validated a method to identify Gram-negative bacteria directly from positive blood culture medium using the Bruker MALDI Biotyper and to rapidly perform susceptibility testing using the BD Phoenix.

Free full text available from Pubmed PMID: 22518850

K) Rapid Diagnosis of Infection in the Critically Ill, a Multicenter Study of Molecular Detection in Bloodstream Infections, Pneumonia, and Sterile Site Infections Vincent JL, Brealey D, Libert N, et al. Rapid Diagnosis of Infection in the Critically Ill, a Multicenter Study of Molecular Detection in Bloodstream Infections, Pneumonia, and Sterile Site Infections. Crit Care Med. 2015; 43(11): 2283-2291.

Summary: OBJECTIVE: Early identification of causative microorganism(s) in patients with severe infection is crucial to optimize antimicrobial use and patient survival. However, current culture-based pathogen identification is slow and unreliable such that broad-spectrum antibiotics are often used to insure coverage of all potential organisms, carrying risks of overtreatment, toxicity, and selection of multidrug-resistant bacteria. We compared the results obtained using a novel, culture-independent polymerase chain reaction/electrospray ionization-mass spectrometry technology with those obtained by standard microbiological testing and evaluated the potential clinical implications of this technique. DESIGN: Observational study. SETTING: Nine ICUs in six European countries. PATIENTS: Patients admitted between October 2013 and June 2014 with suspected or proven bloodstream infection, pneumonia, or sterile fluid and tissue infection were considered for inclusion. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We tested 616 bloodstream infection, 185 pneumonia, and 110 sterile fluid and tissue specimens from 529 patients. From the 616 bloodstream infection samples, polymerase chain reaction/electrospray ionization-mass spectrometry identified a pathogen in 228 cases (37%) and culture in just 68 (11%). Culture was positive and polymerase chain reaction/electrospray ionization-mass spectrometry negative in 13 cases, and both were negative in 384 cases, giving polymerase chain reaction/electrospray ionization-mass spectrometry a sensitivity of 81%, specificity of 69%, and negative predictive value of 97% at 6 hours from sample acquisition. The distribution of organisms was similar with both techniques. Similar observations were made for pneumonia and sterile fluid and tissue specimens. Independent clinical analysis of results suggested that polymerase chain reaction/electrospray ionization-mass spectrometry technology could potentially have resulted in altered treatment in up to 57% of patients. CONCLUSIONS: Polymerase chain reaction/electrospray ionization-mass spectrometry provides rapid pathogen identification in critically ill patients. The ability to rule out infection within 6 hours has potential clinical and economic benefits.

Free full text available from PubMed

PMID: 26327198

L) Rapid Laboratory Diagnosis for Respiratory Infectious Diseases by Using MALDI-TOF Mass Spectrometry Wang YF, Fu J. Rapid Laboratory Diagnosis for Respiratory Infectious Diseases by Using MALDI-TOF Mass Spectrometry. J Thorac Dis. 2014; 6(5): 507-511.

Summary: It is still challenging to prevent and treat respiratory infectious diseases. One critical step in the successful treatment of respiratory infections is rapid diagnosis by identifying the causative microorganisms in a timely fashion. However, traditional methods for identification of causative agents could not satisfy the need for rapid and accurate testing due to the limitations of technology-used. In recent years, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) has been validated and used for rapid identification of microorganism and for potential discovery of diseases associated biomarkers. We reviewed recent advances of MALDI-TOF-MS as the laboratory diagnostic tool for the rapid laboratory diagnosis of microorganisms associated with respiratory infectious diseases, with the focus on rapid identification of pathogenic bacteria and molecular markers discovery using MALDI-TOF-MS. With the advanced technologies such as MALDI-TOF, early and targeted therapies based on rapid identification of pathogens and could lead to quick and effective treatment of respiratory infections and better patient management.

Free full text available from PubMed PMID: 24822111

M) Application of Mass Spectrometry to Molecular Diagnostics of Viral Infections Ganova-Raeva LM, Khudyakov YE. Application of mass spectrometry to molecular diagnostics of viral infections. Expert Rev Mol Diagn. 2013; 13(4): 377-388.

Summary: Mass spectrometry (MS) has found numerous applications in life sciences. It has high accuracy, sensitivity and wide dynamic range in addition to medium- to high-throughput capabilities. These features make MS a superior platform for analysis of various biomolecules including proteins, lipids, nucleic acids and carbohydrates. Until recently, MS was applied for protein detection and characterization. During the last decade, however, MS has successfully been used for molecular diagnostics of microbial and viral infections with the most notable applications being identification of pathogens, genomic sequencing, mutation detection, DNA methylation analysis, tracking of transmissions, and characterization of genetic

heterogeneity. These new developments vastly expand the MS application from experimental research to public health and clinical fields. Matching of molecular techniques with specific requirements of the major MS platforms has produced powerful technologies for molecular diagnostics, which will further benefit from coupling with computational tools for extracting clinical information from MS-derived data.

Full text available from Expert Review of Molecular Diagnostics (USD 89.00 for 24 hour access) PMID: 23638820

1.2 Immunology as an Emerging Area for Precision Medicine

This section includes the following topics:

• Immunology & Transplantation (Section 1.2.1); and • HLA Genotyping (Section 1.2.1.1).

1.2.1 Immunology & Transplantation

Immunogenomics is at the intersection of genomics and immunology, and is one of the youngest, but fastest developing areas in genomic sciences. Considerable progress has been made in the analysis of the polymorphisms of various cytokines and immune response genes that play a pivotal role in susceptibility to infectious diseases and in the immune surveillance eliminating malignant cells.

Immune deficiency syndromes can now be characterized by means of genomic technologies such as next generation sequencing. Genomic analysis of the antibody repertoire reveals monoclonal and oligoclonal B-cell proliferation at an unprecedented sensitivity and will likely give rise to new methods for minimal residual disease detection and monitoring in multiple myeloma and other B-cell dyscrasias.

One of the most medically important regions of the human genome is the Major Histocompatibility Complex (MHC) located on chromosome 6. Within the MHC, there are more than 200 genes involved in the immune response, including the Class I and Class II Human Leukocyte Antigen (HLA) genes. The HLA genes are key molecular determinants of the adaptive immune response, and also control

the host immune response to bone marrow and solid-organ transplants. Consistent with a role in the adaptive immune response, the HLA genes are the most polymorphic genes in the human genome with over 12,000 alleles identified as of 2015 (http://hla.alleles.org/alleles/index.html). Due to the high degree of genetic polymorphism, HLA genotyping using standard DNA sequencing techniques is technically challenging. Recent work has demonstrated that next- generation sequencing technologies can improve HLA genotyping by virtue of clonal template amplification and sequencing. Specialized informatics software has also been developed to derive HLA genotyping information from next- generation sequencing data, including exome or whole-genome datasets. Adoption of HLA next-generation sequencing in the clinical laboratory is underway, enabling broader utilization of HLA genetic information for patient care.

1.2.1.1 Hot Topics Spotlight - HLA Genotyping

Hot Topic Next-generation sequencing for HLA genotyping: addressing the challenge Spotlight of extreme genetic diversity written by Matthew W. Anderson, MD, PhD, Medical Director, Diagnostic Laboratories, BloodCenter of Wisconsin, Milwaukee, WI

The human leukocyte antigen (HLA) genes represent the most diverse loci in the human genome with over 14,000 alleles identified as of December 2015.1 The high allelic diversity in the HLA genes reflects HLA protein function in binding and presenting a diverse array of peptide ligands derived from microbial pathogens. As a result of selection pressure from infectious organisms, the HLA system now constitutes one of the major genetic differences between individuals and among different ethnic populations. Therefore, the HLA genes are clinically relevant as key determinants of compatibility in organ and bone marrow transplantation and

in the genetic susceptibility and pathogenesis of immune-mediated diseases. However, the highly polymorphic HLA genes present unique challenges for the development of molecular approaches to genotype HLA alleles. This brief review will summarize the utility of next-generation sequencing (NGS) for HLA genotyping, highlighting the advantages of this approach over other molecular methods for typing HLA alleles.

To accurately determine an HLA genotype, phase (cis/trans) must be established between polymorphic positions both within individual exons and between different exons in the HLA gene. Using standard Sanger sequencing methodology, both alleles of a particular HLA locus are amplified and sequenced together resulting in multiple heterozygous positions in the electropherogram tracing (Figure 1A). As the phase of the polymorphic positions cannot be visually determined, additional steps are required to assign an HLA genotype. These include the use of informatics software to query the IMGT/HLA sequence database and assign the most likely combination of alleles, PCR amplification of only one allele, or the use of sequencing primers which anneal to only one of the two potential HLA alleles. In some instances, alternative allele pairs cannot be excluded, leading to genotype ambiguity. The additional steps required to generate HLA genotypes using Sanger sequencing are laborious and time- consuming, thus increasing the costs associated with HLA genotyping. As a consequence, only a few select exons of an entire HLA gene are routinely sequenced in clinical laboratories to determine a patient’s HLA genotype and ultimately the degree of HLA match between donor and recipient. A common feature of NGS technologies is that each fragment of DNA is amplified and sequenced independently, dramatically reducing the phase ambiguities encountered with Sanger sequencing (Figure 1B). Since 2009, many different approaches for using NGS for HLA genotyping have been reported using a variety of capture strategies and sequencing platforms.2 The first applications of NGS for HLA genotyping utilized the 454 platform as the read length of this technology (~ 250-500 bp) was sufficient to cover the average size of an HLA exon.3 Similar to Sanger approaches, the 454 HLA typing strategies utilized exon-targeted amplification (Figure 2A), which led to challenges in primer design and required numerous PCR reactions during library preparation. Although automation and microfluidic PCR technology were able to mitigate some of these issues, the amplicon-based sequencing approach was gradually replaced by a shotgun sequencing strategy (Figure 2B) in which long-range PCR is used to amplify each HLA locus in a single reaction.4-7 The resulting large PCR amplicons are fragmented to produce appropriately sized sequencing templates, and the short (100-250 bp) sequencing reads are aligned to re-create a full- length HLA sequence. The advantage of the long-range PCR and shotgun sequencing approach is that primers can be designed to anneal in less polymorphic regions of the HLA genes (5’ and 3’ UTR’s, for example), and more

of the HLA genetic sequence can be captured and sequenced. Paired-end sequencing can also be utilized to bioinformatically phase HLA sequence data over longer genetic distances (Figure 2C), including between exons. Multiple commercial assays that utilize the shotgun approach for HLA NGS are now available for the Ion Torrent (Thermo Fisher Scientific) and Illumina sequencing platforms.

One of the inherent limitations to the shotgun sequencing approach are the bioinformatic challenges associated with clustering and alignment of highly homologous, yet polymorphic sequence data. For example, novel sequence variants (representing HLA alleles not yet characterized in the IMGT/HLA sequence database) may lead to errors in alignment, producing incorrect HLA genotyping calls due to mismatches with the reference. To solve this problem, long-read sequencing technologies such as the PacBio RS II platform have been used to generate long (>3 kb) HLA sequence reads to create a consensus full- gene HLA sequence that can then be aligned to a reference database to generate an HLA genotype.8

Finally, bioinformatic approaches have also been developed to produce HLA genotyping information from targeted capture (exome) and non-targeted whole- genome sequence data.9 While these analysis pipelines perform generally well, a major hurdle is the relative lack of sequence coverage from the HLA region obtained through exome capture technologies and sequence read misalignment caused by pseudogene sequences. Despite these challenges, improvements to capture strategies and HLA genotyping software may soon offer the ability to generate accurate HLA genotyping from exome and whole-genome datasets. With the development of NGS and companion bioinformatic approaches for HLA genotyping, we now have the ability to better define genes within the HLA region, identifying polymorphisms that may elucidate the evolutionary history of HLA alleles, contribute to gene expression, and control disease risk.

Spotlight References: 1. Robinson J, Halliwell JA, Hayhurst JH, Flicek P, Parham P, Marsh SGE. The IPD and IMGT/HLA database: allele variant databases. Nucleic Acids Research. 2015; 43:D423-431. 2. Erlich, HA. HLA typing using next generation sequencing: an overview. Hum. Immunol. 2015. http://dx.doi.org/10.1016/j.humimm.2015.03.001. 3. Bentley G, Higuchi R, Hoglund B, Goodridge D, Sayer D, Trachtenberg EA, Erlich HA. High-resolution, high-throughput HLA genotyping by next- generation sequencing. Tissue Antigens. 2009; 74(5):393–403. 4. Lind C, Ferriola D, Mackiewicz S, Heron S, Rogers M, Slavich L, et al. Next- generation sequencing: the solution for high-resolution, unambiguous human leukocyte antigen typing. Hum. Immunol. 2010; 71(10):1033–42. 5. Shiina T, Suzuki S, Ozaki Y, Taira H, Kikkawa E, Shigenari A, et al. Super high resolution for single molecule-sequence-based typing of classical HLA loci at the 8-digit level using next generation sequencers. Tissue Antigens. 2012; 80(4):305–16. 6. Wang C, Krishnakumar S, Wilhelmy J, Babrzadeh F, Stepanyan L, Su LF., et al. High-throughput, high-fidelity HLA genotyping with deep sequencing. Proc. Natl. Acad Sci. U.S.A. 2012; 109(22):8676–81. 7. Hosomichi K, Jinam TA, Mitsunaga S, Nakaoka H, and Inoue I. Phase- defined complete sequencing of the HLA genes by next-generation sequencing. BMC Genomics. 2013; 14:355. 8. Mayor NP, Robinson J, McWhinnie, AJM, Ranade S, Eng, K, Midwinter, W, et al. HLA typing for the next generation. PLOS ONE. 2015; 10(5):e0127153. 9. Major E, Rigo K, Hague T, Berces A, Juhos S. HLA typing from 1000 genomes whole genome and whole exome Illumina data. PLOS ONE. 2013; 8(11):e78410.

Additional Resources:

A) HLA Typing by Next-Generation Sequencing - Getting Closer to Reality Gabriel C, Fürst D, Faé I, et al. HLA typing by next-generation sequencing - getting closer to reality. Tissue Antigens. 2014; 83(2): 65-75.

Summary: Next generation sequencing (NGS) denotes novel sequencing technologies that enable the generation of a large number of clonal sequences in a single sequencing run. NGS was initially introduced for whole genome sequencing and for quantitation of viral variants or genetic mutations

in tumor tissues; more recently, the potential for high resolution HLA typing and high throughput analyses has been explored. It became clear that the complexity of the HLA system implicates new challenges, especially for bioinformatics. From an economical point of view, NGS is becoming increasingly attractive for HLA typing laboratories currently relying on Sanger based sequencing. Realizing the full potential of NGS will require the development of specifically adapted typing strategies and software algorithms. In the present review, three laboratories that were among the first to perform HLA-typing using different NGS platforms, the Roche 454, the Illumina Miseq and the Ion Torrent system, respectively, give an overview of these applications and point out advantages and limitations.

Full text available from Tissue Antigens (USD 6.00-38.00) PMID: 24447174

B) Cost-Efficient High-Throughput HLA Typing by MiSeq Amplicon Sequencing Lange V, Böhme I, Hofmann J, et al. Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing. BMC Genomics. 2014;15:63.

Summary: BACKGROUND: A close match of the HLA alleles between donor and recipient is an important prerequisite for successful unrelated hematopoietic stem cell transplantation. To increase the chances of finding an unrelated donor, registries recruit many hundred thousands of volunteers each year. Many registries with limited resources have had to find a trade-off between cost and resolution and extent of typing for newly recruited donors in the past. Therefore, we have taken advantage of recent improvements in NGS to develop a workflow for low-cost, high-resolution HLA typing. RESULTS: We have established a straightforward three-step workflow for high-throughput HLA typing: Exons 2 and 3 of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 are amplified by PCR on Fluidigm Access Array microfluidic chips. Illumina sequencing adapters and sample specific tags are directly incorporated during PCR. Upon pooling and cleanup, 384 samples are sequenced in a single Illumina MiSeq run. We developed "neXtype" for streamlined data analysis and HLA allele assignment. The workflow was validated with 1140 samples typed at 6 loci. All neXtype results were concordant with the Sanger sequences, demonstrating error-free typing of more than 6000 HLA loci. Current capacity in routine operation is 12,000 samples per week. CONCLUSIONS: The workflow presented proved to be a cost-efficient alternative to Sanger sequencing for high-throughput HLA typing. Despite the focus on cost efficiency, resolution exceeds the current standards of Sanger typing for donor registration.

Free full text available from PubMed PMID: 24460756

C) An Integrated Tool to Study MHC Region: Accurate SNV Detection and HLA Genes Typing in Human MHC Region Using Targeted High- Throughput Sequencing Cao H, Wu J, Wang Y, et al. An integrated tool to study MHC region: accurate SNV detection and HLA genes typing in human MHC region using targeted high-throughput sequencing. PLoS One. 2013; 8(7): e69388.

Summary: The major histocompatibility complex (MHC) is one of the most variable and gene-dense regions of the human genome. Most studies of the MHC, and associated regions, focus on minor variants and HLA typing, many of which have been demonstrated to be associated with human disease susceptibility and metabolic pathways. However, the detection of variants in the MHC region, and diagnostic HLA typing, still lacks a coherent, standardized, cost effective and high coverage protocol of clinical quality and reliability. In this paper, we presented such a method for the accurate detection of minor variants and HLA types in the human MHC region, using high-throughput, high-coverage sequencing of target regions. A probe set was designed to template upon the 8 annotated human MHC haplotypes, and to encompass the 5 megabases (Mb) of the extended MHC region. We deployed our probes upon three, genetically diverse human samples for probe set evaluation, and sequencing data show that approximately 97% of the MHC region, and over 99% of the genes in MHC region, are covered with sufficient depth and good evenness. 98% of genotypes called by this capture sequencing prove consistent with established HapMap genotypes. We have concurrently developed a one-step pipeline for calling any HLA type referenced in the IMGT/HLA database from this target capture sequencing data, which shows over 96% typing accuracy when deployed at 4 digital resolution. This cost-effective and highly accurate approach for variant detection and HLA typing in the MHC region may lend further insight into immune-mediated diseases studies, and may find clinical utility in transplantation medicine research. This one-step pipeline is released for general evaluation and use by the scientific community.

Free full text available from PubMed PMID: 23894464

D) 16(th) IHIW: Review of HLA Typing by NGS De Santis D, Dinauer D, Duke J, et al. 16(th) IHIW: review of HLA typing by NGS. Int J Immunogenet. 2013; 40(1): 72-76.

Summary: Human leucocyte antigen (HLA) genes play an important role in the success of organ transplantation and are associated with autoimmune and infectious diseases. Current DNA-based genotyping methods, including Sanger sequence-based typing (SSBT), have identified a high degree of polymorphism. This level of polymorphism makes high-resolution HLA genotyping challenging, resulting in ambiguous typing results due to an inability to resolve phase and/or defining polymorphisms lying outside the region amplified. Next-generation sequencing (NGS) may resolve the issue through the combination of clonal amplification, which provides phase information, and the ability to sequence larger regions of genes, including introns, without the additional effort or cost associated with current methods. The NGS HLA sequencing project of the 16IHIW aimed to discuss the different approaches to (i) template preparation including short- and long- range PCR amplicons, exome capture and whole genome; (ii) sequencing platforms, including GS 454 FLX, Ion Torrent PGM, Illumina MiSeq/HiSeq and Pacific Biosciences SMRT; (iii) data analysis, specifically allele-calling software. The pilot studies presented at the workshop demonstrated that although individual sequencers have very different performance characteristics, all produced sequence data suitable for the resolution of HLA genotyping ambiguities. The developments presented at this workshop clearly highlight the potential benefits of NGS in the HLA laboratory.

Full text available from International Journal of Immunogenetics (USD 6.00- 38.00) PMID: 23302098

1.3 The Microbiome

This section includes the following topics:

• Insights from Adopter - Lynn Bry (Section 1.3.1)

1.3.1 Insights from Adopter - Lynn Bry, MD, PhD, FCAP

Lynn Bry, MD, PhD, FCAP, Directs the Massachusetts Host-Microbiome Center and is an Associate Professor of Pathology at Harvard Medical School. Clinically, she is Associate Medical Director in the BWH Clinical Microbiology Laboratory and an attending Pathologist in the Center for Advanced Molecular Diagnostics where she oversees the Pathogen WGS program and attends on the Clinical Pathology Molecular service. Dr. Bry also serves on national panels for the FDA

and College of American Pathologists to develop NGS for clinical diagnostics. Her group has also implemented NGS to identify resistance determinants and predict their mobilization capacity to aid in hospital infection control and national surveillance programs. Dr. Bry’s group was the first CLIA lab to join the FDA’s GenomeTrakr network, and through this program supports an ongoing prospective pathogen surveillance program across 4 hospitals, 2 cancer centers and multiple outpatient practices in the greater Boston area.

Dr. Bry’s research focuses on study of the dynamic interactions among host, pathogens and commensals, and has developed integrated human clinical trials with animal model “avatars” to identify protective and dysbiotic communities that contribute to a variety of conditions, including allergic and auto-immune diseases, and host metabolism of dietary and toxic compounds.

The Massachusetts Host-Microbiome Center supports >70 groups evaluating contributions of the host’s microbiota in health and disease. With a focus on function, Dr. Bry’s group and that of other Center faculty have developed defined microbial communities for therapeutic applications, and CLIA-level testing to help direct the use of therapies that manipulate the microbiota. In 2015, the Center was awarded a $5 million grant from the Massachsuetts Life Sciences Center to expand its resources in support of academic-industry collaborations. This funding is expanding the Center’s gnotobiotic (germfree) resources to a 60-isolator facility plus 600 high-throughput cage containment system, enhancing automation for microbiology, metabolite and sequence-based methods, and providing additional computational infrastructure to support the center’s development of principled computational models to study microbiota dynamics.

Dr. Bry’s insights for the next wave of adopters (June 2016):

1 The field is still very Diagnostic applications using or incorporating much in its infancy. microbiota signatures will arise as we move from descriptive associations of microbes and host outcomes to understanding what communities are causative of a particular effect. At that point we have new opportunities to therapeutic interventions and for diagnostics to understand when to treat and how to follow responses to therapies.

2 Microbiome studies Thus, the two are often linked. However, for have been enabled CLIA labs, infectious disease applications may by NGS. be the first examples by which a pathology practice would implement NGS-based methods to study microbes. Applications include looking at viral populations (HIV or HCV genotyping), bacterial genomes (ID of slow-growing species, resistance prediction and surveillance), and WGS metagenomics to broadly survey for pathogens in specific populations such as transplant and other immunocompromised patients. 3 Pathologists will Microbiome studies evaluating community need to be more effects require principled models to assess the versed on dynamics of complex ecosystems, often computational comprised of hundreds of species, and to be analyses, able to predict a disease outcome from communicating well microbiome and related host information – be it with computer clinical, biochemical, immunologic, etc. scientists and Research and eventual clinical diagnostics that computational look at broad community signatures will need biologists sophisticated computational methods for analyses, methods that are more likely to have been developed by non-Pathologists. As such, Pathologists will need to be more versed with these types of computational analyses, how to communicate with computer scientists and computational biologists developing such methods, and how to implement and manage them in a CLIA environment. 4 Support of As we understand causative effects of the “microbiome microbiota in vivo, the most sensitive and applications” need specific assays will focus on the pathways and not be with only NGS products of importance from microbial methods. communities. For example, some measures of dysbiosis may look at bile acid profiles or the host’s absorption of microbially-produced products rather than indirect measures such as microbial community structure or metagenomes. Consider what assays already performed in your practice could be leveraged to help identify microbiota-mediated effects in vivo.

5 Consider the role of In terms of using therapeutic microbiota (“bugs the Pathologist in as drugs”) – consider the role of the blood banking Pathologist in blood banking, so might Pathology ClinMicro labs be extended to manage the therapeutic use of microbial communities, whether the therapeutic materials originate from vendors or other sources. 6 Microbiome factors As genomics has permeated all branches of will become clinical medicine to further understand of integrated in our pathophysiologic contributions to disease, so understanding of microbiome factors will become integrated in health and disease our understanding of health and disease. The importance of the microbiota in areas outside of the exposure bodily surfaces and the alimentary tract has already been shown, including effects on brain development, autoimmune diseases, and drug pharmacology.

Dr. Bry’s suggested reading on this topic:

A) The Colonic Microbiota in Health and Disease Shanahan F. The Colonic Microbiota in Health and Disease. Curr Opin Gastroenterol. 2013; 29(1): 49-54.

Summary: PURPOSE OF REVIEW: Diverse research interests have converged on the gut microbiota because of its contribution to immune development, mucosal homeostasis and to the pathogenesis of a diversity of intestinal and extraintestinal disorders. Recent landmark findings are addressed here. RECENT FINDINGS: The impact of lifestyle, including dietary changes and antibiotics, on the microbiota has been mechanistically linked with disease risk. Microbial, immune and metabolic signalling are mutually interactive, with each of these being regulated by diet. Although changes in the microbiota have been found in several disorders and may have important therapeutic implications, some components of the commensal microbiota may behave like pathogens (pathobionts) depending on the context and host susceptibility. SUMMARY: Advances in understanding host-microbe interactions in the gut continue apace, they are relevant to a diversity of infectious, inflammatory, neoplastic and metabolic disorders and are poised for clinical translation.

Free full text available from Current Opinion in Gastroenterology PMID: 23041677

B) The Skin Microbiome Grice EA,Segre JA. The Skin Microbiome. Nat Rev Microbiol. 2011; 9(4): 244-253.

Summary: The skin is the human body's largest organ, colonized by a diverse milieu of microorganisms, most of which are harmless or even beneficial to their host. Colonization is driven by the ecology of the skin surface, which is highly variable depending on topographical location, endogenous host factors and exogenous environmental factors. The cutaneous innate and adaptive immune responses can modulate the skin microbiota, but the microbiota also functions in educating the immune system. The development of molecular methods to identify microorganisms has led to an emerging view of the resident skin bacteria as highly diverse and variable. An enhanced understanding of the skin microbiome is necessary to gain insight into microbial involvement in human skin disorders and to enable novel promicrobial and antimicrobial therapeutic approaches for their treatment.

Free full text available from PubMed PMID: 21407241

C) Gut Biogeography of the Bacterial Microbiota Donaldson GP, Lee SM,Mazmanian SK. Gut Biogeography of the Bacterial Microbiota. Nat Rev Microbiol. 2016; 14(1): 20-32.

Summary: Animals assemble and maintain a diverse but host-specific gut microbial community. In addition to characteristic microbial compositions along the longitudinal axis of the intestines, discrete bacterial communities form in microhabitats, such as the gut lumen, colonic mucus layers and colonic crypts. In this Review, we examine how the spatial distribution of symbiotic bacteria among physical niches in the gut affects the development and maintenance of a resilient microbial ecosystem. We consider novel hypotheses for how nutrient selection, immune activation and other mechanisms control the biogeography of bacteria in the gut, and we discuss the relevance of this spatial heterogeneity to health and disease.

Free full text available from Nature PMID: 26499895

D) Systematic Review of Intestinal Microbiota Transplantation (Fecal Bacteriotherapy) for Recurrent Clostridium Difficile Infection Gough E, Shaikh H, Manges AR. Systematic Review of Intestinal Microbiota Transplantation (Fecal Bacteriotherapy) for Recurrent Clostridium Difficile Infection. Clin Infect Dis. 2011; 53(10): 994-1002.

Summary: Clostridium difficile infection (CDI) is a gastrointestinal disease believed to be causally related to perturbations to the intestinal microbiota. When standard treatment has failed, intestinal microbiota transplantation (IMT) is an alternative therapy for patients with CDI. IMT involves infusing intestinal microorganisms (in a suspension of healthy donor stool) into the intestine of a sick patient to restore the microbiota. However, protocols and reported efficacy for IMT vary. We conducted a systematic literature review of IMT treatment for recurrent CDI and pseudomembranous colitis. In 317 patients treated across 27 case series and reports, IMT was highly effective, showing disease resolution in 92% of cases. Effectiveness varied by route of instillation, relationship to stool donor, volume of IMT given, and treatment before infusion. Death and adverse events were uncommon. These findings can guide physicians interested in implementing the procedure until better designed studies are conducted to confirm best practices.

Free full text available from Clinical Infectious Disease PMID: 22002980

E) Intestinal Microbial Metabolism of Phosphatidylcholine and Cardiovascular Risk Tang WH, Wang Z, Levison BS, et al. Intestinal Microbial Metabolism of Phosphatidylcholine and Cardiovascular Risk. N Engl J Med. 2013; 368(17): 1575-1584.

Summary: BACKGROUND: Recent studies in animals have shown a mechanistic link between intestinal microbial metabolism of the choline moiety in dietary phosphatidylcholine (lecithin) and coronary artery disease through the production of a proatherosclerotic metabolite, trimethylamine-N- oxide (TMAO). We investigated the relationship among intestinal microbiota- dependent metabolism of dietary phosphatidylcholine, TMAO levels, and adverse cardiovascular events in humans. METHODS: We quantified plasma and urinary levels of TMAO and plasma choline and betaine levels by means of liquid chromatography and online tandem mass spectrometry after a phosphatidylcholine challenge (ingestion of two hard-boiled eggs and deuterium [d9]-labeled phosphatidylcholine) in healthy participants before and after the suppression of intestinal microbiota with oral broad-spectrum antibiotics. We further examined the relationship between fasting plasma levels of TMAO and incident major adverse cardiovascular events (death, myocardial infarction, or stroke) during 3 years of follow-up in 4007 patients undergoing elective coronary angiography. RESULTS: Time-dependent increases in levels of both TMAO and its d9 isotopologue, as well as other choline metabolites, were detected after the phosphatidylcholine challenge. Plasma levels of TMAO were markedly suppressed after the administration of antibiotics and then reappeared after withdrawal of antibiotics. Increased plasma levels of TMAO were associated with an increased risk of a major

adverse cardiovascular event (hazard ratio for highest vs. lowest TMAO quartile, 2.54; 95% confidence interval, 1.96 to 3.28; P<0.001). An elevated TMAO level predicted an increased risk of major adverse cardiovascular events after adjustment for traditional risk factors (P<0.001), as well as in lower-risk subgroups. CONCLUSIONS: The production of TMAO from dietary phosphatidylcholine is dependent on metabolism by the intestinal microbiota. Increased TMAO levels are associated with an increased risk of incident major adverse cardiovascular events. (Funded by the National Institutes of Health and others.).

Free full text available from PubMed PMID: 23614584

F) The Role of Microbiota in Cancer Therapy Perez-Chanona E, Trinchieri G. The Role of Microbiota in Cancer Therapy. Curr Opin Immunol. 2016; 39: 75-81.

Summary: The relationship between the host and the commensal microbiota regulates physiological functions including inflammation and immunity and it has been scrutinized in the context of cancer. While viruses and bacterial species have been implicated in oncogenesis, commensal microbes also have a beneficial role in the fight against cancer. Therapy efficacy, including adoptive T cell transfer, alkylating agents and immune checkpoint blockers, relies on immunity that receives its education from the gut microbiota. In cancer therapy with immunostimulating oligonucleotides and platinum salts, the microbiota also modulates the response by priming for the release of proinflammatory factors and reactive oxygen species, respectively. This new information offers promising clinical possibilities of modulating cancer therapy and its toxic side effects by targeting the microbiota.

Full text available from Current Opinion in Immunology (USD 35.95) PMID: 26820225

G) Of Bugs and Drugs: How the Microbiome Controls Responsiveness to Antitumor Immunotherapy Ford M. Of Bugs and Drugs: How the Microbiome Controls Responsiveness to Antitumor Immunotherapy. American Journal of Transplantation. 2016; 16(1): 3.

Summary: A pair of new studies reveals that the composition of the microbiome alters efficacy of immune checkpoint blockade in mouse cancer models.

Free full text available from American Journal of Transplantation

Return to Table of Contents Section 1

H) Mind-Altering Microorganisms: The Impact of the Gut Microbiota on Brain and Behaviour Cryan JF, Dinan TG. Mind-Altering Microorganisms: The Impact of the Gut Microbiota on Brain and Behaviour. Nat Rev Neurosci. 2012; 13(10): 701- 712.

Summary: Recent years have witnessed the rise of the gut microbiota as a major topic of research interest in biology. Studies are revealing how variations and changes in the composition of the gut microbiota influence normal physiology and contribute to diseases ranging from inflammation to obesity. Accumulating data now indicate that the gut microbiota also communicates with the CNS--possibly through neural, endocrine and immune pathways--and thereby influences brain function and behaviour. Studies in germ-free animals and in animals exposed to pathogenic bacterial infections, probiotic bacteria or antibiotic drugs suggest a role for the gut microbiota in the regulation of anxiety, mood, cognition and pain. Thus, the emerging concept of a microbiota-gut-brain axis suggests that modulation of the gut microbiota may be a tractable strategy for developing novel therapeutics for complex CNS disorders.

Full text available from Nature Reviews Neuroscience (USD 32.00) PMID: 22968153

Additional Resources:

A) An Integrated Catalog of Reference Genes in the Human Gut Microbiome Li J, Jia H, Cai X, et al. An Integrated Catalog of Reference Genes in the Human Gut Microbiome. Nat Biotechnol. 2014; 32(8): 834-841.

Summary: Many analyses of the human gut microbiome depend on a catalog of reference genes. Existing catalogs for the human gut microbiome are based on samples from single cohorts or on reference genomes or protein sequences, which limits coverage of global microbiome diversity. Here we combined 249 newly sequenced samples of the Metagenomics of the Human Intestinal Tract (MetaHit) project with 1,018 previously sequenced samples to create a cohort from three continents that is at least threefold larger than cohorts used for previous gene catalogs. From this we established the integrated gene catalog (IGC) comprising 9,879,896 genes. The catalog includes close-to-complete sets of genes for most gut microbes, which are also of considerably higher quality than in previous catalogs. Analyses of a group of samples from Chinese and Danish individuals using the catalog revealed country-specific gut microbial signatures. This expanded catalog should facilitate quantitative characterization of metagenomic,

metatranscriptomic and metaproteomic data from the gut microbiome to understand its variation across populations in human health and disease.

Full text available from Nature Biotechnology (USD 32.00) PMID: 24997786

B) Structure, Function and Diversity of the Healthy Human Microbiome Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature. 2012;486(7402):207-14. doi:10.1038/nature11234.

Summary: Studies of the human microbiome have revealed that even healthy individuals differ remarkably in the microbes that occupy habitats such as the gut, skin and vagina. Much of this diversity remains unexplained, although diet, environment, host genetics and early microbial exposure have all been implicated. Accordingly, to characterize the ecology of human- associated microbial communities, the Human Microbiome Project has analysed the largest cohort and set of distinct, clinically relevant body habitats so far. We found the diversity and abundance of each habitat's signature microbes to vary widely even among healthy subjects, with strong niche specialization both within and among individuals. The project encountered an estimated 81-99% of the genera, enzyme families and community configurations occupied by the healthy Western microbiome. Metagenomic carriage of metabolic pathways was stable among individuals despite variation in community structure, and ethnic/racial background proved to be one of the strongest associations of both pathways and microbes with clinical metadata. These results thus delineate the range of structural and functional configurations normal in the microbial communities of a healthy population, enabling future characterization of the epidemiology, ecology and translational applications of the human microbiome.

Free full text available from Nature PMID: 22699609 NOTE: Also cited in Section 5.2.2.4

C) Rapid Metagenomic Identification of Viral Pathogens in Clinical Samples by Real-Time Nanopore Sequencing Analysis Greninger AL, Naccache SN, Federman S, et al. Rapid Metagenomic Identification of Viral Pathogens in Clinical Samples by Real-Time Nanopore Sequencing Analysis. Genome Med. 2015; 7(1): 99.

Summary: We report unbiased metagenomic detection of chikungunya virus (CHIKV), Ebola virus (EBOV), and hepatitis C virus (HCV) from four human blood samples by MinION nanopore sequencing coupled to a newly developed, web-based pipeline for real-time bioinformatics analysis on a computational server or laptop (MetaPORE). At titers ranging from 10(7)- 10(8) copies per milliliter, reads to EBOV from two patients with acute hemorrhagic fever and CHIKV from an asymptomatic blood donor were detected within 4 to 10 min of data acquisition, while lower titer HCV virus (1 x 10(5) copies per milliliter) was detected within 40 min. Analysis of mapped nanopore reads alone, despite an average individual error rate of 24 % (range 8-49 %), permitted identification of the correct viral strain in all four isolates, and 90 % of the genome of CHIKV was recovered with 97-99 %

accuracy. Using nanopore sequencing, metagenomic detection of viral pathogens directly from clinical samples was performed within an unprecedented <6 hr sample-to-answer turnaround time, and in a timeframe amenable to actionable clinical and public health diagnostics.

Free full text available from PubMed PMID: 26416663

D) Disentangling Type 2 Diabetes and Metformin Treatment Signatures in the Human Gut Microbiota Forslund K, Hildebrand F, Nielsen T, et al. Disentangling Type 2 Diabetes and Metformin Treatment Signatures in the Human Gut Microbiota. Nature. 2015; 528(7581): 262-266.

Summary: In recent years, several associations between common chronic human disorders and altered gut microbiome composition and function have been reported. In most of these reports, treatment regimens were not controlled for and conclusions could thus be confounded by the effects of various drugs on the microbiota, which may obscure microbial causes, protective factors or diagnostically relevant signals. Our study addresses disease and drug signatures in the human gut microbiome of type 2 diabetes mellitus (T2D). Two previous quantitative gut metagenomics studies of T2D patients that were unstratified for treatment yielded divergent conclusions regarding its associated gut microbial dysbiosis. Here we show, using 784 available human gut metagenomes, how antidiabetic medication confounds these results, and analyse in detail the effects of the most widely used antidiabetic drug metformin. We provide support for microbial mediation of the therapeutic effects of metformin through short-chain fatty acid production, as well as for potential microbiota-mediated mechanisms behind known intestinal adverse effects in the form of a relative increase in abundance of Escherichia species. Controlling for metformin treatment, we report a unified signature of gut microbiome shifts in T2D with a depletion of butyrate- producing taxa. These in turn cause functional microbiome shifts, in part alleviated by metformin-induced changes. Overall, the present study emphasizes the need to disentangle gut microbiota signatures of specific human diseases from those of medication.

Full text available from Nature (USD 32.00) PMID: 26633628

E) Update on Fecal Microbiota Transplantation 2015: Indications, Methodologies, Mechanisms, and Outlook Kelly CR, Kahn S, Kashyap P, et al. Update on Fecal Microbiota Transplantation 2015: Indications, Methodologies, Mechanisms, and Outlook. Gastroenterology. 2015; 149(1): 223-237.

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Summary: The community of microorganisms within the human gut (or microbiota) is critical to health and functions with a level of complexity comparable to that of an organ system. Alterations of this ecology (or dysbiosis) have been implicated in a number of disease states, and the prototypical example is Clostridium difficile infection (CDI). Fecal microbiota transplantation (FMT) has been demonstrated to durably alter the gut microbiota of the recipient and has shown efficacy in the treatment of patients with recurrent CDI. There is hope that FMT may eventually prove beneficial for the treatment of other diseases associated with alterations in gut microbiota, such as inflammatory bowel disease, irritable bowel syndrome, and metabolic syndrome, to name a few. Although the basic principles that underlie the mechanisms by which FMT shows therapeutic efficacy in CDI are becoming apparent, further research is needed to understand the possible role of FMT in these other conditions. Although relatively simple to perform, questions regarding both short-term and long- term safety as well as the complex and rapidly evolving regulatory landscape has limited widespread use. Future work will focus on establishing best practices and more robust safety data than exist currently, as well as refining FMT beyond current "whole-stool" transplants to increase safety and tolerability. Encapsulated formulations, full-spectrum stool-based products, and defined microbial consortia are all in the immediate future.

Free full text available from Gastroenterology PMID: 25982290

F) Diagnosis of Neuroinvasive Astrovirus Infection in an Immunocompromised Adult with Encephalitis by Unbiased Next- Generation Sequencing Naccache SN, Peggs KS, Mattes FM, et al. Diagnosis of Neuroinvasive Astrovirus Infection in an Immunocompromised Adult with Encephalitis by Unbiased Next-Generation Sequencing. Clin Infect Dis. 2015; 60(6): 919- 923.

Summary: Metagenomic next-generation sequencing (NGS) was used to diagnose an unusual and fatal case of progressive encephalitis in an immunocompromised adult presenting at disease onset as bilateral hearing loss. The sequencing and confirmatory studies revealed neuroinvasive infection of the brain by an astrovirus belonging to a recently discovered VA/HMO clade.

Free full text available from PubMed PMID: 25572898

G) New Insight into the Gut Microbiome Through Metagenomics Ji B, Nielsen J. New insight into the gut microbiome through metagenomics. Advances in Genomics and Genetics. 2015; 5:77-91.

Free full text available from Advances in Genomics and Genetics

H) Comparison of Three Next-Generation Sequencing Platforms for Metagenomic Sequencing and Identification of Pathogens in Blood Frey KG, Herrera-Galeano JE, Redden CL, et al. Comparison of three next- generation sequencing platforms for metagenomic sequencing and identification of pathogens in blood. BMC Genomics. 2014; 15: 96.

Summary: BACKGROUND: The introduction of benchtop sequencers has made adoption of whole genome sequencing possible for a broader community of researchers than ever before. Concurrently, metagenomic sequencing (MGS) is rapidly emerging as a tool for interrogating complex samples that defy conventional analyses. In addition, next-generation sequencers are increasingly being used in clinical or related settings, for instance to track outbreaks. However, information regarding the analytical sensitivity or limit of detection (LoD) of benchtop sequencers is currently lacking. Furthermore, the specificity of sequence information at or near the LoD is unknown. RESULTS: In the present study, we assess the ability of three next-generation sequencing platforms to identify a pathogen (viral or bacterial) present in low titers in a clinically relevant sample (blood). Our results indicate that the Roche-454 Titanium platform is capable of detecting Dengue virus at titers as low as 1X102.5 pfu/mL, corresponding to an estimated 5.4X104 genome copies/ml maximum. The increased throughput of the benchtop sequencers, the Ion Torrent PGM and Illumina MiSeq platforms, enabled detection of viral genomes at concentrations as low as 1X104 genome copies/mL. Platform-specific biases were evident in sequence read distributions as well as viral genome coverage. For bacterial samples, only the MiSeq platform was able to provide sequencing reads that could be unambiguously classified as originating from Bacillus anthracis. CONCLUSION: The analytical sensitivity of all three platforms approaches that of standard qPCR assays. Although all platforms were able to detect pathogens at the levels tested, there were several noteworthy differences. The Roche-454 Titanium platform produced consistently longer reads, even when compared with the latest chemistry updates for the PGM platform. The MiSeq platform produced consistently greater depth and breadth of coverage, while the Ion Torrent was unequaled for speed of sequencing. None of the platforms were able to verify a single nucleotide polymorphism responsible for antiviral resistance in an Influenza A strain isolated from the 2009 H1N1 pandemic. Overall, the benchtop platforms perform well for

identification of pathogens from a representative clinical sample. However, unlike identification, characterization of pathogens is likely to require higher titers, multiple libraries and/or multiple sequencing runs.

Free full text available from PubMed PMID: 24495417

I) Actionable Diagnosis of Neuroleptospirosis by Next-Generation Sequencing Wilson MR, Naccache SN, Samayoa E, et al. Actionable Diagnosis of Neuroleptospirosis by Next-Generation Sequencing. N Engl J Med. 2014; 370(25):2408-17.

Summary: A 14-year-old boy with severe combined immunodeficiency presented three times to a medical facility over a period of 4 months with fever and headache that progressed to hydrocephalus and status epilepticus necessitating a medically induced coma. Diagnostic workup including brain biopsy was unrevealing. Unbiased next-generation sequencing of the cerebrospinal fluid identified 475 of 3,063,784 sequence reads (0.016%) corresponding to leptospira infection. Clinical assays for leptospirosis were negative. Targeted antimicrobial agents were administered, and the patient was discharged home 32 days later with a status close to his premorbid condition. Polymerase-chain-reaction (PCR) and serologic testing at the Centers for Disease Control and Prevention (CDC) subsequently confirmed evidence of Leptospira santarosai infection.

Free full text available from New England Journal of Medicine PMID: 24896819

J) Fecal Microbiota Transplantation in Inflammatory Bowel Disease: Beyond the Excitement Ianiro G, Bibbo S, Scaldaferri F, Gasbarrini A, Cammarota G. Fecal microbiota transplantation in inflammatory bowel disease: beyond the excitement. Medicine (Baltimore). 2014; 93(19): e97.

Summary: The purpose of this article is to perform a systematic review of the literature on the use of fecal microbiota transplantation (FMT) in inflammatory bowel disease (IBD).There is an increasing interest of both physicians and patients in assessing the possible role of the FMT in the treatment of IBD.Electronic and manual bibliographic searches were performed to identify original reports in which subjects with IBD were treated with FMT. Because of the scarcity of studies with adequate sample size, case series and case reports were also considered. A critical appraisal of the

clinical research evidence on the effectiveness, safety, and other parameters related to FMT was made. Data extraction was independently performed by 2 reviewers.We found a total of 31 publications on the use of FMT in IBD. The majority were case reports or case series, whereas 8 publications reported data from open-label trials including a very less number of patients. A total of 133 patients with IBD were managed with FMT. Of these, 57 subjects (43%) had a Clostridium difficile infection. A resolution or reduction of symptoms was reported in 80 of 113 (71%) patients with evaluable IBD. Moreover, FMT does not seem to provide the same safety profile showed for non-IBD individuals with C difficile infection.The available evidence is limited and weak. FMT has the potential to be somehow of help in managing patients with IBD, but considerable further efforts are necessary to make this procedure a valid option for these subjects.

Full text available from Medicine (subscription required) PMID: 25340496

K) Comprehensive Profiling of the Vaginal Microbiome in HIV Positive Women Using Massive Parallel Semiconductor Sequencing Ameur A, Meiring TL, Bunikis I, et al. Comprehensive profiling of the vaginal microbiome in HIV positive women using massive parallel semiconductor sequencing. Sci Rep. 2014;4:4398.

Summary: Infections by HIV increase the risk of acquiring secondary viral and bacterial infections and methods are needed to determine the spectrum of co-infections for proper treatment. We used rolling circle amplification (RCA) and Ion Proton sequencing to investigate the vaginal microbiome of 20 HIV positive women from South Africa. A total of 46 different human papillomavirus (HPV) types were found, many of which are not detected by existing genotyping assays. Moreover, the complete genomes of two novel HPV types were determined. Abundance of HPV infections was highly correlated with real-time PCR estimates, indicating that the RCA-Proton method can be used for quantification of individual pathogens. We also identified a large number of other viral, bacterial and parasitic co-infections and the spectrum of these co-infections varied widely between individuals. Our method provides rapid detection of a broad range of pathogens and the ability to reconstruct complete genomes of novel infectious agents.

Free full text available from PubMed PMID: 24637939

L) Diet Rapidly and Reproducibly Alters the Human Gut Microbiome David LA, Maurice CF, Carmody RN, et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature. 2014; 505(7484): 559-563.

Summary: Long-term dietary intake influences the structure and activity of the trillions of microorganisms residing in the human gut, but it remains unclear how rapidly and reproducibly the human gut microbiome responds to short-term macronutrient change. Here we show that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression. The animal-based diet increased the abundance of bile-tolerant microorganisms (Alistipes, Bilophila and Bacteroides) and decreased the levels of Firmicutes that metabolize dietary plant polysaccharides (Roseburia, Eubacterium rectale and Ruminococcus bromii). Microbial activity mirrored differences between herbivorous and carnivorous mammals, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi and even viruses. Finally, increases in the abundance and activity of Bilophila wadsworthia on the animal-based diet support a link between dietary fat, bile acids and the outgrowth of microorganisms capable of triggering inflammatory bowel disease. In concert, these results demonstrate that the gut microbiome can rapidly respond to altered diet, potentially facilitating the diversity of human dietary lifestyles.

Free full text available from Nature PMID: 24336217

1.4 Regulatory Framework for Precision Medicine

A) Regulatory Considerations for Companion Diagnostic Devices Lee EY, Shen HC. Regulatory Considerations for Companion Diagnostic Devices. Biomark Med. 2015; 9(1): 67-75.

Summary: The emergence of companion diagnostic devices has been spurred by drug discovery and development efforts towards targeted therapies, particularly in oncology. Companion diagnostics and their corresponding therapeutics are often codeveloped, or developed in parallel, to ensure the safe and effective use of the products. The regulatory framework for companion diagnostics has gradually evolved as a result of the essential role of diagnostic tests to identify the intended population for a corresponding treatment. Here, we describe the current regulatory model for companion diagnostics in the US and outline key strategies for a successful codevelopment program from the device perspective. We also discuss how

technological advances and changes in clinical management may challenge the regulatory model in the future.

Full text available from Biomarkers in Medicine (USD 60.00) PMID: 25605456

B) FDA Regulation of Laboratory-Developed Diagnostic Tests: Protect the Public, Advance the Science Sharfstein J. FDA Regulation of Laboratory-Developed Diagnostic Tests: Protect the Public, Advance the Science. JAMA. 2015; 313(7): 667-668.

Summary: Should the FDA regulate laboratory-developed diagnostic tests? —Yes. In April 2014, the Centers for Disease Control and Prevention and the US Food and Drug Administration (FDA) published a warning in Morbidity and Mortality Weekly Report about a commercially available test for Lyme infection. The test returned the result “culture positive,” when in fact the procedure was far more complex than a routine culture. There also were “serious concerns about false-positive results caused by laboratory contamination,” leading to “the potential for misdiagnosis.”1

Full text available from Journal of American Medical Association (USD 30.00) PMID: 25560381

C) Precision Medicine and the FDA's Draft Guidance on Laboratory- Developed Tests Hwang TJ, Lehmann LS, Kesselheim AS. Precision Medicine and the FDA's Draft Guidance on Laboratory-Developed Tests. Nat Biotechnol. 2015; 33(5): 449-451.

Summary: To the Editor: In October 2014, the US Food and Drug Administration (FDA) issued a draft guidance outlining its plan to overhaul the regulation of in vitro diagnostics, which are tests conducted outside of a living body to detect or diagnose diseases, conditions and infections1, 2. Most in vitro diagnostics, specifically…

Full text available from Nature Biotechnology (USD 18.00) PMID: 25965745

D) Cutting the Gordian Helix - Regulating Genomic Testing in the Era of Precision Medicine Lander ES. Cutting the Gordian Helix - Regulating Genomic Testing in the Era of Precision Medicine. N Engl J Med. 2015;372(13):1185-6.

Summary: In his 2015 State of the Union address, President Barack Obama announced a new Precision Medicine Initiative (PMI), a national investment

in research on approaches to disease treatment and prevention that take into account individual variability in each person's genes, environment, and lifestyle. In a recent Perspective article, Francis Collins and Harold Varmus sketched out initial ideas for the PMI research plans as envisioned by the National Institutes of Health (NIH).1 But scientific progress alone won't guarantee that the public reaps the full benefits of precision medicine - an achievement that, as Collins and Varmus note, "will also require advancing…”

Full text available from New England Journal of Medicine (USD 20.00) PMID: 25689017

E) Revisiting Oversight and Regulation of Molecular-based Laboratory- Developed Tests: A Position Statement of the Association for Molecular Pathology Ferreira-Gonzalez A, Emmadi R, Day SP, et al. Revisiting oversight and regulation of molecular-based laboratory-developed tests: a position statement of the Association for Molecular Pathology. J Mol Diagn. 2014; 16(1): 3-6.

Summary: Since 2006, the US Food and Drug Administration, Congress, and other policymakers have explored the appropriate way to guarantee the clinical and analytical validity of laboratory-developed tests. In the past, the Association for Molecular Pathology has publicly urged the Food and Drug Administration to exercise caution in implementing regulatory changes that could potentially hinder innovation or interfere with the practice of medicine. In 2012, the Association for Molecular Pathology Professional Relations Committee chose to develop this paper with the goal of outlining the best methods for ensuring appropriate oversight and validation of molecular diagnostic procedures. At the conclusion of this process, the workgroup reaffirmed the Association's previous position that the Centers for Medicare and Medicaid Services Clinical Laboratory Improvement Amendments program can provide the appropriate level of oversight for the vast majority of diagnostic tests.

Free full text available from Journal of Molecular Diagnostics PMID: 24331365

F) United States FDA's Emergency Use Authorization of Ebola Virus Diagnostics: Current Impact and Lessons for the Future Whitehouse CA, Bavari S, Perkins MD. United States FDA's Emergency Use Authorization of Ebola Virus Diagnostics: Current Impact and Lessons for the Future. Expert Rev Mol Diagn. 2015; 15(10): 1231-1235.

Summary: The Ebola outbreak that took hold in West Africa in 2014 outran the epidemic response capacity of many organizations. Five months after the epidemic was first declared, there were still only two laboratories in West Africa with the capacity to confirm Ebola virus infection. In the summer of 2014, before the first case of imported Ebola occurred in the USA, the US FDA announced it would issue Emergency Use Authorizations for Ebola virus in vitro diagnostics to speed their availability. Between October 2014 and March 2015, the FDA issued Emergency Use Authorizations for nine diagnostic products. The actions of the FDA not only allowed nationwide deployment of Ebola virus testing capacity in the USA but also established an attractive regulatory goalpost for companies developing assays for use in West Africa. Here, we comment on the diagnostic assays for which the FDA has issued emergency authorizations and their fitness for purpose.

Free full text available from Expert Review of Molecular Diagnostics PMID: 26394699

G) CAP’s Brief Leaders in Genomic Medicine

Summary: Although researchers have just begun exploring disease at the molecular level, CAP recognizes the importance of personalized medicine to patient care, and the impact it will have on the future of pathology. In addition to R&D roles and changes in diagnostics, pathologists are the stewards of medical test data, including molecular and genomic analytics, and the CAP pursues a policy agenda that retains the pathologist’s physician role in genomic testing and analysis, and data management.

Free full text available from CAP

1.5 Reimbursement

A) Molecular Coding and Reimbursement http://www.cap.org/apps/docs/practice_management/molecular_pathology_h cpcs_g_code.pdf

B) G-code for Medicare Molecular Pathology Physician Interpretation CAP reaffirms that Medicare physicians who interpret molecular tests and prepare written reports above and beyond laboratory results should already be billing for that service using the physician fee schedule (PFS) HCPCS G- code G0452 (molecular pathology procedure; physician interpretation and report).

“We encourage anyone performing these services to consult with your billing personnel to ensure the G-code is being used where appropriate,” said Economic Affairs Committee Chair Jonathan Myles, MD, FCAP. “CMS has indicated that it would monitor utilization of the code and reassess the need for the code based on utilization. It’s important to capture this information as accurately as possible.”

CMS announced the creation of the G-code in the 2013 PFS, to be used when physician interpretation of a molecular pathology test is medically necessary to provide a clinically meaningful, beneficiary-specific result. However, there have been reports that some institutions still have not added the G-code G0452 to their billing system.

CMS’ announcement in the 2013 final rule also included specific instruction and criteria associated with the new physician interpretation code:

This professional component-only HCPCS G-code will be considered a “clinical laboratory interpretation service” which is one of the current categories of PFS pathology services under the definition of physician pathology services at §415.130(b)(4).

Section §415.130(b)(4) of the regulations and section 60 of the Claims Processing Manual (IOM 100-04, Ch. 12, section 60.E.) specify certain requirements for billing the professional component of certain clinical laboratory services including that the interpretation (1) must be requested by the patient’s attending physician, (2) must result in a written narrative report included in the patient’s medical record, and (3) requires the exercise of medical judgment by the consultant physician.

We note that a hospital’s standing order policy can be used as a substitute for the individual request by a patient’s attending physician.

We will monitor the utilization of this service and collect data on billing patterns to ensure that G0452 is only being used when interpretation and report by a physician is medically necessary and is not duplicative of laboratory reporting paid under the CLFS. In the near future, we intend to reassess whether this HCPCS code is necessary, and if so, in conjunction with which molecular pathology tests.

For those providing molecular pathology physician interpretation services for Medicare beneficiaries who meet the above CMS criteria, G0452 should be reported.

Free text available from CAP's Statline Archives

C) Clinical Pathology Consultations on Molecular Tests The 2013 Physician Fee Schedule Final Rule also provided Medicare guidance on the provision of clinical pathology consultation services in conjunction with molecular testing as follows:

We note that physicians can continue to receive payment for the current clinical pathology consultation CPT codes 80500 (Clinical pathology consultation; limited, without review of a patient's history and medical records) and 80502 (Clinical pathology consultation; comprehensive, for a complex diagnostic problem, with review of patient's history and medical records) if the pathology consultation services relating to a molecular pathology test meet the definition of those codes.

For more information, see page 68999 Federal Register / Vol. 77, No. 222 of the 2013 Final Rule

D) 2015 National Correct Coding Initiative (NCCI) Policy for Medicare Services for Clinical Pathology Consultations and G0452 molecular pathology physician interpretation services:

Chapter 10 for Pathology and Laboratory Services of the 2015 NCCI Manual provides additional instruction for physicians providing clinical pathology consultations and G0452:

Clinical Pathology Consultation

CPT codes 80500 and 80502 describe clinical pathology consultation services. CMS has specific rules for reporting these services. There must be a written order for the clinical pathology consultation from the treating physician. A standing order is not an acceptable substitute for an individual written order by the treating physician. (Federal Register, Volume 62, Number 211, October 31, 1997, Page 59077)

The consultation must be related to an abnormal test result that requires medical judgment by a physician (M.D. or D.O.). Since the clinical pathology consultation requires that medical judgment be exercised, the nature of the consultation must include information that could not be

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provided by a laboratory scientist, technologist, or technician. A written report documenting the consultation must appear in the medical record. A clinical pathology consultation does not require face-to-face patient contact. If face-to-face contact is medically reasonable and necessary, an evaluation and management (E&M) CPT code may be reported in lieu of a clinical pathology consultation code. Since E&M services include interpretation of laboratory test results, a clinical pathology consultation code should never be reported with an E&M code on the same date of service. CPT codes 80500 and 80502 should never be reported for consultation related to a pathology or laboratory service that includes a physician interpretation.

G-code for Medicare Molecular Pathology Physician Interpretation On January 1, 2013 HCPCS code G0452 (molecular pathology procedure; physician interpretation and report) was implemented to report medically reasonable and necessary interpretations of molecular pathology procedures by physicians (M.D. or D.O.).

This code may be reported if: (1) the interpretation is requested by the attending physician; (2) the interpretation results in a written narrative report; and (3) the interpretation requires the exercise of medical judgment. This code may not be reported for an interpretation by a laboratory scientist. This code may be reported with a maximum of one (1) unit of service (UOS) for a Tier 1 molecular pathology procedure CPT code for each distinct source of a specimen. For example, if separate interpretations and reports for the same CPT coded Tier 1 molecular pathology procedure are reported for testing on a bone marrow specimen and a lymph node specimen, two UOS may be reported for the G0452. Since each Tier 2 molecular pathology procedure CPT code includes a list of numerous specific molecular pathology procedures, one UOS may be reported for each physician interpretation for each separately listed molecular pathology procedure for each distinct source of a specimen. A physician should not report more than one UOS for any Tier 1 molecular pathology CPT code for testing on a specimen from a single source. For Tier 2 molecular pathology CPT codes, a physician should not report more than one UOS for each listed molecular pathology procedure on a specimen from a single source.

The MUE value for G0452 is “1”. Since claims are adjudicated against this edit on a claim line basis, physicians performing more than one (1) medically reasonable and necessary interpretation may report the code on more than one claim line, the second and additional claim lines with modifier 59, each claim line with one (1) UOS.

As noted by CMS “…a hospital’s standing order policy can be used as a substitute for the individual request by a patient’s attending physician.”

More information about the NCCI Policy Manual is located on the CMS website: http://www.cms.gov/Medicare/Coding/NationalCorrectCodInitEd/index.html

E) Ins & Outs of Coding with the New Molecular Pathology CPT Procedure Codes AMP Bossler AD, Nowak JA. Ins & Outs of Coding with the New Molecular Pathology CPT Procedure Codes AMP 2013 2/26/13

Summary: Ins & Outs of Coding with the New Molecular Pathology CPT Procedure Codes AMP Webinar Presented by the Co-Chairs of the AMP Economic Affairs Committee: Aaron D Bossler, MD, PhD and Jan A Nowak, MD, PhD on 2/26/13

Free text available from Association for Molecular Pathology

F) Whole-Genome Sequencing in Health Care. Recommendations of the European Society of Human Genetics van El CG, Cornel MC, Borry P, et al. Whole-genome sequencing in health care. Recommendations of the European Society of Human Genetics. Eur J Hum Genet. 2013; 21 Suppl 1:S1-5.

Summary: In recent years, the cost of generating genome information has shown a rapid decline.1,2 High-throughput genomic technologies make it possible to sequence the whole exome or genome of a person at a price that is affordable for some health-care systems. More services based on these technologies are now becoming available for patients, raising the issue of how to ensure that these are provided appropriately. In order to determine both the clinical utility of genetic testing and assure a high quality of the analysis, the interpretation and communication of the results must be discussed so that patients can receive appropriate advice and genetic testing. The Public and Professional Policy Committee (PPPC) and the Quality Committee of the European Society of Human Genetics (ESHG) addressed these challenges at a joint workshop in Gothenburg, Sweden, in 2010.3 PPPC also organised workshops in Amsterdam, the Netherlands (January 2011 in collaboration with the EU-funded project TECHGENE, January 2012). A report for the Health Council of the Netherlands served as a background document for the PPPC’s reflections.4 Focusing on the clinical diagnostics setting, this paper is intended to contribute to the discussion and

the development of guidelines in this fast-moving field, and provide recommendations for health-care professionals. The paper and recommendations were posted on the ESHG website from 20 June to 1 August 2012 for comment by the membership. The final version was approved by the ESHG Board in December 2012.

Free full text available from PubMed PMID: 23819146

1.6 Evolving Standards for Precision Medicine

This section includes the following topics:

• Insights from Adopter - Shannon J. McCall (Section 1.6.1); and • Analytical Validity and Clinical Utility (Section 1.6.2).

1.6.1 Insights from Adopter - Shannon J. McCall, MD, FCAP

Shannon McCall, MD, FCAP, is the Director of the College of American Pathologists (CAP)-Accredited Biospecimen Repository & Processing Core Facility at Duke University Medical Center and serves on the Biorepository Accreditation Program Committee. An Assistant Professor at Duke, Dr. McCall specializes in pathology of the gastrointestinal and hepatobiliary tracts. She serves as the Quality Assurance Officer for the Duke Anatomic Pathology Practice and is also a member of the College of American Pathologists’ Quality Practices Committee.

Shannon J. McCall, MD, FCAP Dr. McCall received Bachelor of Science degrees in Chemical Engineering and Biochemistry from North Carolina State University in Raleigh, North Carolina and her medical degree from Duke University in Durham, North Carolina. She also completed her residency training in Anatomic and Clinical Pathology as well as a one year fellowship in Gastrointestinal and Hepatic Pathology at Duke University. She is board-certified in Anatomic and Clinical Pathology. Her research interests include the developing automated quality assurance metric collection for anatomic pathologists for use in laboratory accreditation, credentialing, and new health system or government quality initiatives. Dr. McCall is also involved in the expansion of a novel business and governance model for biobanking at Duke as well as collaborative translational science in gastrointestinal oncology.

Dr. McCall’s Insights for the Next Wave of Adopters:

1 Will Your Specimen The catchphrase “fit for purpose” can be Be “fit for purpose”? translated, “collecting the specimen with the required end assay(s) in mind.” In prior years, anatomic pathology specimens were collected and processed for the end assay of paraffin embedding and histologic evaluation of a slide stained with hematoxylin and eosin. Numerous histochemical and, more recently, immunohistochemical and in situ hybridization staining protocols evolved out of this background, and have been optimized for use with formalin-fixed, paraffin-embedded (FFPE) material. For molecular assays dependent on specimens other than FFPE, however, there is only one chance to optimize collection. While many DNA-based assays demonstrate solid performance using FFPE material, in-situ hybridization (ISH) assays often perform better using touch preparations of tumor cells. RNA- based assays (such as QT-PCR) perform best with frozen tissue, however, almost all have been optimized for FFPE. The current CAP- American Society of Clinical Oncology (ASCO) recommendation for the cold ischemia time, the period between removal of the biospecimen from the patient and its dissection (if necessary) and preservation, is less than 60 minutes. The cold ischemia time (also called “time to fixation”) is becoming a critical variable. It should be included in the surgical pathology report.

2 Know that you’re Acknowledge that when you are utilizing already practicing immunohistochemistry and/or in situ “molecular hybridization to interrogate your tumor samples pathology.” for protein or nucleic acid expression, you are already practicing molecular pathology. It’s a short step from there to performing quantitative assessments of biomarker expression (such as Ki-67, estrogen receptor (ER), progesterone receptor (PgR) and HER2 testing). Some protein expression assays couple with PCR- based assays (mismatch repair protein expression and microsatellite instability testing, for example). Expanding your practice to include interrogating your tumor samples via PCR assays or even next-generation sequencing builds on and adds value to your current practice. 3 Add a few more data Guidelines for molecular biomarker testing in points to your ER/PgR, published jointly by CAP-ASCO in specimen 2010, and aligned withHER2 guidelines in processing protocols to ensure 2013, helpreduce preanalytic variability by compliance with specifying time to fixation < 1 hour as well as routine molecular minimum and maximum fixation times for oncology testing. breast specimens in 10% neutral phosphate buffered formalin of 6 and72 hours. The time into formalin and estimated “time out of formalin” can be used to calculate the total time the biospecimen was in formalin. These timesshould be documented in the pathology report to assure downstream compliance with the guidelines. When “unusual” or “unexpected” results occur, knowing both the cold ischemia time and the total time in formalin will be very helpful in troubleshooting the potential causes for the discrepancy. 4 Consider adding a Today’s retrospective research samples can few more data points quickly become tomorrow’s validation set for to your specimen the next diagnostic assay. This is the nature of processing protocols to maximize the translational research. Expanded preanalytic downstream value of variables, if not documented at the time of your archival specimen processing, may be very difficult to biospecimens as document at a later time. Documentation of retrospective selected preanalytic variables may

research samples. substantially increase the downstream value of your tissue samples in future clinical or research assays. As an example, prior tumor treatment with chemotherapy and/or radiation should be documented in the pathology report. 5 Don’t throw the baby Once you’ve considered the value of your out with the tissues for downstream clinical molecular bathwater. testing and research, as well as for potential future patient care, you may be tempted make major changes in your processing protocols to accommodate the widest variety of downstream assays. Since the majority of anatomic pathology practices (histology, histochemistry, and immunohistochemistry) have been validated in FFPE however, switching or altering your fixation protocols to include substances like Allprotect or RNAlater could seriously impair your ability to interpret routine IHC slides. Traditional PCR-based molecular diagnostics assays as well as newer next-generation sequencing assays, are also optimized for FFPE samples. 6 Once molecular tests Today’s molecular assays evaluate many have been ordered, different areas. Will the test you need involve refresh yourself on sequencing tumor DNA for a mutation, the basics so that you know the best comparing expansion of microsatellites in sample to provide. tumor and normal, or using fluorescent probes to identify a chromosomal translocation in tumor? There is a difference between submitting a representative tumor block for testing using a notation in the surgical pathology report, which is a good practice, and reviewing the case yourself to select a block specifically for the downstream assay. Traditional DNA sequencing assays could require 30% tumor nuclei and a minimum size. Microsatellite instability assays require samples of both tumor and normal tissue. Consult the molecular lab for specific assay requirements.

Consider submitting a separate block of viable tumor during gross evaluation to facilitate

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downstream molecular testing, if your consenting process and workflow permit. You may also consider placing multiple pieces of both normal and malignant tissue in the same block. This multiple piece technique is useful, because a normal control is simultaneously processed with the test tissue. This technique also helps mitigate the significant problem of biomarker heterogeneity in the test tissue. 7 Treat the disease the Although most molecular assays are thought to patient currently has. maintain concordance between primary and metastatic samples, strict data on the concurrence of these assays in a patient’s tumor foci over time is not available for all assays. Many oncologists prefer to test the patient’s most current tumor (that is, the new metastatic focus), rather than performing molecular testing on a primary tumor sample that may be years old. Be aware of this issue if multiple samples are available for a patient and consult the ordering oncologist if necessary. It is also a good practice to test a stored FFPE tissue with at least one of the originally tested biomarkers, e.g., ER/PgR, to see if the biomolecules have undergone significant degradation with time in storage due to hydrolysis from residual water in the paraffin. 8 Work with your This may mean reviewing a case to ensure an Referral Lab to appropriate test order, communicating with the assist with specimen requesting physician to clarify ambiguous selection for sendout testing. orders, and selecting a paraffin block of tumor and/or normal for the assay. 9 If you have an in- You can add yourself to the workflow by house molecular identifying (“circling”) tumor on the coverslip of laboratory, ask if you the slide for testing, and receiving copies of the can become more involved. test results ordered on your anatomic specimens. In this way, you can learn valuable information about the molecular test ordering patterns of your clinicians, and even suggest ways to streamline ordering or reporting. You may ultimately consider learning to verify in- house molecular assays performed on your samples.

10 Stay abreast of new The CAP has developed the Pathology & guidelines for Laboratory Quality Center as a forum for testing. establishing consensus guidelines and recommendations through the use of expert panels and collaborations between CAP and other national organizations appropriate to the area of study. Recently the CAP partnered with the Association for Molecular Pathology (AMP) and the International Association for the Study of Lung Cancer (IASLC) to codify recommendations for molecular diagnostics testing in lung cancer associated with targeted therapies. These recommendations were presented at the ASCO Annual Meeting in 2013 and jointly published in Archives of Pathology & Laboratory Medicine, the Journal of Thoracic Oncology, and the Journal of Molecular Diagnostics. Future guidelines will include molecular testing of colorectal cancer and gastric cancer.

Dr. McCall’s suggested articles and resources:

A) American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Immunohistochemical Testing of Estrogen and Progesterone Receptors in Breast Cancer (Unabridged Version) Hammond ME, Hayes DF, Dowsett M, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version). Arch Pathol Lab Med. 2010;134(7):e48- 72.

Summary: To develop a guideline to improve the accuracy of immunohistochemical (IHC) estrogen receptor (ER) and progesterone receptor (PgR) testing in breast cancer and the utility of these receptors as predictive markers. METHODS: The American Society of Clinical Oncology and the College of American Pathologists convened an international Expert Panel that conducted a systematic review and evaluation of the literature in partnership with Cancer Care Ontario and developed recommendations for optimal IHC ER/PgR testing performance. RESULTS: Up to 20% of current IHC determinations of ER and PgR testing worldwide may be inaccurate (false negative or false positive). Most of the issues with testing have

occurred because of variation in pre-analytic variables, thresholds for positivity, and interpretation criteria. RECOMMENDATIONS: The Panel recommends that ER and PgR status be determined on all invasive breast cancers and breast cancer recurrences. A testing algorithm that relies on accurate, reproducible assay performance is proposed. Elements to reliably reduce assay variation are specified. It is recommended that ER and PgR assays be considered positive if there are at least 1% positive tumor nuclei in the sample on testing in the presence of expected reactivity of internal (normal epithelial elements) and external controls. The absence of benefit from endocrine therapy for women with ER-negative invasive breast cancers has been confirmed in large overviews of randomized clinical trials.

Free full text available from the CAP’s Archives PMID: 20586616

B) A Call to Standardize Preanalytic Data Elements for Biospecimens Robb JA, Gulley ML, Fitzgibbons PL, et al. A Call to Standardize Preanalytic Data Elements for Biospecimens. Arch Pathol Lab Med. 2014;138(4):526-37.

Summary: Biospecimens must have appropriate clinical annotation (data) to ensure optimal quality for both patient care and research. Clinical preanalytic variables are the focus of this study. Objectives.-To define the essential preanalytic variables (data fields) that should be attached to every collected biospecimen and to provide a complete list of such variables, along with their relative importance, which can vary, depending on downstream use, institutional needs, and information technology capabilities. Design.-The College of American Pathologists Diagnostic Intelligence and Health Information Technology Committee sponsored a Biorepository Working Group to develop a ranked list of the preanalytic variables for annotating biospecimens. Members of the working group were experts in anatomic, clinical, and molecular pathology; biobanking; medical informatics; and accreditation. Several members had experience with federal government programs, such as the National Cancer Institute's Biospecimens and Biorepository Branch and National Cancer Institute's Community Cancer Center Program. Potential preanalytic variables were identified and ranked along with available supporting evidence, definitions, and potential negative effects if the variable was not attached to the biospecimen. Additional national and international stakeholders reviewed the draft manuscript. Results.-The ranked listing of 170 preanalytic variables produced can be used as a guide for site-specific implementation into patient care and/or research biorepository processes. Conclusions.-In our collective experience, it is often difficult to choose which of the many preanalytic variables to attach to any specific set of biospecimens used for patient care and/or research.

The attached ranked list should aid in the selection of preanalytic variables for a given biospecimen collection.

Free full text available from the CAP’s Archives PMID: 23937609

C) Biospecimens and Biorepositories for the Community Pathologist Dash RC, Robb JA, Booker DL, Foo WC, Witte DL, Bry L. Biospecimens and Biorepositories for the Community Pathologist. Arch Pathol Lab Med. 2012;136(6):668-78.

Summary: Pathologists have long served as custodians of human biospecimens collected for diagnostic purposes. Rapid advancements in diagnostic technologies require that pathologists change their practices to optimize patient care. The proper handling of biospecimens creates opportunities for pathologists to improve their diagnoses while assessing prognosis and treatment. In addition, the growing need for high-quality biorepositories represents an opportunity for community pathologists to strengthen their role within the health care team, ensuring that clinical care is not compromised while facilitating research. This article provides a resource to community pathologists learning how to create high-quality biorepositories and participating in emerging opportunities in the biorepository field. While a variety of topics are covered to provide breadth of information, the intent is to facilitate a level of understanding that permits community pathologists to make more informed choices in identifying how best their skills and practice may be augmented to address developments in this field.

Free full text available from the CAP’s Archives PMID: 22646276

D) Preservation of Nucleic Acids and Tissue Morphology in Paraffin Embedded Clinical Samples Comparison of Five Molecular Fixatives Staff S, Kujala P, Karhu R, et al. Preservation of Nucleic Acids and Tissue Morphology in Paraffin-embedded Clinical Samples: Comparison of Five Molecular Fixatives. Journal of Clinical Pathology. 2013;66(9):807-10.

Summary: Formalin fixation preserves tissue morphology at the expense of macromolecule integrity. Freshly frozen samples are the golden standard for DNA and RNA analyses but require laborious deep-freezing and frozen sectioning for morphological studies. Alternative tissue stabilisation methods are therefore needed. We analysed the preservation of nucleic acids, immunohistochemical staining properties and tissue morphology in paraffin- embedded clinical tissue samples fixed with Z7, RCL2, PAXgene, Allprotect

and RNAlater. Formalin-fixed and deep-frozen samples were used as controls. Immunohistochemical analyses showed good preservation of antigenicity in all except Allprotect and RNAlater-fixed samples. RNA quality, based on RNA integrity number value by Bioanalyzer, was comparable with freshly frozen samples only in PAXgene-fixed samples. According to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses, RNA from PAXgene samples yielded results similar to freshly frozen samples. No difference between fixatives was seen in DNA analyses (PCR and real-time PCR). In conclusion, PAXgene seems to be superior to other molecular fixatives and formaldehyde.

Full text available from Journal of Clinical Pathology (USD 37.00 for 24 hour access) PMID: 23750036

E) Factors Influencing the Degradation of Archival Formalin-Fixed Paraffin Embedded Tissue Sections Xie R, Chung JY, Ylaya K, et al. Factors Influencing the Degradation of Archival Formalin-Fixed Paraffin-Embedded Tissue Sections. J Histochem Cytochem. 2011;59(4):356-65.

Summary: The loss of antigenicity in archival formalin-fixed paraffin- embedded (FFPE) tissue sections negatively affects both diagnostic histopathology and advanced molecular studies. The mechanisms underlying antigenicity loss in FFPE tissues remain unclear. The authors hypothesize that water is a crucial contributor to protein degradation and decrement of immunoreactivity in FFPE tissues. To test their hypothesis, they examined fixation time, processing time, and humidity of storage environment on protein integrity and antigenicity by immunohistochemistry, Western blotting, and protein extraction. This study revealed that inadequate tissue processing, resulting in retention of endogenous water in tissue sections, results in antigen degradation. Exposure to high humidity during storage results in significant protein degradation and reduced immunoreactivity, and the effects of storage humidity are temperature dependent. Slides stored under vacuum with desiccant do not protect against the effects of residual water from inadequate tissue processing. These results support that the presence of water, both endogenously and exogenously, plays a central role in antigenicity loss. Optimal tissue processing is essential. The parameters of optimal storage of unstained slides remain to be defined, as they are directly affected by preanalytic variables. Nevertheless, minimization of exposure to water is required for antigen preservation in FFPE tissue sections

Free full text available from PubMed PMID: 21411807

Additional Resources:

A) DNA Yield from Tissue Samples in Surgical Pathology and Minimum Tissue Requirements for Molecular Testing Austin MC, Smith C, Pritchard CC,Tait JF. DNA Yield from Tissue Samples in Surgical Pathology and Minimum Tissue Requirements for Molecular Testing. Arch Pathol Lab Med. 2015.

Summary: Context .- Complex molecular assays are increasingly used to direct therapy and provide diagnostic and prognostic information but can require relatively large amounts of DNA. Objectives .- To provide data to pathologists to help them assess tissue adequacy and provide prospective guidance on the amount of tissue that should be procured. Design .- We used slide-based measurements to establish a relationship between processed tissue volume and DNA yield by A260 from 366 formalin-fixed, paraffin-embedded tissue samples submitted for the 3 most common molecular assays performed in our laboratory (EGFR, KRAS, and BRAF). We determined the average DNA yield per unit of tissue volume, and we used the distribution of DNA yields to calculate the minimum volume of tissue that should yield sufficient DNA 99% of the time. Results .- All samples with a volume greater than 8 mm3 yielded at least 1 mug of DNA, and more than 80% of samples producing less than 1 mug were extracted from less than 4 mm3 of tissue. Nine square millimeters of tissue should produce more than 1 mug of DNA 99% of the time. Conclusions .- We conclude that 2 tissue cores, each 1 cm long and obtained with an 18-gauge needle, will almost always provide enough DNA for complex multigene assays, and our methodology may be readily extrapolated to individual institutional practice.

Free full text available from the CAP’s Archives PMID: 26098132

B) Using Next-Generation Sequencing for High Resolution Multiplex Analysis of Copy Number Variation From Nanogram Quantities of DNA From Formalin-Fixed Paraffin-Embedded Specimens Wood HM, Belvedere O, Conway C, et al. Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens. Nucleic Acids Res. 2010; 38(14):e151.

Summary: The use of next-generation sequencing technologies to produce

genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material.

Free full text available from PubMed PMID: 20525786 NOTE: Also cited in Section 1.2.3.3

1.6.2 Analytical Validity and Clinical Utility

A) Personalized Genomic Analyses for Cancer Mutation Discovery and Interpretation Jones S, Anagnostou V, Lytle K, et al. Personalized Genomic Analyses for Cancer Mutation Discovery and Interpretation. Sci Transl Med. 2015; 7(283): 283ra253.

Summary: Massively parallel sequencing approaches are beginning to be used clinically to characterize individual patient tumors and to select therapies based on the identified mutations. A major question in these analyses is the extent to which these methods identify clinically actionable alterations and whether the examination of the tumor tissue alone is sufficient or whether matched normal DNA should also be analyzed to accurately identify tumor-specific (somatic) alterations. To address these issues, we comprehensively evaluated 815 tumor-normal paired samples from patients of 15 tumor types. We identified genomic alterations using next-generation sequencing of whole exomes or 111 targeted genes that were validated with sensitivities >95% and >99%, respectively, and specificities >99.99%. These analyses revealed an average of 140 and 4.3 somatic mutations per exome

and targeted analysis, respectively. More than 75% of cases had somatic alterations in genes associated with known therapies or current clinical trials. Analyses of matched normal DNA identified germline alterations in cancer- predisposing genes in 3% of patients with apparently sporadic cancers. In contrast, a tumor-only sequencing approach could not definitively identify germline changes in cancer-predisposing genes and led to additional false- positive findings comprising 31% and 65% of alterations identified in targeted and exome analyses, respectively, including in potentially actionable genes. These data suggest that matched tumor-normal sequencing analyses are essential for precise identification and interpretation of somatic and germline alterations and have important implications for the diagnostic and therapeutic management of cancer patients.

Free full text available from PubMed PMID: 25877891 NOTE: Also cited in Section 7.1.1

B) Performance of Common Analysis Methods for Detecting Low- Frequency Single Nucleotide Variants in Targeted Next-Generation Sequence Data Spencer DH, Tyagi M, Vallania F, et al. Performance of Common Analysis Methods for Detecting Low-Frequency Single Nucleotide Variants in Targeted Next-Generation Sequence Data. J Mol Diagn. 2014; 16(1): 75-88.

Summary: Next-generation sequencing (NGS) is becoming a common approach for clinical testing of oncology specimens for mutations in cancer genes. Unlike inherited variants, cancer mutations may occur at low frequencies because of contamination from normal cells or tumor heterogeneity and can therefore be challenging to detect using common NGS analysis tools, which are often designed for constitutional genomic studies. We generated high-coverage (>1000x) NGS data from synthetic DNA mixtures with variant allele fractions (VAFs) of 25% to 2.5% to assess the performance of four variant callers, SAMtools, Genome Analysis Toolkit, VarScan2, and SPLINTER, in detecting low-frequency variants. SAMtools had the lowest sensitivity and detected only 49% of variants with VAFs of approximately 25%; whereas the Genome Analysis Toolkit, VarScan2, and SPLINTER detected at least 94% of variants with VAFs of approximately 10%. VarScan2 and SPLINTER achieved sensitivities of 97% and 89%, respectively, for variants with observed VAFs of 1% to 8%, with >98% sensitivity and >99% positive predictive value in coding regions. Coverage analysis demonstrated that >500x coverage was required for optimal performance. The specificity of SPLINTER improved with higher coverage, whereas VarScan2 yielded more false positive results at high coverage levels, although this effect was abrogated by removing low-quality reads

before variant identification. Finally, we demonstrate the utility of high- sensitivity variant callers with data from 15 clinical lung cancers.

Free full text available from PubMed PMID: 24211364 NOTE: Also cited in Section 2.1.1.3

C) Performance Comparison of Four Exome Capture Systems for Deep Sequencing Chilamakuri CS, Lorenz S, Madoui MA, et al. Performance Comparison of Four Exome Capture Systems for Deep Sequencing. BMC Genomics. 2014; 15:449.

Summary: BACKGROUND: Recent developments in deep (next-generation) sequencing technologies are significantly impacting medical research. The global analysis of protein coding regions in genomes of interest by whole exome sequencing is a widely used application. Many technologies for exome capture are commercially available; here we compare the performance of four of them: NimbleGen's SeqCap EZ v3.0, Agilent's SureSelect v4.0, Illumina's TruSeq Exome, and Illumina's Nextera Exome, all applied to the same human tumor DNA sample. RESULTS: Each capture technology was evaluated for its coverage of different exome databases, target coverage efficiency, GC bias, sensitivity in single nucleotide variant detection, sensitivity in small indel detection, and technical reproducibility. In general, all technologies performed well; however, our data demonstrated small, but consistent differences between the four capture technologies. Illumina technologies cover more bases in coding and untranslated regions. Furthermore, whereas most of the technologies provide reduced coverage in regions with low or high GC content, the Nextera technology tends to bias towards target regions with high GC content. CONCLUSIONS: We show key differences in performance between the four technologies. Our data should help researchers who are planning exome sequencing to select appropriate exome capture technology for their particular application.

Free full text available from PubMed PMID: 24912484

D) Validation of a Next-Generation Sequencing Assay for Clinical Molecular Oncology Cottrell CE, Al-Kateb H, Bredemeyer AJ, et al. Validation of a Next- Generation Sequencing Assay for Clinical Molecular Oncology. J Mol Diagn. 2014; 16(1): 89-105.

Summary: Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance.

Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (>/= 1000x average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4- 100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of >/= 15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management.

Free full text available from Journal of Molecular Diagnostics PMID: 24211365

E) Variant Validity (Selected vs. General Population) Billings PR, Raghavachari N, Senthil G. Variant validity (selected vs. general population). Institute of Medicine (Perspective Series). January 22, 2015.

Summary: Advances in genome sequencing represent an unparalleled opportunity to examine the genomic landscape of an individual and potentially identify genetic variants that are relevant for the diagnosis and treatment of disease. However, the identification and interpretation of variants as being causative for disease and relevant to treatment choice can be a significant challenge for the proper application of sequencing in clinical practice. This discussion paper, the second of seven individually authored commentaries that explores the evidence needed to support the use of genome sequencing in the clinic, examines the validation and interpretation of variants from a next-generation sequencing-based test.

Free full text available from Institute of Medicine

F) Analytic Validity of Genomic Testing Pratt V, Leonard DG. Analytic Validity of Genomic Testing. Institute of Medicine. January 15, 2015.

Summary: The College of American Pathologists (CAP) defines analytic validity as a test’s ability to accurately measure the analyte of interest.3 In the context of genomic testing using nextgeneration sequencing (NGS), analytic validation of every potential variant (e.g., single nucleotide variants, insertion/deletions, copy number variants, structural chromosomal rearrangements) is not feasible. Therefore, professional guidelines recommend that NGS test validation assures the accuracy of the entire pathway for different types of variants rather than all specific variants (Aziz et al., 2014; Rehm et al., 2013). Currently, there is an FDA-cleared instrument and reagents, but at this time there is no approved test kit for whole genome sequencing (FDA, 2013). Of note, using present-day NGS technologies, we cannot fully sequence the exome or genome because of an inability to generate high-quality data for regions of high GC content (e.g., 5’ end of the gene) and for pseudogenes or repetitive regions. Anticipated technology advancements should provide more complete coverage of the exome or genome in the future. During test validation, reference materials are used to determine the false negative, false positive, and accuracy rates. Only recently have the National Institute of Standards and Technology4 and the Centers for Disease Control and Prevention focused on reference material for NGS.5 As with any clinical test, other testing methods are used to confirm novel results. Novel NGS-based test results can be confirmed by focused Sanger sequencing, pyrosequencing, or realtime polymerase chain reaction methods. For genetic tests, novel variant(s) may be confirmed by testing of parents or other informative family members. Because the false positive rate for NGSbased tests is fairly high, especially for exome or genome sequencing, verification of novel results remains important.

Free full text available from Institute of Medicine NOTE: Also cited in Section 1.2.3.3

G) Evaluating the Clinical Utility of Genomic Variants Derived from Next- Generation Sequencing for Opportunistic Disease Screening and Risk Assessment: Evidence Gaps and Priorities Khoury MJ, Cashion A, Billings PR. Evaluating the clinical utility of genomic variants derived from next-generation sequencing for opportunistic disease screening and risk assessment: evidence gaps and priorities. Institute of Medicine (Perspective Series). February 5, 2015.

Summary: Next-generation sequencing is increasingly used in the diagnosis and management of suspected Mendelian diseases of unknown cause and in precision medicine approaches to cancer treatment. In the course of sequencing, additional genetic information can be generated that is unrelated to the initial reason sequencing was performed. This incidental information

presents an opportunity to screen individuals for current asymptomatic disease or future risk of disease. This discussion paper, the fourth of seven individually authored commentaries that explores the evidence needed to support the use of genome sequencing in the clinic, examines the evidence gaps and requirements for use of incidental findings for the purpose of opportunistic disease screening and risk assessment.

Free full text available from Institute of Medicine NOTE: Also cited in Section 4.1.1

H) Clinical Utility: Informing Treatment Decisions by Changing the Paradigm McCormack R, Billings PR. Clinical Utility: Informing Treatment Decisions by Changing the Paradigm. Institute of Medicine. January 29, 2015.

Summary: Next-generation sequencing has provided significant insight into the basic biology of cancer, allowing for the development of new targeted therapeutics and the identification of biomarkers that can direct treatment course. However, the evidence needed to demonstrate that next-generation sequencing-based tests lead to clinical decisions that improve outcomes can be challenging for test developers and laboratories to generate. This discussion paper, the third of seven individually authored commentaries that explores the evidence needed to support the use of genome sequencing in the clinic, examines the challenges involved in and opportunities for demonstrating the clinical utility of a next-generation sequencing-based test.

Free full text available from Institute of Medicine NOTE: Also cited in Sections 1.2.1 & 1.2.2

I) SeqReporter: Automating Next-Generation Sequencing Result Interpretation and Reporting Workflow in a Clinical Laboratory Roy S, Durso MB, Wald A, Nikiforov YE, Nikiforova MN. SeqReporter: Automating Next-Generation Sequencing Result Interpretation and Reporting Workflow in a Clinical Laboratory. J Mol Diagn. 2014; 16(1): 11-22.

Summary: A wide repertoire of bioinformatics applications exist for next- generation sequencing data analysis; however, certain requirements of the clinical molecular laboratory limit their use: i) comprehensive report generation, ii) compatibility with existing laboratory information systems and computer operating system, iii) knowledgebase development, iv) quality management, and v) data security. SeqReporter is a web-based application developed using ASP.NET framework version 4.0. The client-side was designed using HTML5, CSS3, and Javascript. The server-side processing

(VB.NET) relied on interaction with a customized SQL server 2008 R2 database. Overall, 104 cases (1062 variant calls) were analyzed by SeqReporter. Each variant call was classified into one of five report levels: i) known clinical significance, ii) uncertain clinical significance, iii) pending pathologists' review, iv) synonymous and deep intronic, and v) platform and panel-specific sequence errors. SeqReporter correctly annotated and classified 99.9% (859 of 860) of sequence variants, including 68.7% synonymous single-nucleotide variants, 28.3% nonsynonymous single- nucleotide variants, 1.7% insertions, and 1.3% deletions. One variant of potential clinical significance was re-classified after pathologist review. Laboratory information system-compatible clinical reports were generated automatically. SeqReporter also facilitated quality management activities. SeqReporter is an example of a customized and well-designed informatics solution to optimize and automate the downstream analysis of clinical next- generation sequencing data. We propose it as a model that may envisage the development of a comprehensive clinical informatics solution.

Free full text available from Journal of Molecular Diagnostics PMID: 24220144 NOTE: Also cited in Section 9.1.1

J) Technical and Implementation Issues in Using Next-Generation Sequencing of Cancers in Clinical Practice Ulahannan D, Kovac MB, Mulholland PJ, Cazier JB,Tomlinson I. Technical and implementation issues in using next-generation sequencing of cancers in clinical practice. Br J Cancer. 2013; 109(4): 827-835.

Summary: Next-generation sequencing (NGS) of cancer genomes promises to revolutionise oncology, with the ability to design and use targeted drugs, to predict outcome and response, and to classify tumours. It is continually becoming cheaper, faster and more reliable, with the capability to identify rare yet clinically important somatic mutations. Technical challenges include sequencing samples of low quality and/or quantity, reliable identification of structural and copy number variation, and assessment of intratumour heterogeneity. Once these problems are overcome, the use of the data to guide clinical decision making is not straightforward, and there is a risk of premature use of molecular changes to guide patient management in the absence of supporting evidence. Paradoxically, NGS may simply move the bottleneck of personalised medicine from data acquisition to the identification of reliable biomarkers. Standardised cancer NGS data collection on an international scale would be a significant step towards optimising patient care.

Free full text available from PubMed PMID: 23887607 NOTE: Also cited in Section 2.2.4

K) Variant Discovery in Targeted Resequencing Using Whole Genome Amplified DNA Indap AR, Cole R, Runge CL, Marth GT, Olivier M. Variant discovery in targeted resequencing using whole genome amplified DNA. BMC Genomics. 2013; 14: 468.

Summary: BACKGROUND: Next generation sequencing and advances in genomic enrichment technologies have enabled the discovery of the full spectrum of variants from common to rare alleles in the human population. The application of such technologies can be limited by the amount of DNA available. Whole genome amplification (WGA) can overcome such limitations. Here we investigate applicability of using WGA by comparing SNP and INDEL variant calls from a single genomic/WGA sample pair from two capture separate experiments: a 50 Mbp whole exome capture and a custom capture array of 4 Mbp region on chr12. RESULTS: Our results comparing variant calls derived from genomic and WGA DNA show that the majority of variant SNP and INDEL calls are common to both callsets, both at the site and genotype level and suggest that allele bias plays a minimal role when using WGA DNA in re-sequencing studies. CONCLUSIONS: Although the results of this study are based on a limited sample size, they suggest that using WGA DNA allows the discovery of the vast majority of variants, and achieves high concordance metrics, when comparing to genomic DNA calls.

Free full text available from PubMed PMID: 23837845

L) Assessment of Clinical Analytical Sensitivity and Specificity of Next- Generation Sequencing for Detection of Simple and Complex Mutations Chin EL, da Silva C, Hegde M. Assessment of clinical analytical sensitivity and specificity of next-generation sequencing for detection of simple and complex mutations. BMC Genet. 2013; 14: 6.

Summary: BACKGROUND: Detecting mutations in disease genes by full gene sequence analysis is common in clinical diagnostic laboratories. Sanger dideoxy terminator sequencing allows for rapid development and implementation of sequencing assays in the clinical laboratory, but it has limited throughput, and due to cost constraints, only allows analysis of one or at most a few genes in a patient. Next-generation sequencing (NGS), on the other hand, has evolved rapidly, although to date it has mainly been used for

large-scale genome sequencing projects and is beginning to be used in the clinical diagnostic testing. One advantage of NGS is that many genes can be analyzed easily at the same time, allowing for mutation detection when there are many possible causative genes for a specific phenotype. In addition, regions of a gene typically not tested for mutations, like deep intronic and promoter mutations, can also be detected. RESULTS: Here we use 20 previously characterized Sanger-sequenced positive controls in disease- causing genes to demonstrate the utility of NGS in a clinical setting using standard PCR based amplification to assess the analytical sensitivity and specificity of the technology for detecting all previously characterized changes (mutations and benign SNPs). The positive controls chosen for validation range from simple substitution mutations to complex deletion and insertion mutations occurring in autosomal dominant and recessive disorders. The NGS data was 100% concordant with the Sanger sequencing data identifying all 119 previously identified changes in the 20 samples. CONCLUSIONS: We have demonstrated that NGS technology is ready to be deployed in clinical laboratories. However, NGS and associated technologies are evolving, and clinical laboratories will need to invest significantly in staff and infrastructure to build the necessary foundation for success.

Free full text available from PubMed PMID: 23418865

1.7 Precision Medicine Approaches in Health Care Systems

A) Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus Recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology Richards S, Aziz N, Bale S, et al. Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus Recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015; 17(5): 405-424.

Summary: The American College of Medical Genetics and Genomics (ACMG) previously developed guidance for the interpretation of sequence variants.(1) In the past decade, sequencing technology has evolved rapidly with the advent of high-throughput next-generation sequencing. By adopting and leveraging next-generation sequencing, clinical laboratories are now performing an ever-increasing catalogue of genetic testing spanning

genotyping, single genes, gene panels, exomes, genomes, transcriptomes, and epigenetic assays for genetic disorders. By virtue of increased complexity, this shift in genetic testing has been accompanied by new challenges in sequence interpretation. In this context the ACMG convened a workgroup in 2013 comprising representatives from the ACMG, the Association for Molecular Pathology (AMP), and the College of American Pathologists to revisit and revise the standards and guidelines for the interpretation of sequence variants. The group consisted of clinical laboratory directors and clinicians. This report represents expert opinion of the workgroup with input from ACMG, AMP, and College of American Pathologists stakeholders. These recommendations primarily apply to the breadth of genetic tests used in clinical laboratories, including genotyping, single genes, panels, exomes, and genomes. This report recommends the use of specific standard terminology-"pathogenic," "likely pathogenic," "uncertain significance," "likely benign," and "benign"-to describe variants identified in genes that cause Mendelian disorders. Moreover, this recommendation describes a process for classifying variants into these five categories based on criteria using typical types of variant evidence (e.g., population data, computational data, functional data, segregation data). Because of the increased complexity of analysis and interpretation of clinical genetic testing described in this report, the ACMG strongly recommends that clinical molecular genetic testing should be performed in a Clinical Laboratory Improvement Amendments-approved laboratory, with results interpreted by a board-certified clinical molecular geneticist or molecular genetic pathologist or the equivalent.

Free full text available from PubMed PMID: 25741868

B) A Classification System for Clinical Relevance of Somatic Variants Identified in Molecular Profiling of Cancer Sukhai MA, Craddock KJ, Thomas M, et al. A Classification System for Clinical Relevance of Somatic Variants Identified in Molecular Profiling of Cancer. Genet Med. 2016; 18(2): 128-136.

Summary: PURPOSE: Interpretation systems for clinical laboratory reporting of genetic variants for inherited conditions have been widely published. By contrast, there are no existing systems for interpretation and classification of somatic variants found from molecular testing of cancer. METHODS: We designed an assessment protocol and classification system for somatic variants identified through next-generation sequencing molecular profiling of tumor-derived samples and applied these to a pilot dataset of somatic variants found by next-generation sequencing profiling of 158 tumor samples derived from advanced cancer patients examined at the Princess Margaret Cancer Centre. RESULTS: We present a classification system to interpret the significance of genetic variants in molecular analysis of cancer, including the following key factors: (i) known or predicted pathogenicity of the variant; (ii) primary site and tumor histology in which the variant is found; (iii) recurrence of the variant; and (iv) evidence of clinical actionability. We used these factors to develop a five-category somatic variant classification for simplified reporting of variant interpretations to treating oncologists. CONCLUSION: Our somatic variant classification can be of practical value to

other clinical molecular laboratories performing cancer genetic profiling by promoting consistent reporting of somatic variants and permitting harmonization of variant data among laboratories and clinical studies.

Free full text available from Genetics in Medicine PMID: 25880439

C) Breast Cancer Experience of the Molecular Tumor Board at the University of California, San Diego Moores Cancer Center Parker BA, Schwaederle M, Scur MD, et al. Breast Cancer Experience of the Molecular Tumor Board at the University of California, San Diego Moores Cancer Center. J Oncol Pract. 2015; 11(6): 442-449.

Summary: PURPOSE: Multiplex genomic tests are enabling oncologists to interrogate the DNA of their patients. However, few oncologists are proficient with respect to the implications of complex molecular diagnostics. We initiated a Molecular Tumor Board that focused on individual patients with advanced cancer whose tumors underwent genomic profiling, and here report our experience with breast cancer. METHODS: A multidisciplinary team that included physicians, scientists, geneticists, and bioinformatics/pathway specialists attended. All molecular tests were performed in a Clinical Laboratory Improvement Amendments environment (next-generation sequencing, 182 or 236 genes). RESULTS: Forty of 43 patients (93%; mean age, 59 years) had at least one theoretically actionable aberration (mean, 4.79 anomalies/patient). Median time from ordering to report was 27 days (median of approximately 11 days for specimen acquisition and approximately 14 days for diagnostic processing). Even if we considered distinct abnormalities in a gene as the same, there were only two patients with an identical molecular profile. Seventy-three genes (206 abnormalities; 119 distinct) were aberrant. Seventeen of the 43 patients (40%; median, seven previous therapies in the metastatic setting) were treated in a manner consistent with Molecular Tumor Board discussions; seven (16% of 43, or 41% of 17) achieved stable disease for 6 or more months (n = 2) or partial remission (n = 5). Lack of access to targeted medication was the most common reason that patients could not be treated. CONCLUSION: Multidisciplinary molecular tumor boards may help to optimize the management of patients with advanced, heavily pretreated breast cancer who have undergone genomic testing. Facilitating availability of appropriately targeted drugs and clinical trials is needed.

Free full text available from Journal of Oncology Practice PMID: 26243651

D) Development of a Center for Personalized Cancer Care at a Regional Cancer Center: Feasibility Trial of an Institutional Tumor Sequencing Advisory Board Lane BR, Bissonnette J, Waldherr T, et al. Development of a Center for Personalized Cancer Care at a Regional Cancer Center: Feasibility Trial of an Institutional Tumor Sequencing Advisory Board. J Mol Diagn. 2015; 17(6): 695-704.

Summary: Next-generation sequencing (NGS) capabilities can affect therapeutic decisions in patients with complex, advanced, or refractory cancer. We report the feasibility of a tumor sequencing advisory board at a regional cancer center. Specimens were analyzed for approximately 2800 mutations in 50 genes. Outcomes of interest included tumor sequencing advisory board function and processes, timely discussion of results, and proportion of reports having potentially actionable mutations. NGS results were successfully generated for 15 patients, with median time from tissue processing to reporting of 11.6 days (range, 5 to 21 days), and presented at a biweekly multidisciplinary tumor sequencing advisory board. Attendance averaged 19 participants (range, 12 to 24) at 20 days after patient enrollment (range, 10 to 30 days). Twenty-seven (range, 1 to 4 per patient) potentially actionable mutations were detected in 11 of 15 patients: TP53 (n = 6), KRAS (n = 4), MET (n = 3), APC (n = 3), CDKN2A (n = 2), PTEN (n = 2), PIK3CA,

FLT3, NRAS, VHL, BRAF, SMAD4, and ATM. The Hotspot Panel is now offered as a clinically available test at our institution. NGS results can be obtained by in-house high-throughput sequencing and reviewed in a multidisciplinary tumor sequencing advisory board in a clinically relevant manner. The essential components of a center for personalized cancer care can support clinical decisions outside the university.

Full text available from Journal of Molecular Diagnostics (USD 31.50) PMID: 26331835

E) The Evolving Role of the Laboratory Professional in the Age of Genome Sequencing: A Vision of the Association for Molecular Pathology Schrijver I, Farkas DH, Gibson JS, Lyon E, AMP Executive Committee. The Evolving Role of the Laboratory Professional in the Age of Genome Sequencing: A Vision of the Association for Molecular Pathology. J Mol Diagn. 2015; 17(4): 335-338.

Summary: The Association for Molecular Pathology emphasizes the need for proactive engagement of laboratory directors with clinicians, patients, and professional organizations as genomic sequence analysis gains importance in diagnostic medicine.

Free full text available from Journal of Molecular Diagnostics PMID: 26047767

F) Building the Foundation for Genomics in Precision Medicine Aronson SJ, Rehm HL. Building the Foundation for Genomics in Precision Medicine. Nature. 2015; 526(7573): 336-342.

Summary: Precision medicine has the potential to profoundly improve the practice of medicine. However, the advances required will take time to implement. Genetics is already being used to direct clinical decision-making and its contribution is likely to increase. To accelerate these advances, fundamental changes are needed in the infrastructure and mechanisms for data collection, storage and sharing. This will create a continuously learning health-care system with seamless cycling between clinical care and research. Patients must be educated about the benefits of sharing data. The building blocks for such a system are already forming and they will accelerate the adoption of precision medicine.

Free full text available from Nature PMID: 26469044

1.8 Pathologists Education in Molecular Pathology and Genomics

A) Progress and Potential: Training in Genomic Pathology Haspel RL, Olsen RJ, Berry A, et al. Progress and potential: training in genomic pathology. Arch Pathol Lab Med. 2014; 138(4): 498-504.

Summary: CONTEXT: Genomic medicine is revolutionizing patient care. Physicians in areas as diverse as oncology, obstetrics, and infectious disease have begun using next-generation sequencing assays as standard diagnostic tools. OBJECTIVE: To review the role of pathologists in genomic testing as well as current educational programs and future training needs in genomic pathology. DATA SOURCES: Published literature as well as personal experience based on committee membership and genomic pathology curricular design. CONCLUSIONS: Pathologists, as the directors of the clinical laboratories, must be prepared to integrate genomic testing into their practice. The pathology community has made significant progress in genomics-related education. A continued coordinated and proactive effort will ensure a future vital role for pathologists in the evolving health care system and also the best possible patient care.

Free full text available from PubMed PMID: 24678680

B) Teaching Residents Genomic Pathology: A Novel Approach for New Technology Haspel RL. Teaching residents genomic pathology: a novel approach for new technology. Adv Anat Pathol. 2013; 20(2): 125-129.

Summary: Genomics-based diagnostics have become part of patient care. As pathologists have the expertise in clinical laboratory testing as well as access to patient samples, all genomic medicine is genomic pathology. This article will review the evidence that there is a critical need for pathology resident training in genomics. Several individual program curricula are described as well as the progress of the Training Residents in Genomics Working Group. This group has made significant advances toward developing, implementing, and evaluating a national curriculum in genomics for pathology residents. The novel approach of the Training Residents in Genomics Working Group can be used as a model for training pathology professionals in any new technology.

Free full text available from PubMed

PMID: 23399798

C) Integration of Genomic Medicine into Pathology Residency Training: The Stanford Open Curriculum Schrijver I, Natkunam Y, Galli SJ, Boyd S. Integration of Genomic Medicine into Pathology Residency Training: The Stanford Open Curriculum. J Mol Diagn. 2013; 15(2): 141-8.

Summary: Next-generation sequencing methods provide an opportunity for molecular pathology laboratories to perform genomic testing that is far more comprehensive than single-gene analyses. Genome-based test results are expected to develop into an integral component of diagnostic clinical medicine and to provide the basis for individually tailored health care. To achieve these goals, rigorous interpretation of high-quality data must be informed by the medical history and the phenotype of the patient. The discipline of pathology is well positioned to implement genome-based testing and to interpret its results, but new knowledge and skills must be included in the training of pathologists to develop expertise in this area. Pathology residents should be trained in emerging technologies to integrate genomic test results appropriately with more traditional testing, to accelerate clinical studies using genomic data, and to help develop appropriate standards of data quality and evidence-based interpretation of these test results. We have created a genomic pathology curriculum as a first step in helping pathology residents build a foundation for the understanding of genomic medicine and its implications for clinical practice. This curriculum is freely accessible online.

Free full text available from Journal of Molecular Diagnostics PMID: 23313248

D) The Training Residents in Genomics Working Group Curriculum Training Residents in Genomics (TRIG) Working Group. Training Residents in Genomics Lecture Materials. 2013. Intersociety Council for Pathology Information Web site. http://www.pathologytraining.org/trainees/TRIG_lecture_materials.cfm. Accessed June 12, 2015.

Summary: A working group of the Pathology Residency Directors Section of the Association of Pathology Chairs developed this curriculum. Members include experts in molecular genetic pathology, genetic counseling and medical education. The website includes a book of references as well as PowerPoint lectures with notes. The lectures cover test methodology,

application of testing to patient care, and communication of results to physicians and patients.

Free full text available from Intersociety Council for Pathology Information website

E) Stanford Open Curriculum in Genomic Medicine Stanford School of Medicine. Stanford University Open Curriculum in Genomic Medicine. 2012. Stanford University genomic lectures YouTube channel. http://www.youtube.com/playlist?feature=plcp&list=PLfTljtR5bxMcTg8hgQp9 sA4YQwicpSAQv. Accessed January 25, 2013.

Summary: A series of 10 YouTube videos of lectures included in the Stanford University pathology resident genomics curriculum. Topics include genomics methods and microarrays as well as specific applications of molecular pathology and genomics in hematology, histocompatibility antigen testing, pharmacogenomics, solid tumors and inherited disorders. There is also a lecture on ethical, regulatory and commercial issues related to personal genomics.

Free full presentations available from YouTube

F) Genomic Medicine Initiative at Beth Israel Deaconess Medical Center Beth Israel Deaconess Medical Center. Genomic medicine initiative web site. http://www.genomicmedicineinitiative.org. Accessed July 7, 2015.

Summary: Lectures available for use and repurpose at http://genomicmedicineinitiative.org/. This resource is especially useful for pathology resident education.

Archived presentations available from Genomic Medicine Initiative website

This section includes the following topics:

• The Basics (Section 2.1); • Breast Cancer (Section 2.2); • Central Nervous System Tumors (Section 2.3); • Gastrointestinal Cancer (Section 2.4); • Lung Cancer (Section 2.5); • Melanoma (Section 2.6); • Thyroid Cancer (Section 2.7); • Bone & Soft Tissue (Section 2.8); • Head & Neck (Section 2.9); • Genitourinary Cancer (Section 2.10); • Cancer of Unknown Primary Origin (Section 2.11); and • Hematopoietic Neoplasms (Section 2.12).

2.1 The Basics

Genetic abnormalities in cancer have been discovered at an accelerated rate since the advent of genomic technologies. Array Comparative Genomic Hybridization (aCGH) and Single Nucleotide Polymorphism array (SNParray) analysis and whole genome, whole exome, and whole transcriptome sequencing are important discovery tools that have led to the identification of previously unknown genetic abnormalities in many types of cancer. Many of these alterations are now utilized as important diagnostic and prognostic clinical markers or therapeutic targets for personalized therapy. SNParray and whole exome and targeted gene panels utilizing next-generation sequencing are now moving into the mainstream of clinical oncology testing. Gene expression panels and miRNA testing are also emerging in the clinical testing arena for molecular subclassification of tumors.

While genomic testing approaches are yielding an unprecedented amount of information, genetic testing in cancer is also subject to some unique challenges. Most solid tumors are fixed in formalin to preserve tissue histology, but formalin reduces the quality of retrievable nucleic acids in the sample. NGS oncology

assay design must take into consideration limitations associated with DNA extracted from formalin-fixed specimens. Tumor specimens are usually a mixture of tumor and non-tumor cells, and tumor-specific variants may be missed if the tumor content is too low. For sequencing based assays, a critical factor is sufficient depth of coverage to allow for the detection of low frequency mutations in heterogeneous samples. Tumor heterogeneity, in which different samples of a tumor have different mutation profiles, is another potential complicating factor. One must also consider how to first verify that a variant is somatic in nature instead of germline (i.e should corresponding non-cancerous DNA also be tested) and secondly classify somatic variants according to clinical significance (e.g. detrimental, unknown significance, benign). High quality bioinformatic analysis is extremely important in helping to resolve some of these issues.

This section includes the following topics:

• Insights from Adopters (Section 2.1.1); and • Quick Reference Table: Selected Tests by Tumor Type in Solid Tumors (Section 2.1.2).

A) CAP Short Presentations on Emerging Concepts (SPEC): Emerging Concepts in Next-Generation Sequencing and Cancer Genomics [PowerPoint slides] Chandra PK. CAP Short Presentations on Emerging Concepts (SPECs): Emerging Concepts on Next-Generation Sequencing and Cancer Genomics (v 2.0e rev 9/2/16) [PowerPoint slides]. Anderson MW, Crothers J, Misialek MJ, Vance GH, Zutter MM, eds. Northfield, IL: College of American Pathologists; 2016.

Access the slides

B) The Cancer Genome Atlas Pan-Cancer Analysis Project Cancer Genome Atlas Research Network, Weinstein JN, Collisson EA, et al. The Cancer Genome Atlas Pan-Cancer analysis project. Nat Genet. 2013; 45(10): 1113-1120.

Summary: The Cancer Genome Atlas (TCGA) Research Network has profiled and analyzed large numbers of human tumors to discover molecular aberrations at the DNA, RNA, protein and epigenetic levels. The resulting rich data provide a major opportunity to develop an integrated picture of commonalities, differences and emergent themes across tumor lineages. The Pan-Cancer initiative compares the first 12 tumor types profiled by TCGA. Analysis of the molecular aberrations and their functional roles across tumor

types will teach us how to extend therapies effective in one cancer type to others with a similar genomic profile.

Free full text available from PubMed PMID: 24071849

C) Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing Gerlinger M, Rowan AJ, Horswell S, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 2012; 366(10):883–892.

Summary: BACKGROUND: Intratumor heterogeneity may foster tumor evolution and adaptation and hinder personalized-medicine strategies that depend on results from single tumor-biopsy samples. METHODS: To examine intratumor heterogeneity, we performed exome sequencing, chromosome aberration analysis, and ploidy profiling on multiple spatially separated samples obtained from primary renal carcinomas and associated metastatic sites. We characterized the consequences of intratumor heterogeneity using immunohistochemical analysis, mutation functional analysis, and profiling of messenger RNA expression. RESULTS: Phylogenetic reconstruction revealed branched evolutionary tumor growth, with 63 to 69% of all somatic mutations not detectable across every tumor region. Intratumor heterogeneity was observed for a mutation within an autoinhibitory domain of the mammalian target of rapamycin (mTOR) kinase, correlating with S6 and 4EBP phosphorylation in vivo and constitutive activation of mTOR kinase activity in vitro. Mutational intratumor heterogeneity was seen for multiple tumor-suppressor genes converging on loss of function; SETD2, PTEN, and KDM5C underwent multiple distinct and spatially separated inactivating mutations within a single tumor, suggesting convergent phenotypic evolution. Gene-expression signatures of good and poor prognosis were detected in different regions of the same tumor. Allelic composition and ploidy profiling analysis revealed extensive intratumor heterogeneity, with 26 of 30 tumor samples from four tumors harboring divergent allelic-imbalance profiles and with ploidy heterogeneity in two of four tumors. CONCLUSIONS: Intratumor heterogeneity can lead to underestimation of the tumor genomics landscape portrayed from single tumor-biopsy samples and may present major challenges to personalized- medicine and biomarker development. Intratumor heterogeneity, associated with heterogeneous protein function, may foster tumor adaptation and therapeutic failure through Darwinian selection. (Funded by the Medical Research Council and others.).

Free full text available from New England Journal of Medicine PMID: 22397650

D) Next-Generation Companion Diagnostics: Promises, Challenges, and Solutions Khoury JD,Catenacci DV. Next-Generation Companion Diagnostics: Promises, Challenges, and Solutions. Arch Pathol Lab Med. 2015; 139(1):11- 3. doi: 10.5858/arpa.2014-0063-ED.

No summary available.

Free full text available from the CAP’s Archives PMID: 25166874

E) Mutational Landscape and Significance across 12 Major Cancer Types Kandoth C, McLellan MD, Vandin F, et al. Mutational Landscape and Significance across 12 Major Cancer Types. Nature. 2013; 502(7471): 333- 339.

Summary: The Cancer Genome Atlas (TCGA) has used the latest sequencing and analysis methods to identify somatic variants across thousands of tumours. Here we present data and analytical results for point mutations and small insertions/deletions from 3,281 tumours across 12 tumour types as part of the TCGA Pan-Cancer effort. We illustrate the distributions of mutation frequencies, types and contexts across tumour types, and establish their links to tissues of origin, environmental/carcinogen influences, and DNA repair defects. Using the integrated data sets, we identified 127 significantly mutated genes from well-known (for example, mitogen-activated protein kinase, phosphatidylinositol-3-OH kinase, Wnt/beta-catenin and receptor tyrosine kinase signalling pathways, and cell cycle control) and emerging (for example, histone, histone modification, splicing, metabolism and proteolysis) cellular processes in cancer. The average number of mutations in these significantly mutated genes varies across tumour types; most tumours have two to six, indicating that the number of driver mutations required during oncogenesis is relatively small. Mutations in transcriptional factors/regulators show tissue specificity, whereas histone modifiers are often mutated across several cancer types. Clinical association analysis identifies genes having a significant effect on survival, and investigations of mutations with respect to clonal/subclonal architecture delineate their temporal orders during tumorigenesis. Taken together, these results lay the groundwork for developing new diagnostics and individualizing cancer treatment.

Free full text available from PubMed

PMID: 24132290

F) Somatic Mutation in Cancer and Normal Cells Martincorena I,Campbell PJ. Somatic Mutation in Cancer and Normal Cells. Science. 2015; 349(6255): 1483-1489.

Summary: Spontaneously occurring mutations accumulate in somatic cells throughout a person's lifetime. The majority of these mutations do not have a noticeable effect, but some can alter key cellular functions. Early somatic mutations can cause developmental disorders, whereas the progressive accumulation of mutations throughout life can lead to cancer and contribute to aging. Genome sequencing has revolutionized our understanding of somatic mutation in cancer, providing a detailed view of the mutational processes and genes that drive cancer. Yet, fundamental gaps remain in our knowledge of how normal cells evolve into cancer cells. We briefly summarize a number of the lessons learned over 5 years of cancer genome sequencing and discuss their implications for our understanding of cancer progression and aging.

Full text available from Science (USD 30.00 for 24 hour access) PMID: 26404825

G) The Cancer Genomics Resource List 2014 Zutter MM, Bloom KJ, Cheng L, et al. The Cancer Genomics Resource List 2014. Arch Pathol Lab Med. 2015; 139(8):989-1008.

Summary: Context .- Genomic sequencing for cancer is offered by commercial for-profit laboratories, independent laboratory networks, and laboratories in academic medical centers and integrated health networks. The variability among the tests has created a complex, confusing environment. Objective .- To address the complexity, the Personalized Health Care (PHC) Committee of the College of American Pathologists proposed the development of a cancer genomics resource list (CGRL). The goal of this resource was to assist the laboratory pathology and clinical oncology communities. Design .- The PHC Committee established a working group in 2012 to address this goal. The group consisted of site-specific experts in cancer genetic sequencing. The group identified current next- generation sequencing (NGS)-based cancer tests and compiled them into a usable resource. The genes were annotated by the working group. The annotation process drew on published knowledge, including public databases and the medical literature. Results .- The compiled list includes NGS panels offered by 19 laboratories or vendors, accompanied by annotations. The list has 611 different genes for which NGS-based mutation testing is offered. Surprisingly, of these 611 genes, 0 genes were listed in every panel, 43

genes were listed in 4 panels, and 54 genes were listed in 3 panels. In addition, tests for 393 genes were offered by only 1 or 2 institutions. Table 1 provides an example of gene mutations offered for breast cancer genomic testing with the annotation as it appears in the CGRL 2014. Conclusions .- The final product, referred to as the Cancer Genomics Resource List 2014, is available as supplemental digital content.

Free full text available from the CAP’s Archives PMID: 25436904 NOTE: Also cited in Section 1.1.1

H) Clinical Validation of a Next-Generation Sequencing Screen for Mutational Hotspots in 46 Cancer-Related Genes Singh RR, Patel KP, Routbort MJ, et al. Clinical validation of a next- generation sequencing screen for mutational hotspots in 46 cancer-related genes. J Mol Diagn. 2013; 15(5): 607-622.

Summary: Transfer of next-generation sequencing technology to a Clinical Laboratory Improvement Amendments-certified laboratory requires vigorous validation. Herein, we validated a next-generation sequencing screen interrogating 740 mutational hotspots in 46 cancer-related genes using the Ion Torrent AmpliSeq cancer panel and Ion Torrent Personal Genome Machine (IT-PGM). Ten nanograms of FFPE DNA was used as template to amplify mutation hotspot regions of 46 genes in 70 solid tumor samples, including 22 archival specimens with known mutations and 48 specimens sequenced in parallel with alternate sequencing platforms. In the archival specimens, the IT-PGM detected expected nucleotide substitutions (n = 29) and four of six insertions/deletions; in parallel, 66 variants were detected. These variants, except a single nucleotide substitution, were confirmed by alternate platforms. Repeated sequencing of progressively diluted DNA from two cancer cell lines with known mutations demonstrated reliable sensitivity at 10% variant frequency for single nucleotide variants with high intrarun and inter-run reproducibility. Manual library preparation yielded relatively superior sequencing performance compared with the automated Ion Torrent OneTouch system. Overall, the IT-PGM platform with the ability to multiplex and simultaneously sequence multiple patient samples using low amounts of FFPE DNA was specific and sensitive for single nucleotide variant mutation analysis and can be incorporated easily into the clinical laboratory for routine testing.

Free full text available from Journal of Molecular Diagnostics PMID: 23810757

2.1.1 Insights from Adopters

This section includes insights from the following adopters:

• David G. Hicks (Section 2.1.1.1); • Colin Pritchard (Section 2.1.1.2); and • Eric J. Duncavage (Section 2.1.1.3).

2.1.1.1 Insights from David G. Hicks, MD, FCAP

David G. Hicks, MD, FCAP, is currently the director of Surgical Pathology at the University of Rochester Medical Center. Dr. Hicks earned his medical degree from the University of Rochester School of Medicine and Dentistry. He works with breast cancer patients, with the goal of identifying new biomarkers to help better understand the prognosis and guide the therapeutic management of this disease. Dr. Hicks participated in the ASCO/CAP ER/PgR Guideline panel and is currently serving as Co-Chair of the ASCO-CAP HER2 Testing in Breast Cancer Committee. David G. Hicks, MD, FCAP Dr. Hicks has co-authored over 140 peer reviewed articles that have appeared in a variety of journals, including Clinical Cancer Research, The American Journal of Pathology, Cancer, and the American Journal of Surgical Pathology. He also serves on the editorial boards of the Archives of Pathology and Laboratory Medicine, Biotechnic and Histochemistry and Applied Immunohistochemistry and Molecular Morphology. As part of the working group for the CAP BPFT AP3 Program, Dr. Hicks has contributed significantly to the overall direction and development of the BPFT curriculum and assessments.

Dr. Hicks’ Insights for the Next Wave of Adopters:

1 Stay informed on Seek out and review new literature related to treatment the molecular analysis of clinical samples and information related how these studies help inform treatment to molecular decisions. In doing so, you will be better analysis and breast equipped to discuss the benefits and limitations cancer care of molecular testing in the clinical setting. There are excellent articles and educational offerings from the College as well as other pathology organizations on this topic that can help make the pathologist a ‘clinical consultant on the biology of disease’ for other members of the multidisciplinary care team.

2 Help ensure the Provide leadership and guide your institution’s biologic quality of efforts to review procedures related to tissue tissue samples for collection and fixation with a movement biomarker studies towards standardizing pre-analytic variables. As molecular analysis of tissue samples becomes increasingly applicable to clinical care, the accuracy, reliability and relevance of this approach, and the tissue requirements for testing need to be addressed. There is a growing awareness that variable tissue handling and prolonged cold ischemic times can adversely affect the quality of breast tissue samples for ER, PR and HER2 testing and other molecular analysis. 3 Provide clear, Develop a firm understanding of how your concise, and reports are used to make clinical decisions and comprehensive make sure the information is presented in a reports clear, concise and understandable manner. Pathology reports need to be comprehensive in addressing all issues relevant to the patient’s care as well as provide summary-level diagnostic information (including molecular test results) and recommendations that are patient/tumor specific. 4 Become an active Attend tumor boards and/or multidisciplinary participant and treatment planning discussions. Be available consultant to the as a diagnostic consultant and actively multidisciplinary participate in these discussions and share care team information on how molecular testing can be used to inform clinical diagnosis and treatment decisions.

5 Check that the Review all results to verify that the molecular biomarker results tests or profile are a reasonable fit with the correlate with the clinical features for each breast cancer patient clinical profile for regardless of whether the testing (either single each patient marker studies or multigene panels) is done in your laboratory or sent out. There is strong evidence in the literature that the histopathologic features of a breast cancer tumor correlate with its molecular profile. Low grade tumors with a low proliferative index typically will be ER/PR positive, HER2 negative and have a low recurrence score by the Oncotype DX test. High grade tumors with a high proliferative index are more likely to show low levels or an absence of ER expression, over-express HER2 and have a high recurrence score by the Oncotype DX test. If the molecular testing is dramatically different from the histopathologic features of the tumor, the case should be thoroughly investigated by the pathologist in collaboration with medical oncology before decisions on adjuvant treatment are made.

Dr. Hicks’ thoughts on molecular testing:

A) CAP Short Presentations in Emerging Concepts (SPECS): Emerging Concepts in Molecular Testing in Breast Cancer [PowerPoint slides] Hicks DG. CAP Short Presentations on Emerging Concepts (SPECS): Emerging Concepts in Molecular Testing in Breast Cancer (v 4.0e rev 7/7/15) [PowerPoint slides]. Caughron SK, Misialek MJ, Moyer AM, Nowak JA, eds. Northfield, IL: College of American Pathologists; 2015.

Access the slides

Dr. Hicks’ suggested articles and resources:

A) Prediction of the Oncotype DX Recurrence Score: Use of Pathology- Generated Equations Derived by Linear Regression Analysis Klein ME, Dabbs DJ, Shuai Y, et al. Prediction of the Oncotype DX recurrence score: use of pathology-generated equations derived by linear regression analysis. Mod Pathol. 2013;26(5):658-64.

Summary: Oncotype DX is a commercial assay frequently used for making chemotherapy decisions in estrogen receptor (ER)-positive breast cancers. The result is reported as a recurrence score ranging from 0 to 100, divided into low-risk (<18), intermediate-risk (18-30), and high-risk (≥31) categories. Our pilot study showed that recurrence score can be predicted by an equation incorporating standard morphoimmunohistologic variables (referred to as original Magee equation). Using a data set of 817 cases, we formulated three additional equations (referred to as new Magee equations 1, 2, and 3) to predict the recurrence score category for an independent set of 255 cases. The concordance between the risk category of Oncotype DX and our equations was 54.3%, 55.8%, 59.4%, and 54.4% for original Magee equation, new Magee equations 1, 2, and 3, respectively. When the intermediate category was eliminated, the concordance increased to 96.9%, 100%, 98.6%, and 98.7% for original Magee equation, new Magee equations 1, 2, and 3, respectively. Even when the estimated recurrence score fell in the intermediate category with any of the equations, the actual recurrence score was either intermediate or low in more than 80% of the cases. Any of the four equations can be used to estimate the recurrence score depending on available data. If the estimated recurrence score is clearly high or low, the oncologists should not expect a dramatically different result from Oncotype DX, and the Oncotype DX test may not be needed. Conversely, an Oncotype DX result that is dramatically different from what is expected based on standard morphoimmunohistologic variables should be thoroughly investigated.

Free full text available from PubMed PMID: 23503643

2.1.1.2 Insights from Colin Pritchard, MD, PhD, FCAP

Colin Pritchard, MD, PhD, FCAP, is an assistant professor of laboratory medicine and the associate director of genetics and solid tumors at University of Washington in Seattle where he earned his MD and PhD. This clinical laboratory performs many different genetic tests including tests that use next-generation sequencing technology for the University of Washington patient population. His interests are largely in molecular oncology, including cancer based genetics. He has worked to bring on a test menu for somatic mutations based biomarkers for

Colin Pritchard, MD, guiding precision therapy, cancer based testing. Additionally, he has collaborated PhD, FCAP with researchers at the University of Washington on some of the inherited cancer mutation testing, looking at cancer predispositions in terms of inherited mutations.

Dr. Pritchard’s insights focused on pathologists bringing in NGS cancer panels tests or also pathologists who are ordering these tests (May 2014):

1 Understand the From a pathologist standpoint, understanding difference between the methodology and difference between hot hot spot and spot and comprehensive panels is really comprehensive important. There are strengths and limitations sequencing panels. of both. A hot spot panel may test 200 genes but only test 1 mutation each from those 200 genes. Whereas a comprehensive panel may also test 200 genes but test up to 1000x more spots, maybe sequencing all the coding exons from the 200 genes. It could also detect copy number variation amplifications, deletions of certain genes or translocation which may be clinically important. 2 Hot spot panels are It may be somewhat easier to set up in house often the best place and the data interpretation is easier—there’s to start. even off the shelf software which will assist you. You often already know what those hot spots mean in terms of their clinical significance.

Hot spot panels generally use methodology of some kind of multiplex PCR to enrich the genes that you are testing. There are multiple PCR reactions that are done to amplify the genes of interest and then those genes are sequenced. The turnaround time tends to be faster but it’s not as comprehensive. A hot spot panel may only detect half of the things that are there that you care about. There may be a very good chance that you are missing a key thing in that tumor that would help that patient.

Interpretation of hot spot panels is pretty straight forward. You can usually just make a list of the (hundreds of) variants being tested by the technology and then the interpretation is attached for each of those variants.

3 A comprehensive Comprehensive panels usually take longer and panel has the cost more and are more technically challenging advantage of to perform. In comprehensive panels, there’s a detecting most of the whole host of different methods but a common things that you care method used is called “capture” and that could about. be done with hybridized based capture with DNA or RNA probes. 4 Pathologists will Hotspot panels will be around for a while but eventually be doing they won’t be around forever. However, exome or genome hotspot panels are the best place to start for sequencing. NGS cancer testing. 5 For pathologists that A lab needs a way to track all the mutations are considering and variants that have been detected with your bringing NGS caner assay. It will help you understand the kinds of testing in house it is mutations or variants that you are seeing in critical to have your your patient population. Tracking your variants own in house is a way of quickly filtering out any potential database analytical false positives artifacts that are specific to your exact assay, which tend to be recurrent in a large percentage of cases. This is a very useful tool for pathologists who are trying to interpret the data and wondering if the particular mutation in x gene (which is important) is real without having to go through a lot of trouble to analytically validate by an alternative method.

Another advantage of the database is to understand the variant frequencies among your population, which could be different than what’s in the published literature 6 Collaborate with the Set up meetings with the oncologists to plan clinicians, especially what is going to be on the panel based on the oncologists, for clinical need. Get the clinicians who are going tumor based testing. to be using this technology (to make decisions for their patients) involved at a very early stage of the planning. Via this close collaboration, you’ve established relationships between the pathologists and the ordering providers which may help down the road in facilitating of results.

7 Collaborate with the Ask what’s going to be helpful for them and ordering clinician as what’s not. This is going to vary by institution. to what they want on Develop clear, concise and easy to understand the NGS report. pathology reports for NGS. 8 Communicate test When there’s a clear course of action to be results by phone to taken, I call the expert clinician. It ensures that the ordering the critical results to the patient have been provider, especially adequately communicated. Also, the when there is a pathologist will start to get a better handle on critically important how this information is being used in treatment result where it is decision making and the things that are obvious there is a important for patient care but are not reflected clear course of in the data (psychosocial issues or insurance action to be taken for reimbursement barriers). For instance, you the patient. may have a great result but the patient can’t get the drug because the insurance won’t cover it. One can write a beautiful report but it’s completely irrelevant if that treatment is not possible for that patient 9 There are benefits to There’s a benefit for patient care to having a bringing NGS tests local expert who can talk one-on-one to the in house. ordering provider and have access to the same medical records as opposed to send out. 10 Work with the It’s worth contacting the payers who will be payers. servicing the population and get a handle on potential reimbursement issues. Unexpected large bills can be a barrier to patient care. Discuss with payers the clinical utility and validity of the test that is being done. Provide some education. It is helpful to show the evidence for clinical utility and validity. Also provide recent national guidelines. Educate payers that it’s an evolving technology but it’s becoming the standard of care, especially for certain cancer types.

11 For interpretation, Having people available on the team who are having expertise in involved in the interpretation and reporting of both inherited cancer the tests with expertise in both areas is very genetics and tumor important. It helps to understand the types of genetics is very mutations in tumors and what they mean for useful. patients if you’re doing inherited and vice versa. Having that knowledge base or having access to someone who is an expert in that particular aspect is critical for the accurate interpretation of the data. 12 Set realistic Turnaround time depends on what approach is expectations of being taken (hot spot vs. comprehensive turnaround time panel). Hot spot is faster but still usually a (TAT). couple of weeks. Comprehensive panels are usually slower – 3-12 weeks depending on the specific considerations and the lab, etc. Have a realistic appraisal of turnaround time. Under promise and over deliver. 13 Share the data. If you’re an academic or a larger private pathology group who is bringing NGS in house there’s an upside with sharing interpretation data. It helps everyone learn. There are just so many variants out there; it’s just not possible for one lab to be up on all these. Putting your heads together really helps everyone. 14 Invest in Bioinformatics is especially important for bioinformatics, not comprehensive panels as well as the people just monetary but in who help you crunch the data for the final terms of effort. variant list. The investment is not just monetary but director’s time and effort. Invest in frequent meetings with the people who are crunching the data especially when a test is being developed. This will help the pathologists get more understanding of how the bioinformatics works.

Even if you’re using an off the shelf software, it’s critical to dig in deep and understand how it’s working: strengths, weaknesses of software and bioinformatics. It’s no longer possible for a pathologist to manually review all of the raw data like in the past. Have the lab director meet with the bioinformaticists, giving examples, to

make sure that what’s being done meets the needs. 15 Don’t rely solely on Bioinformatics helps you review the data for automated output for everything you’re testing. However, there is interpretation. value in looking at the raw data. For any variant that we put into a clinical report, we have multiple directors review the raw data for the specific variants looking for quality metrics. It’s not possible to do this for everything but you can do this in a targeted way. Always have a healthy level of skepticism. No bioinformatics or software approach is perfect. 16 Build flexibility into Remembering NGS is different than anything the NGS platform. else we’ve ever done. You’re not like “I’m doing Consider a platform this one test but I need to build my platform to that’s flexible and include 20 more things in a year”. Building in can accommodate flexibility is something worth thinking about adding new genes or otherwise a lot of effort could be put into dropping existing something that will be obsolete very soon. genes that are Consider a platform that’s flexible and can irrelevant in accommodate adding new genes or dropping relatively short time existing genes that are irrelevant in relatively frames. short time frames. Keep in mind clinical validation that needs to be done for any change to the test.

Dr. Pritchard’s suggested articles:

A) Existing and Emerging Technologies for Tumor Genomic Profiling MacConaill LE. Existing and emerging technologies for tumor genomic profiling. J Clin Oncol. 2013; 31(15): 1815-1824.

Summary: Ongoing global genome characterization efforts are revolutionizing our knowledge of cancer genomics and tumor biology. In parallel, information gleaned from these studies on driver cancer gene alterations--mutations, copy number alterations, translocations, and/or chromosomal rearrangements--an be leveraged, in principle, to develop a cohesive framework for individualized cancer treatment. These possibilities have been enabled, to a large degree, by revolutionary advances in genomic technologies that facilitate systematic profiling for hallmark cancer genetic alterations at increasingly fine resolutions. Ongoing innovations in existing genomics technologies, as well as the many emerging technologies, will

likely continue to advance translational cancer genomics and precision cancer medicine.

Free full text available from PubMed PMID: 23589546

B) Cancer Genomics and Inherited Risk Stadler ZK, Schrader KA, Vijai J, Robson ME, Offit K. Cancer genomics and inherited risk. J Clin Oncol. 2014; 32(7): 687-698.

Summary: Next-generation sequencing (NGS) has enabled whole-exome and whole-genome sequencing of tumors for causative mutations, allowing for more accurate targeting of therapies. In the process of sequencing the tumor, comparisons to the germline genome may identify variants associated with susceptibility to cancer as well as other hereditary diseases. Already, the combination of massively parallel sequencing and selective capture approaches has facilitated efficient simultaneous genetic analysis (multiplex testing) of large numbers of candidate genes. As the field of oncology incorporates NGS approaches into tumor and germline analyses, it has become clear that the ability to achieve high-throughput genotyping surpasses our current ability to interpret and appropriately apply the vast amounts of data generated from such technologies. A review of the current state of knowledge of rare and common genetic variants associated with cancer risk or treatment outcome reveals significant progress, as well as a number of challenges associated with the clinical translation of these discoveries. The combined efforts of oncologists, genetic counselors, and cancer geneticists will be required to drive the paradigm shift toward personalized or precision medicine and to ensure the incorporation of NGS technologies into the practice of preventive oncology.

Full text available from Journal of Clinical Oncology (subscription required) PMID: 24449244

C) Criteria for the Use of Omics-Based Predictors in Clinical Trials McShane LM, Cavenagh MM, Lively TG, et al. Criteria for the use of omics- based predictors in clinical trials. Nature. 2013; 502(7471): 317-320.

Summary: The US National Cancer Institute (NCI), in collaboration with scientists representing multiple areas of expertise relevant to 'omics'-based test development, has developed a checklist of criteria that can be used to determine the readiness of omics-based tests for guiding patient care in clinical trials. The checklist criteria cover issues relating to specimens, assays, mathematical modelling, clinical trial design, and ethical, legal and

regulatory aspects. Funding bodies and journals are encouraged to consider the checklist, which they may find useful for assessing study quality and evidence strength. The checklist will be used to evaluate proposals for NCI- sponsored clinical trials in which omics tests will be used to guide therapy.

Free full text available from PubMed PMID: 24228635

2.1.1.3 Insights from Eric J. Duncavage, MD, FCAP

Eric J. Duncavage, MD, FCAP, is currently an assistant professor of pathology and immunology at Washington University in Saint Louis where he is the director of the molecular genetic pathology fellowship. He trained in anatomic and clinical pathology at Washington University and completed fellowships in molecular pathology and hematopathology.

Eric J. Duncavage, MD, Washington University Genomics and Pathology Services (WU-GPS) was one of FCAP the first clinical labs to launch an NGS-based cancer panel three years ago. Dr. Duncavage helped develop some of the early informatics tools and methods for clinical grade sequencing analysis as well as annotating identified variants. To date, WU-GPS has signed-out over 2,000 clinical cancer cases using their custom targeted cancer panel and informatics pipeline. The WU-GPS laboratory focuses primarily on cancer, but has recently started to expand into constitutional sequencing for inherited disorders such as cardiomyopathies and complement deficiencies.

Dr. Duncavage’s insights for the next wave of adopters (June 2014):

1 Expect the adoption NGS based cancer sequencing panels are rate of clinical NGS going to be a larger and larger part of what we cancer panels to do as pathologists. There’s a lot of additional continue to grow information from sequencing that you can’t get rapidly from tumor morphology alone. Eventually, the majority of cancers diagnosed in the U.S. will be sequenced at diagnosis to identify prognostically or therapeutically significant mutations. Oncologists are becoming increasingly dependent on the identification of gene mutations to guide their therapy and aid risk assessment. I expect this trend to continue.

2 The trend is to use We first started to sequence patients with very NGS cancer panels high-stage disease, patients that had failed first for patients with and second line chemotherapy, for which there earlier staged was no therapeutic alternative. We are now disease. seeing cancer panels being used for patients with earlier-stage disease where the oncologists can develop a rational therapy approach based on the identification of specific mutations. 3 NGS cancer panels In recent years, several vendors have are easier to developed well-optimized ‘off the shelf’ cancer implement now than panel reagents. These vendor panels cover in prior years. most of the relevant cancer-related genes and make the wet-lab part of building an NGS assay much simpler. Many vendor panels also include optimized analysis software. 4 The cost of entry There’s been rapid growth in ‘clinical-scale’ now has been sequencers that are faster and much less significantly lowered expensive than previous versions geared to the point where toward sequencing whole genomes. It has most academic become very cost-attractive to perform NGS laboratories and assays in the clinical laboratory because you large private labs will can test for multiple genes at once, reducing adopt NGS for their the need for divergent testing platforms. NGS- clinical lab based methods can also detect copy number variants and rearrangements and may one day replace FISH and microarrays for routine use. 5 The cost of the NGS assays generate lots of data, often computational hundreds of gigabytes a week. This data hardware is going to requires a significant computational be equivalent to the infrastructure to analyze and store. Most cost of the laboratories will find that they need a modestly sequencing sized computational cluster for NGS data hardware. analysis. In general, smaller, less expensive sequencers generate less data and require less computational hardware to analyze data. Plan on spending as much on computer hardware as you do on the sequencer.

6 The rule of thumb The number of bioinformaticists you need that we tell people is depends on what type of analysis that you that you probably want to do. Most labs are going to require at need at least as least one informaticist. If you are only hiring many one informaticist for a small lab, you should bioinformaticists as probably stick with a vendor-provided panel wet-lab and associated software. If you are doing a technologists. more complex analysis like calling translocations or copy number variants, you may need additional bioinformatics personnel. 7 Don’t get stuck in the Validation of NGS panels is an area that many validation step. labs struggle with. While the guidelines have improved, there is still ambiguity with regards to what constitutes validating an NGS-based assay. To determine analytical sensitivity and specificity (which is required for any assay), we use mixtures of publicly-available HapMap cell lines. We’ve found this to be a powerful tool rather than relying on patient samples with only a single mutation. To determine the sensitivity and specificity of an assay, we create artificial mixes of cell lines to simulate various variant allele frequencies. Since you know what variants should be present, you can calculate the sensitivity at various dilution points; likewise since you know what variants shouldn’t be there you can calculate specificity. Cell lines offer another advantage in that they are renewable resources and can be use over time to compare the performance of different methods and ensure QC. The National Institute of Standards and Technology (NIST) NA12878 cell line is particularly useful as it has been sequenced on multiple platforms. 8 NGS variant calling It is important for laboratories to try out various tools have different variant calling tools using their own data to sensitivity and determine which tools provide the best balance specificity levels. between sensitivity and specificity.

9 It is a misconception Insurance companies are often willing to pay that NGS is not for NGS testing because it’s cheaper to reimbursed. sequence a set of 10 or more genes than it is to run multiple stand-alone molecular assays. NGS-based testing can also detect a full range of variants including copy number alterations, making sequencing an alternative to FISH and chromosomal arrays.

Dr. Duncavage’s suggested articles:

A) Detection of Gene Rearrangements in Targeted Clinical Next- Generation Sequencing Abel HJ, Al-Kateb H, Cottrell CE, et al. Detection of Gene Rearrangements in Targeted Clinical Next-Generation Sequencing. J Mol Diagn. 2014;16(4):405- 17.

Summary: The identification of recurrent gene rearrangements in the clinical laboratory is the cornerstone for risk stratification and treatment decisions in many malignant tumors. Studies have reported that targeted next-generation sequencing assays have the potential to identify such rearrangements; however, their utility in the clinical laboratory is unknown. We examine the sensitivity and specificity of ALK and KMT2A (MLL) rearrangement detection by next-generation sequencing in the clinical laboratory. We analyzed a series of seven ALK rearranged cancers, six KMT2A rearranged leukemias, and 77 ALK/KMT2A rearrangement-negative cancers, previously tested by fluorescence in situ hybridization (FISH). Rearrangement detection was tested using publicly available software tools, including Breakdancer, ClusterFAST, CREST, and Hydra. Using Breakdancer and ClusterFAST, we detected ALK rearrangements in seven of seven FISH-positive cases and KMT2A rearrangements in six of six FISH-positive cases. Among the 77 ALK/KMT2A FISH-negative cases, no false-positive identifications were made by Breakdancer or ClusterFAST. Further, we identified one ALK rearranged case with a noncanonical intron 16 breakpoint, which is likely to affect its response to targeted inhibitors. We report that clinically relevant chromosomal rearrangements can be detected from targeted gene panel- based next-generation sequencing with sensitivity and specificity equivalent to that of FISH while providing finer-scale information and increased efficiency for molecular oncology testing.

Free full text available from Journal of Molecular Diagnostics PMID: 24813172

B) Detection of Structural DNA Variation from Next Generation Sequencing Data: A Review of Informatic Approaches Abel HJ, Duncavage EJ. Detection of structural DNA variation from next generation sequencing data: a review of informatic approaches. Cancer Genet. 2013; 206(12): 432-40.

Summary: Next generation sequencing (NGS), or massively paralleled sequencing, refers to a collective group of methods in which numerous sequencing reactions take place simultaneously, resulting in enormous amounts of sequencing data for a small fraction of the cost of Sanger sequencing. Typically short (50-250 bp), NGS reads are first mapped to a reference genome, and then variants are called from the mapped data. While most NGS applications focus on the detection of single nucleotide variants (SNVs) or small insertions/deletions (indels), structural variation, including translocations, larger indels, and copy number variation (CNV), can be identified from the same data. Structural variation detection can be performed from whole genome NGS data or "targeted" data including exomes or gene panels. However, while targeted sequencing greatly increases sequencing coverage or depth of particular genes, it may introduce biases in the data that require specialized informatic analyses. In the past several years, there have been considerable advances in methods used to detect structural variation, and a full range of variants from SNVs to balanced translocations to CNV can now be detected with reasonable sensitivity from either whole genome or targeted NGS data. Such methods are being rapidly applied to clinical testing where they can supplement or in some cases replace conventional fluorescence in situ hybridization or array-based testing. Here we review some of the informatics approaches used to detect structural variation from NGS data.

Free full text available from Cancer Genetics PMID: 24405614

C) Performance of Common Analysis Methods for Detecting Low- Frequency Single Nucleotide Variants in Targeted Next-Generation Sequence Data Spencer DH, Tyagi M, Vallania F, et al. Performance of common analysis methods for detecting low-frequency single nucleotide variants in targeted next-generation sequence data. J Mol Diagn. 2014; 16(1): 75-88.

heterogeneity and can therefore be challenging to detect using common NGS analysis tools, which are often designed for constitutional genomic studies. We generated high-coverage (>1000×) NGS data from synthetic DNA mixtures with variant allele fractions (VAFs) of 25% to 2.5% to assess the performance of four variant callers, SAMtools, Genome Analysis Toolkit, VarScan2, and SPLINTER, in detecting low-frequency variants. SAMtools had the lowest sensitivity and detected only 49% of variants with VAFs of approximately 25%; whereas the Genome Analysis Toolkit, VarScan2, and SPLINTER detected at least 94% of variants with VAFs of approximately 10%. VarScan2 and SPLINTER achieved sensitivities of 97% and 89%, respectively, for variants with observed VAFs of 1% to 8%, with >98% sensitivity and >99% positive predictive value in coding regions. Coverage analysis demonstrated that >500× coverage was required for optimal performance. The specificity of SPLINTER improved with higher coverage, whereas VarScan2 yielded more false positive results at high coverage levels, although this effect was abrogated by removing low-quality reads before variant identification. Finally, we demonstrate the utility of high- sensitivity variant callers with data from 15 clinical lung cancers.

Free full text available from Journal of Molecular Diagnostics PMID: 24211364 NOTE: Also cited in Section 1.6.2

D) Validation and Implementation of Targeted Capture and Sequencing for the Detection of Actionable Mutation, Copy Number Variation, and Gene Rearrangement in Clinical Cancer Specimens Pritchard CC, Salipante SJ, Koehler K, et al. Validation and implementation of targeted capture and sequencing for the detection of actionable mutation, copy number variation, and gene rearrangement in clinical cancer specimens. J Mol Diagn. 2014; 16(1): 56-67.

Summary: Recent years have seen development and implementation of anticancer therapies targeted to particular gene mutations, but methods to assay clinical cancer specimens in a comprehensive way for the critical mutations remain underdeveloped. We have developed UW-OncoPlex, a clinical molecular diagnostic assay to provide simultaneous deep-sequencing information, based on >500× average coverage, for all classes of mutations in 194 clinically relevant genes. To validate UW-OncoPlex, we tested 98 previously characterized clinical tumor specimens from 10 different cancer types, including 41 formalin-fixed paraffin-embedded tissue samples. Mixing studies indicated reliable mutation detection in samples with ≥ 10% tumor cells. In clinical samples with ≥ 10% tumor cells, UW-OncoPlex correctly identified 129 of 130 known mutations [sensitivity 99.2%, (95% CI, 95.8%-

99.9%)], including single nucleotide variants, small insertions and deletions, internal tandem duplications, gene copy number gains and amplifications, gene copy losses, chromosomal gains and losses, and actionable genomic rearrangements, including ALK-EML4, ROS1, PML-RARA, and BCR-ABL. In the same samples, the assay also identified actionable point mutations in genes not previously analyzed and novel gene rearrangements of MLL and GRIK4 in melanoma, and of ASXL1, PIK3R1, and SGCZ in acute myeloid leukemia. To best guide existing and emerging treatment regimens and facilitate integration of genomic testing with patient care, we developed a framework for data analysis, decision support, and reporting clinically actionable results.

Free full text available from Journal of Molecular Diagnostics PMID: 24189654

E) The Next-Generation Sequencing Revolution and Its Impact on Genomics Koboldt DC, Steinberg KM, Larson DE, Wilson RK, Mardis ER. The next- generation sequencing revolution and its impact on genomics. Cell. 2013; 155(1): 27-38.

Summary: Genomics is a relatively new scientific discipline, having DNA sequencing as its core technology. As technology has improved the cost and scale of genome characterization over sequencing's 40-year history, the scope of inquiry has commensurately broadened. Massively parallel sequencing has proven revolutionary, shifting the paradigm of genomics to address biological questions at a genome-wide scale. Sequencing now empowers clinical diagnostics and other aspects of medical care, including disease risk, therapeutic identification, and prenatal testing. This Review explores the current state of genomics in the massively parallel sequencing era. Free full text available from Cell PMID: 24074859

2.1.2 Quick Reference Table: Selected Tests by Tumor Type in Solid Tumors

This table is for quick reference only. Clinical decision making, including diagnosis and therapy, should not be based solely on this information. The information should be considered in conjunction with clinical information, imaging, and laboratory studies. Additional reading and investigation should be

undertaken regarding the tabular entries before information is used in the clinical setting.

Tumor Type Gene/ Somatic Clinical Use References Loci Alteration Colorectal Adenocarcinoma KRAS Mutation Lack of response to 2-3, 11-12, 14, 24, codons EGFR monoclonal 31, 49, 52 12, 13, 59, antibodies (p.G13D 61, 117, may be an 146 exception) NRAS Mutation Lack of response to 11, 14, 52 codons EGFR monoclonal 12, 13, 59, antibodies 61, 117, 146 BRAF p.V600E MSI stratification, 3, 10-11, 31, 44, 46 mutation prognostic factor, possible reduced response to EGFR monoclonal antibodies but insufficient evidence MLH1 Promoter Indicates sporadic 3, 10 methylation MSI Tumor PIK3CA Mutation Possible improved 13, 29 survival with post- operative aspirin therapy Lung Adenocarcinoma EGFR Mutation Response to EGFR 5, 32, 35, 37, 38 exons 18- inhibitors 21 EGFR p.T790M Resistance to EGFR 5, 26, 30, 39, 53 and some inhibitors exon 20 insertion mutations KRAS Mutation Exclusion of EGFR 5, 8, 30, 42 codons mutation 12,13,61

BRAF Mutation Possible response 40 p.V600E to BRAF inhibitor ALK Rearrange- Response to TKI 5, 8, 28, 30 ment RET Rearrange- Response to TKI 15, 17 ment ROS1 Rearrange- Response to TKI 4, 8 ment MET Amplification Resistance to EGFR 5, 8, 16 inhibitors Breast Carcinoma HER2/ Amplification Response to HER2 18, 51 ERBB2 monoclonal antibodies Gastric Adenocarcinoma HER2/ Amplification Response to HER2 45 ERBB2 monoclonal antibodies Thyroid Carcinoma Papillary Thyroid BRAF p.V600E Pre-operative FNA 9, 36, 43 Carcinoma / mutation diagnosis and Anaplastic prognosis, Thyroid Cancer potential therapeutic target NRAS, Mutation Pre-operative FNA 36 HRAS, diagnosis KRAS RET-PTC Rearrange- Pre-operative FNA 36 ment diagnosis Follicular Thyroid NRAS, Mutation Pre-operative FNA 36 Carcinoma HRAS, diagnosis KRAS PAX8- Rearrange- Pre-operative FNA 36 PPAR ment diagnosis Melanoma Cutaneous & BRAF Mutation Response to BRAF 19-20, 33 Mucosal codon 600 inhibitors KIT Mutation Response to TKI 7 Uveal GNAQ or Mutation Diagnostic 50 GNA11 Chromo- Loss Unfavorable 23 some 3 (monosomy) prognosis

GIST KIT Mutation Response to TKI 41 PDGFRA Mutation Response to TKI 41 BRAF Mutation Possible imatinib 1, 34 p.V600E resistance CNS Neoplasms Glioma MGMT Promoter Favorable response 21 methylation to alkylating agents IDH1 and Mutation Distinguishes 27, 54 IDH2 reactive gliosis from glioma, favorable prognosis Oligodendro- Chromo- Co-deletion Favorable prognosis 6, 22 glioma some 1p and response to and 19q therapy Pilocytic BRAF Duplication Diagnostic 27, 47 Astrocytoma / fusion and p.V600E mutation (extracere- bellar) Pleomorphic BRAF p.V600E Diagnostic 47 Xanthoastro- mutation cytoma and Ganglioglioma Cholangiocarcinoma/Pancreatic Carcinoma KRAS Mutation Pre-operative bile 25 codons duct brushing 12, 13, 61 diagnosis Oropharyngeal Squamous Cell Carcinoma HR HPV- Positive Favorable response 48 related detection to chemoradiation therapy Source: Allison M. Cushman-Vokoun, MD, PhD

MSI = Microsatellite Instability; TKI = Tyrosine-Kinase Inhibitors; HR HPV= High Risk Human Papillomavirus This table is meant to be a list of selected tests and is not comprehensive.

References: 1. Agaram NP, Wong GC, Guo T, et al. Novel V600E BRAF Mutations in Imatinib-Naive and Imatinib-Resistant Gastrointestinal Stromal Tumors. Genes Chromosomes Cancer. 2008; 47(10): 853-859. 2. Amado RG, Wolf M, Peeters M, et al. Wild-Type KRAS Is Required for Panitumumab Efficacy in Patients with Metastatic Colorectal Cancer. J Clin Oncol. 2008; 26(10): 1626-1634.

3. Bartley AN, Hamilton SR, Alsabeh R, et al. Template for Reporting Results of Biomarker Testing of Specimens from Patients with Carcinoma of the Colon and Rectum. Arch Pathol Lab Med. 2014; 138(2): 166-170. 4. Bergethon K, Shaw AT, Ou SH, et al. ROS1 Rearrangements Define a Unique Molecular Class of Lung Cancers. J Clin Oncol. 2012; 30(8): 863-870. 5. Cagle PT, Sholl LM, Lindeman NI, et al. Template for Reporting Results of Biomarker Testing of Specimens from Patients with Non-Small Cell Carcinoma of the Lung. Arch Pathol Lab Med. 2014; 138(2): 171-174. 6. Cairncross JG, Ueki K, Zlatescu MC, et al. Specific Genetic Predictors of Chemotherapeutic Response and Survival in Patients with Anaplastic Oligodendrogliomas. J Natl Cancer Inst. 1998; 90(19): 1473-1479. 7. Carvajal RD, Antonescu CR, Wolchok JD, et al. KIT as a Therapeutic Target in Metastatic Melanoma. JAMA. 2011; 305(22): 2327-2334. 8. Dacic S. Molecular Genetic Testing for Lung Adenocarcinomas: A Practical Approach to Clinically Relevant Mutations and Translocations. J Clin Pathol. 2013; 66(10): 870-874. 9. Dadu R, Shah K, Busaidy NL, et al. Efficacy and Tolerability of Vemurafenib in Patients with BRAF(V600E) -Positive Papillary Thyroid Cancer: M.D. Anderson Cancer Center Off Label Experience. J Clin Endocrinol Metab. 2015; 100(1): E77-81. 10. de la Chapelle A, Hampel H. Clinical Relevance of Microsatellite Instability in Colorectal Cancer. J Clin Oncol. 2010; 28(20): 3380-3387. 11. De Roock W, Claes B, Bernasconi D, et al. Effects of KRAS, BRAF, NRAS, and PIK3CA Mutations on the Efficacy of Cetuximab Plus Chemotherapy in Chemotherapy-Refractory Metastatic Colorectal Cancer: A Retrospective Consortium Analysis. Lancet Oncol. 2010; 11(8): 753-762 12. De Roock W, Jonker DJ, Di Nicolantonio F, et al. Association of KRAS p.G13D Mutation with Outcome in Patients with Chemotherapy-Refractory Metastatic Colorectal Cancer Treated with Cetuximab. JAMA. 2010; 304(16): 1812-1820. 13. Domingo E, Church DN, Sieber O, et al. Evaluation of PIK3CA Mutation as a Predictor of Benefit from Nonsteroidal Anti-Inflammatory Drug Therapy in Colorectal Cancer. J Clin Oncol. 2013; 31(34): 4297-4305. 14. Douillard JY, Oliner KS, Siena S, et al. Panitumumab-FOLFOX4 Treatment and RAS Mutations in Colorectal Cancer. N Engl J Med. 2013; 369(11): 1023-1034. 15. Drilon A, Wang L, Hasanovic A, et al. Response to Cabozantinib in Patients with RET Fusion- Positive Lung Adenocarcinomas. Cancer Discov. 2013; 3(6): 630-635. 16. Engelman JA, Zejnullahu K, Mitsudomi T, et al. MET Amplification Leads to Gefitinib Resistance in Lung Cancer by Activating ERBB3 Signaling. Science. 2007; 316(5827): 1039-1043. 17. Falchook GS, Ordonez NG, Bastida CC, et al. Effect of the RET Inhibitor Vandetanib in a Patient with RET Fusion-Positive Metastatic Non-Small-Cell Lung Cancer. J Clin Oncol. 2016; 34(15): e141-144. 18. Fitzgibbons PL, Dillon DA, Alsabeh R, et al. Template for Reporting Results of Biomarker Testing of Specimens from Patients with Carcinoma of the Breast. Arch Pathol Lab Med. 2014; 138(5): 595-601. 19. Flaherty KT, Infante JR, Daud A, et al. Combined BRAF and MEK Inhibition in Melanoma with BRAF V600 Mutations. N Engl J Med. 2012; 367(18): 1694-1703. 20. Flaherty KT, Puzanov I, Kim KB, et al. Inhibition of Mutated, Activated BRAF in Metastatic Melanoma. N Engl J Med. 2010; 363(9): 809-819. 21. Hegi ME, Diserens AC, Gorlia T, et al. MGMT Gene Silencing and Benefit from Temozolomide in Glioblastoma. N Engl J Med. 2005; 352(10): 997-1003. 22. Intergroup Radiation Therapy Oncology Group Trial 9402, Cairncross G, Berkey B, et al. Phase III Trial of Chemotherapy Plus Radiotherapy Compared with Radiotherapy Alone for Pure and Mixed Anaplastic Oligodendroglioma: Intergroup Radiation Therapy Oncology Group Trial 9402. J Clin Oncol. 2006; 24(18): 2707-2714. 23. Jovanovic P, Mihajlovic M, Djordjevic-Jocic J, Vlajkovic S, Cekic S, Stefanovic V. Ocular Melanoma: An Overview of the Current Status. Int J Clin Exp Pathol. 2013; 6(7): 1230-1244.

24. Karapetis CS, Khambata-Ford S, Jonker DJ, et al. K-RAS Mutations and Benefit from Cetuximab in Advanced Colorectal Cancer. N Engl J Med. 2008; 359(17): 1757-1765. 25. Kipp BR, Fritcher EG, Clayton AC, et al. Comparison of KRAS Mutation Analysis and FISH for Detecting Pancreatobiliary Tract Cancer in Cytology Specimens Collected During Endoscopic Retrograde Cholangiopancreatography. J Mol Diagn. 2010; 12(6): 780-786. 26. Kobayashi S, Boggon TJ, Dayaram T, et al. EGFR Mutation and Resistance of Non-Small-Cell Lung Cancer to Gefitinib. N Engl J Med. 2005; 352(8): 786-792. 27. Korshunov A, Meyer J, Capper D, et al. Combined Molecular Analysis of BRAF and IDH1 Distinguishes Pilocytic Astrocytoma from Diffuse Astrocytoma. Acta Neuropathol. 2009; 118(3): 401-405. 28. Kwak EL, Bang YJ, Camidge DR, et al. Anaplastic Lymphoma Kinase Inhibition in Non-Small- Cell Lung Cancer. N Engl J Med. 2010; 363(18): 1693-1703. 29. Liao X, Lochhead P, Nishihara R, et al. Aspirin Use, Tumor PIK3CA Mutation, and Colorectal- Cancer Survival. N Engl J Med. 2012; 367(17): 1596-1606. 30. Lindeman NI, Cagle PT, Beasley MB, et al. Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors: Guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. J Thorac Oncol. 2013;8(7):823-59. 31. Loupakis F, Ruzzo A, Cremolini C, et al. KRAS Codon 61, 146 and BRAF Mutations Predict Resistance to Cetuximab Plus Irinotecan in KRAS Codon 12 and 13 Wild-Type Metastatic Colorectal Cancer. Br J Cancer. 2009; 101(4): 715-721. 32. Lynch TJ, Bell DW, Sordella R, et al. Activating Mutations in the Epidermal Growth Factor Receptor Underlying Responsiveness of Non-Small-Cell Lung Cancer to Gefitinib. N Engl J Med. 2004; 350(21): 2129-2139. 33. McArthur GA, Chapman PB, Robert C, et al. Safety and Efficacy of Vemurafenib in BRAF(V600E) and BRAF(V600K) Mutation-Positive Melanoma (BRIM-3): Extended Follow-up of a Phase 3, Randomised, Open-Label Study. Lancet Oncol. 2014; 15(3): 323-332. 34. Miranda C, Nucifora M, Molinari F, et al. KRAS and BRAF Mutations Predict Primary Resistance to Imatinib in Gastrointestinal Stromal Tumors. Clin Cancer Res. 2012; 18(6): 1769-1776. 35. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or Carboplatin-Paclitaxel in Pulmonary Adenocarcinoma. N Engl J Med. 2009; 361(10): 947-957. 36. Nikiforov YE. Molecular Diagnostics of Thyroid Tumors. Arch Pathol Lab Med. 2011; 135(5): 569-577. 37. Paez JG, Janne PA, Lee JC, et al. EGFR Mutations in Lung Cancer: Correlation with Clinical Response to Gefitinib Therapy. Science. 2004; 304(5676): 1497-1500. 38. Pao W, Miller V, Zakowski M, et al. EGF Receptor Gene Mutations Are Common in Lung Cancers from "Never Smokers" and Are Associated with Sensitivity of Tumors to Gefitinib and Erlotinib. Proc Natl Acad Sci U S A. 2004; 101(36): 13306-13311. 39. Pao W, Miller VA, Politi KA, et al. Acquired Resistance of Lung Adenocarcinomas to Gefitinib or Erlotinib Is Associated with a Second Mutation in the EGFR Kinase Domain. PLoS Med. 2005; 2(3): e73. 40. Peters S, Michielin O, Zimmermann S. Dramatic Response Induced by Vemurafenib in a BRAF V600E-Mutated Lung Adenocarcinoma. J Clin Oncol. 2013; 31(20): e341-344. 41. Rammohan A, Sathyanesan J, Rajendran K, et al. A Gist of Gastrointestinal Stromal Tumors: A Review. World J Gastrointest Oncol. 2013; 5(6): 102-112. 42. Roberts PJ, Stinchcombe TE. KRAS Mutation: Should We Test for It, and Does It Matter? J Clin Oncol. 2013; 31(8): 1112-1121. 43. Rosove MH, Peddi PF, Glaspy JA. BRAF V600E Inhibition in Anaplastic Thyroid Cancer. N Engl J Med. 2013; 368(7): 684-685. 44. Roth AD, Tejpar S, Delorenzi M, et al. Prognostic Role of KRAS and BRAF in Stage II and III Resected Colon Cancer: Results of the Translational Study on the PETACC-3, EORTC 40993, SAKK 60-00 Trial. J Clin Oncol. 2010; 28(3): 466-474. 45. Ruschoff J, Hanna W, Bilous M, et al. HER2 Testing in Gastric Cancer: A Practical Approach. Mod Pathol. 2012; 25(5): 637-650.

46. Samowitz WS, Sweeney C, Herrick J, et al. Poor Survival Associated with the BRAF V600E Mutation in Microsatellite-Stable Colon Cancers. Cancer Res. 2005; 65(14): 6063-6069. 47. Schindler G, Capper D, Meyer J, et al. Analysis of BRAF V600E Mutation in 1,320 Nervous System Tumors Reveals High Mutation Frequencies in Pleomorphic Xanthoastrocytoma, Ganglioglioma and Extra-Cerebellar Pilocytic Astrocytoma. Acta Neuropathol. 2011; 121(3): 397- 405. 48. Syrjanen S. The Role of Human Papillomavirus Infection in Head and Neck Cancers. Ann Oncol. 2010; 21 Suppl 7:vii243-245. 49. Tejpar S, Celik I, Schlichting M, Sartorius U, Bokemeyer C, Van Cutsem E. Association of KRAS G13D Tumor Mutations with Outcome in Patients with Metastatic Colorectal Cancer Treated with First-Line Chemotherapy with or without Cetuximab. J Clin Oncol. 2012; 30(29): 3570-3577. 50. Van Raamsdonk CD, Griewank KG, Crosby MB, et al. Mutations in GNA11 in Uveal Melanoma. N Engl J Med. 2010; 363(23): 2191-2199. 51. Wolff AC, Hammond ME, Hicks DG, et al. Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Update. J Clin Oncol. 2013; 31(31): 3997- 4013. 52. Wong NA, Gonzalez D, Salto-Tellez M, et al. RAS Testing of Colorectal Carcinoma-a Guidance Document from the Association of Clinical Pathologists Molecular Pathology and Diagnostics Group. J Clin Pathol. 2014; 67(9): 751-757. 53. Wu JY, Wu SG, Yang CH, et al. Lung Cancer with Epidermal Growth Factor Receptor Exon 20 Mutations Is Associated with Poor Gefitinib Treatment Response. Clin Cancer Res. 2008; 14(15): 4877-4882. 54. Yan H, Parsons DW, Jin G, et al. IDH1 and IDH2 Mutations in Gliomas. N Engl J Med. 2009; 360(8): 765-773.

2.2 Breast Cancer

This section includes articles on the following topics:

• Articles on Breast Cancer (Section 2.2.1); and • Short Presentations on Emerging Concepts: Breast Cancer (Section 2.2.2).

2.2.1 Articles on Breast Cancer

A) Theranostic and Molecular Classification of Breast Cancer Cornejo KM, Kandil D, Khan A,Cosar EF. Theranostic and molecular classification of breast cancer. Arch Pathol Lab Med. 2014; 138(1): 44-56.

Summary: CONTEXT: Despite advances in breast cancer management, women continue to relapse and die of breast cancer. Traditionally, evaluation for hormone receptors (estrogen and progesterone), as well as HER2 overexpression, have guided therapy-related decision-making because they are both prognostic and predictive indicators. However, there are limitations

with those studies, which can lead to improper treatment. Gene signatures have recently been shown to be of value in identifying molecular portraits of breast carcinoma and are beginning to play role in management and treatment algorithms. OBJECTIVE: To provide a summary of the prognostic and predictive indicators of breast cancer, such as hormone receptors, HER2, and molecular gene signatures that currently help guide clinical decision making. DATA SOURCES: Published articles from peer-reviewed journals in PubMed (US National Library of Medicine). CONCLUSIONS: Emerging evidence shows promise that, in addition to hormone receptors and HER2 studies, evaluating tumors with gene expression profiling can provide additional prognostic and predictive information, further aiding clinical management and leading to a more personalized approach to treating breast cancer.

Free full text available from the CAP’s Archives PMID: 24377811

B) Molecular Classification of Breast Carcinomas by Immunohistochemical Analysis: Are We Ready? Tang P, Skinner KA, Hicks DG. Molecular classification of breast carcinomas by immunohistochemical analysis: are we ready? Diagn Mol Pathol. 2009;18(3):125-32.

Summary: Gene expression profiling with breast carcinomas has allowed further classification of these tumors into 5 distinct subtypes (luminal A, luminal B, HER2-overexpression, basal-like, and normal-like) with unique clinical outcomes. Subsequent studies have shown that breast carcinomas can also be divided into 5 similar subgroups using immunohistochemical (IHC) analysis with a limited panel of molecular markers (including estrogen receptor, progesterone receptor, HER2, CK5/6, and epidermal growth factor receptor). These subgroups have distinguishing features closely associated with subtypes defined by gene expression profiling, including distinct clinical outcomes. This review aims to present the current data on molecular classification for breast carcinoma, and its clinical significance, with an emphasis on IHC-based studies and the pros and cons of these molecular classifications. We also propose a standardized IHC-based molecular classification, in the hope that it will promote more uniform large multicenter studies, and facilitate its clinical application.

Full text available from Diagnostic Molecular Pathology (subscription required) PMID: 19704256

C) Analysis of Circulating Tumor DNA to Monitor Metastatic Breast Cancer Dawson SJ, Tsui DW, Murtaza M, et al. Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med. 2013; 368(13): 1199-1209.

Summary: The management of metastatic breast cancer requires monitoring of the tumor burden to determine the response to treatment, and improved biomarkers are needed. Biomarkers such as cancer antigen 15-3 (CA 15-3) and circulating tumor cells have been widely studied. However, circulating cell-free DNA carrying tumor-specific alterations (circulating tumor DNA) has not been extensively investigated or compared with other circulating biomarkers in breast cancer. METHODS: We compared the radiographic imaging of tumors with the assay of circulating tumor DNA, CA 15-3, and circulating tumor cells in 30 women with metastatic breast cancer who were receiving systemic therapy. We used targeted or whole-genome sequencing to identify somatic genomic alterations and designed personalized assays to quantify circulating tumor DNA in serially collected plasma specimens. CA 15-3 levels and numbers of circulating tumor cells were measured at identical time points. RESULTS: Circulating tumor DNA was successfully detected in 29 of the 30 women (97%) in whom somatic genomic alterations were identified; CA 15-3 and circulating tumor cells were detected in 21 of 27 women (78%) and 26 of 30 women (87%), respectively. Circulating tumor DNA levels showed a greater dynamic range, and greater correlation with changes in tumor burden, than did CA 15-3 or circulating tumor cells. Among the measures tested, circulating tumor DNA provided the earliest measure of treatment response in 10 of 19 women (53%). CONCLUSIONS: This proof- of-concept analysis showed that circulating tumor DNA is an informative, inherently specific, and highly sensitive biomarker of metastatic breast cancer.

Free full text available from New England Journal of Medicine PMID: 23484797 NOTE: Also cited in Section 3.2.2.1

D) Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Update Wolff AC, Hammond ME, Hicks DG, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. Arch Pathol Lab Med. 2014;138(2):241-56.

Summary: PURPOSE: To update the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer to improve the accuracy of HER2 testing and its utility as a predictive marker in invasive breast cancer. METHODS: ASCO/CAP convened an Update Committee that included coauthors of the 2007 guideline to conduct a systematic literature review and update recommendations for optimal HER2 testing. RESULTS: The Update Committee identified criteria and areas requiring clarification to improve the accuracy of HER2 testing by immunohistochemistry (IHC) or in situ hybridization (ISH). The guideline was reviewed and approved by both organizations. RECOMMENDATIONS: The Update Committee recommends that HER2 status (HER2 negative or positive) be determined in all patients with invasive (early stage or recurrence) breast cancer on the basis of one or more HER2 test results (negative, equivocal, or positive). Testing criteria define HER2-positive status when (on observing within an area of tumor that amounts to >10% of contiguous and homogeneous tumor cells) there is evidence of protein overexpression (IHC) or gene amplification (HER2 copy number or HER2/CEP17 ratio by ISH based on counting at least 20 cells within the area). If results are equivocal (revised criteria), reflex testing should be performed using an alternative assay (IHC or ISH). Repeat testing should be considered if results seem discordant with other histopathologic findings. Laboratories should demonstrate high concordance with a validated HER2 test on a sufficiently large and representative set of specimens. Testing must be performed in a laboratory accredited by CAP or another accrediting entity. The Update Committee urges providers and health systems to cooperate to ensure the highest quality testing.

Free full text available from the CAP’s Archives PMID: 24099077

E) HER2 In Situ Hybridization in Breast Cancer: Clinical Implications of Polysomy 17 and Genetic Heterogeneity Hanna WM, Rüschoff J, Bilous M, et al. HER2 in situ hybridization in breast cancer: clinical implications of polysomy 17 and genetic heterogeneity. Mod Pathol. 2014;27(1):4-18.

Summary: Trastuzumab-containing therapy is a standard of care for patients with HER2+ breast cancer. HER2 status is routinely assigned using in situ hybridization to assess HER2 gene amplification, but interpretation of in situ hybridization results may be challenging in tumors with chromosome 17 polysomy or intratumoral genetic heterogeneity. Apparent chromosome 17 polysomy, defined by increased chromosome enumeration probe 17

(CEP17) signal number, is a common genetic aberration in breast cancer and represents an alternative mechanism for increasing HER2 copy number. Some studies have linked elevated CEP17 count ('polysomy') with adverse clinicopathologic features and HER2 overexpression, although there are numerous discrepancies in the literature. There is evidence that elevated CEP17 ('polysomy') count might account for trastuzumab response in tumors with normal HER2:CEP17 ratios. Nonetheless, recent studies establish that apparent 'polysomy' (CEP17 increase) is usually related to focal pericentromeric gains rather than true polysomy. Assigning HER2 status may also be complex where multiple cell subclones with distinct HER2 amplification characteristics coexist within the same tumor. Such genetic heterogeneity affects up to 40% of breast cancers when assessed according to a College of American Pathologists guideline, although other definitions have been proposed. Recent data have associated heterogeneity with unfavorable clinicopathologic variables and poor prognosis. Genetically heterogeneous tumors harboring HER2-amplified subclones have the potential to benefit from trastuzumab, but this has yet to be evaluated in clinical studies. In this review, we discuss the implications of apparent polysomy 17 and genetic heterogeneity for assigning HER2 status in clinical practice. Among our recommendations, we support the use of mean HER2 copy number rather than HER2:CEP17 ratio to define HER2 positivity in cases where coamplification of the centromere might mask HER2 amplification. We also highlight a need to harmonize in situ hybridization scoring methodology to support accurate HER2 status determination, particularly where there is evidence of heterogeneity.

Free full text available from Modern Pathology PMID: 23807776

F) Pharmacogenomics of Breast Cancer Therapy: An Update Westbrook K, Stearns V. Pharmacogenomics of breast cancer therapy: an update. Pharmacol Ther. 2013; 139(1): 1-11.

degree of estrogen receptor expression and HER2 status. In recent years, there has been great interest in evaluating the role that pharmacogenetics/pharmacogenomics, or variations in germline DNA, play in alteration of drug metabolism and activity, thus leading to disparate outcomes among patients with similar tumor characteristics. The utility of these variations in treatment decision-making remains debated. Here we review the data available to date on genomic variants that may influence response to drugs commonly used to treat breast cancer. While none of the variants reported to date have demonstrated clinical utility, ongoing prospective studies and increasing understanding of pharmacogenetics will allow us to better predict risk of toxicity or likelihood of response to specific treatments and to provide a more personalized therapy.

Free full text available from PubMed PMID: 23500718 NOTE: Also cited in Section 7.3

G) Genome and Transcriptome Sequencing in Prospective Metastatic Triple-Negative Breast Cancer Uncovers Therapeutic Vulnerabilities Craig DW, O'Shaughnessy JA, Kiefer JA, et al. Genome and transcriptome sequencing in prospective metastatic triple-negative breast cancer uncovers therapeutic vulnerabilities. Mol Cancer Ther. 2013; 12(1): 104-116.

Summary: Triple-negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor, progesterone receptor, and HER-2. Thirty percent of patients recur after first-line treatment, and metastatic TNBC (mTNBC) has a poor prognosis with median survival of one year. Here, we present initial analyses of whole genome and transcriptome sequencing data from 14 prospective mTNBC. We have cataloged the collection of somatic genomic alterations in these advanced tumors, particularly those that may inform targeted therapies. Genes mutated in multiple tumors included TP53, LRP1B, HERC1, CDH5, RB1, and NF1. Notable genes involved in focal structural events were CTNNA1, PTEN, FBXW7, BRCA2, WT1, FGFR1, KRAS, HRAS, ARAF, BRAF, and PGCP. Homozygous deletion of CTNNA1 was detected in 2 of 6 African Americans. RNA sequencing revealed consistent overexpression of the FOXM1 gene when tumor gene expression was compared with nonmalignant breast samples. Using an outlier analysis of gene expression comparing one cancer with all the others, we detected expression patterns unique to each patient's tumor. Integrative DNA/RNA analysis provided evidence for deregulation of mutated genes, including the monoallelic expression of TP53 mutations. Finally, molecular alterations in several cancers supported targeted therapeutic intervention on clinical trials with known inhibitors, particularly for

alterations in the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways. In conclusion, whole genome and transcriptome profiling of mTNBC have provided insights into somatic events occurring in this difficult to treat cancer. These genomic data have guided patients to investigational treatment trials and provide hypotheses for future trials in this irremediable cancer.

Free full text available from Molecular Cancer Therapeutics PMID: 23171949

H) Fibroepithelial Tumors of the Breast: Pathologic and Immunohistochemical Features and Molecular Mechanisms Yang X, Kandil D, Cosar EF, Khan A. Fibroepithelial tumors of the breast: pathologic and immunohistochemical features and molecular mechanisms. Arch Pathol Lab Med. 2014; 138(1): 25-36.

Summary: CONTEXT: The 2 main prototypes of fibroepithelial tumors of the breast include fibroadenoma and phyllodes tumor (PT). Although both tumors share some overlapping histologic features, there are significant differences in their clinical behavior and management. Phyllodes tumors have been further divided into clinically relevant subtypes, and there is more than one classification scheme for PT currently in use, suggesting a lack of consistency within different practices. Accurate differentiation between fibroadenoma and PT, as well as the grading of PT, may sometimes be challenging on preoperative core needle biopsy. Some immunohistochemical markers have been suggested to aid in the pathologic classification of these lesions. OBJECTIVE: To discuss the salient histopathologic features of fibroepithelial tumors and review the molecular pathways proposed for the initiation, progression, and metastasis of PTs. Also, to provide an update on immunohistochemical markers that may be useful in their differential diagnosis and outline the practice and experience at our institution from a pathologic perspective. DATA SOURCES: Sources included published articles from peer-reviewed journals in PubMed (US National Library of Medicine). CONCLUSIONS: Fibroepithelial tumor of the breast is a heterogenous group of lesions ranging from fibroadenoma at the benign end of the spectrum to malignant PT. There are overlapping histologic features among various subtypes, and transformation and progression to a more malignant phenotype may also occur. Given the significant clinical differences within various subtypes, accurate pathologic classification is important for appropriate management. Although some immunohistochemical markers may be useful in this differential diagnosis, histomorphology still remains the gold standard.

Free full text available from the CAP’s Archives

PMID: 24377809

2.2.2 Short Presentations on Emerging Concepts: Breast Cancer

CAP’s Pathology Short Presentations in Emerging Concepts (SPECs) are prewritten PowerPoint presentations on selected diseases where molecular tests play a key role in patient management. To learn more about this resource, view Section 9.1 or the Resources & Publications tab on cap.org.

A) CAP Short Presentations on Emerging Concepts (SPEC): Emerging Concepts in Molecular Testing in Breast Cancer [PowerPoint slides] Hicks DG. CAP Short Presentations on Emerging Concepts (SPECS): Emerging Concepts in Molecular Testing in Breast Cancer (v 4.0e rev 7/7/15) [PowerPoint slides]. Caughron SK, Misialek MJ, Moyer AM, Nowak JA, eds. Northfield, IL: College of American Pathologists; 2015.

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B) CAP Short Presentations on Emerging Concepts (SPEC): HER2 Testing in Breast Cancer: 2013 ASCO/CAP HER2 Guideline Update [PowerPoint slides] Hicks DG. CAP Short Presentations on Emerging Concepts (SPECS): HER2 Testing in Breast Cancer: 2013 ASCO/CAP HER2 Guideline Update (v 3.0e rev 7/7/15) [PowerPoint slides]. Caughron SK, Chandra PK, Misialek MJ, Moyer AM, Nowak JA, eds. Northfield, IL: College of American Pathologists; 2015.

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2.3 Central Nervous System Tumors

Clinical and molecular characteristics of subgroups of medulloblastoma Republished with permission of Pharmgenomics Pers Med, from Molecular variants and mutations in medulloblastoma, Schroeder K, Gururangar S468, copyright 2014; 7:43-51 permission conveyed through Copyright Clearance Center, Inc.

A) Molecular Diagnostics of Gliomas Nikiforova MN, Hamilton RL. Molecular diagnostics of gliomas. Arch Pathol Lab Med. 2011; 135(5):558–568.

Summary: Gliomas are the most common primary brain tumors of adults and include a variety of histologic types and morphologies. Histologic evaluation remains the gold standard for glioma diagnosis; however, diagnostic difficulty may arise from tumor heterogeneity, overlapping morphologic features, and tumor sampling. Recently, our knowledge about the genetics of these tumors has expanded, and new molecular markers have been developed. Some of these markers have shown diagnostic value, whereas others are useful prognosticators for patient survival and therapeutic response. Objective.—To review the most clinically useful molecular markers and their detection techniques in gliomas. Data Sources.—Review of the pertinent literature and personal experience with the molecular testing in gliomas. Conclusions.—This article provides an overview of the most common molecular markers in neurooncology, including 1p/19q codeletion in oligodendroglial tumors, mutations in the isocitrate dehydrogenase 1 and 2

genes in diffuse gliomas, hypermethylation of the O6-methylguanine-DNA methyltransferase gene promoter in glioblastomas and anaplastic gliomas, alterations in the epidermal growth factor receptor and phosphatase and tensin homolog genes in high-grade gliomas, as well as BRAF alterations in pilocytic astrocytomas. Molecular testing of gliomas is increasingly used in routine clinical practice and requires that neuropathologists be familiar with these genetic markers and the molecular diagnostic techniques for their detection.

Full free article available from the CAP’s Archives PMID: 21526954

B) Molecular Variants and Mutations in Medulloblastoma Schroeder K, Gururangan S. Molecular variants and mutations in medulloblastoma. Pharmgenomics Pers Med. 2014; 7:43-51

Summary: Medulloblastoma is the commonest malignant brain tumor in children. Treatment with surgery, irradiation, and chemotherapy has improved outcomes in recent years, but patients are frequently left with devastating neurocognitive and other sequelae following such therapy. While the prognosis has traditionally been based on conventional histopathology and clinical staging (based on age, extent of resection, and presence or absence of metastasis), it has become apparent in recent years that the inherent biology of the tumor plays a significant part in predicting survival and sometimes supersedes clinical or pathologic risk factors. The advent of deep sequencing gene technology has provided invaluable clues to the molecular makeup of this tumor and allowed neuro-oncologists to understand that medulloblastoma is an amalgamation of several distinct disease entities with unique clinical associations and behavior. This review is a concise summary of the pathology, genetic syndromes, recent advances in molecular subgrouping, and the associated gene mutations and copy number variations in medulloblastoma. The association of molecular alterations with patient prognosis is also discussed, but it should be remembered that further validation is required in prospective clinical trials utilizing uniform treatment approaches.

Free full text available from PubMed PMID: 24523595

C) BRAF V600E Mutation Identifies a Subset of Low-Grade Diffusely Infiltrating Gliomas in Adults Chi AS, Batchelor TT, Yang D, et al. BRAF V600E mutation identifies a subset of low-grade diffusely infiltrating gliomas in adults. J Clin Oncol. 2013; 31(14): e233-236.

Summary: BRAF V600 hotspot mutations have been reported to be rare in adult low-grade, diffusely infiltrating gliomas (WHO grade 2 diffuse astrocytomas, oligodendrogliomas, and oligoastrocytomas), with only two (0.8%) documented in 251 such tumors compiled across several systematic studies.1–4 BRAF V600 point mutations are more commonly observed in other low-grade glioma subtypes, such as pleomorphic xanthoastrocytoma,5 ganglioglioma,4 and pilocytic astrocytoma.4 At our institution, patient tumors are prospectively genotyped using a targeted sequencing platform (SNaPshot assay; Applied Biosystems, Foster City, CA) as part of routine clinical care.6,7 During the course of tumor genotyping, we encountered an unexpectedly high number of low-grade, diffuse astrocytic tumors with this mutation. These tumors exhibited unusual radiographic and/or histologic features. Herein, we report that BRAF V600E mutations may identify a subset of grade 2 diffuse gliomas with atypical clinical characteristics.

Free full text available from Journal of Clinical Oncology PMID: 23547069

D) Analysis of BRAF V600E Mutation in 1,320 Nervous System Tumors Reveals High Mutation Frequencies in Pleomorphic Xanthoastrocytoma, Ganglioglioma and Extra-Cerebellar Pilocytic Astrocytoma Schindler G, Capper D, Meyer J, et al. Analysis of BRAF V600E mutation in 1,320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma, ganglioglioma and extra-cerebellar pilocytic astrocytoma. Acta Neuropathol. 2011; 121(3): 397-405.

Summary: Missense mutations of the V600E type constitute the vast majority of tumor-associated somatic alterations in the v-RAF murine sarcoma viral oncogene homolog B1 (BRAF) gene. Initially described in melanoma, colon and papillary thyroid carcinoma, these alterations have also been observed in primary nervous system tumors albeit at a low frequency. We analyzed exon 15 of BRAF spanning the V600 locus by direct sequencing in 1,320 adult and pediatric tumors of the nervous system including various types of glial, embryonal, neuronal and glioneuronal, meningeal, adenohypophyseal/sellar, and peripheral nervous system tumors. A total of 96 BRAF mutations were detected; 93 of the V600E type and 3

cases with a three base pair insertion between codons 599 and 600. The highest frequencies of BRAF (V600E) mutations were found in WHO grade II pleomorphic xanthoastrocytomas (42/64; 66%) and pleomorphic xanthoastrocytomas with anaplasia (15/23; 65%), as well as WHO grade I gangliogliomas (14/77; 18%), WHO grade III anaplastic gangliogliomas (3/6) and pilocytic astrocytomas (9/97; 9%). In pilocytic astrocytomas BRAF (V600E) mutation was strongly associated with extra-cerebellar location (p = 0.009) and was most frequent in diencephalic tumors (4/12; 33%). Glioblastomas and other gliomas were characterized by a low frequency or absence of mutations. No mutations were detected in non-glial tumors, including embryonal tumors, meningiomas, nerve sheath tumors and pituitary adenomas. The high mutation frequencies in pleomorphic xanthoastrocytomas, gangliogliomas and extra-cerebellar pilocytic astrocytomas implicate BRAF (V600E) mutation as a valuable diagnostic marker for these rare tumor entities. Future clinical trials should address whether BRAF (V600E) mutant brain tumor patients will benefit from BRAF (V600E)-directed targeted therapies.

Full text available from Acta Neuropathologica (USD 39.95) PMID: 21274720 NOTE: Also cited in Section 2.1.2

E) Comprehensive Genomic Characterization Defines Human Glioblastoma Genes and Core Pathways The Cancer Genome Atlas (TCGA) Research Network. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature. 2008; 455(7216): 1061-1068.

Summary: Human cancer cells typically harbour multiple chromosomal aberrations, nucleotide substitutions and epigenetic modifications that drive malignant transformation. The Cancer Genome Atlas (TCGA) pilot project aims to assess the value of large-scale multi-dimensional analysis of these molecular characteristics in human cancer and to provide the data rapidly to the research community. Here we report the interim integrative analysis of DNA copy number, gene expression and DNA methylation aberrations in 206 glioblastomas--the most common type of adult brain cancer--and nucleotide sequence aberrations in 91 of the 206 glioblastomas. This analysis provides new insights into the roles of ERBB2, NF1 and TP53, uncovers frequent mutations of the phosphatidylinositol-3-OH kinase regulatory subunit gene PIK3R1, and provides a network view of the pathways altered in the development of glioblastoma. Furthermore, integration of mutation, DNA methylation and clinical treatment data reveals a link between MGMT promoter methylation and a hypermutator phenotype consequent to

mismatch repair deficiency in treated glioblastomas, an observation with potential clinical implications. Together, these findings establish the feasibility and power of TCGA, demonstrating that it can rapidly expand knowledge of the molecular basis of cancer.

Free full text available from PubMed PMID: 18772890

F) Glioma Groups Based on 1p/19q, IDH, and TERT Promoter Mutations in Tumors Eckel-Passow JE, Lachance DH, Molinaro AM, et al. Glioma Groups Based on 1p/19q, IDH, and TERT Promoter Mutations in Tumors. N Engl J Med. 2015; 372(26): 2499-2508.

Summary: BACKGROUND: The prediction of clinical behavior, response to therapy, and outcome of infiltrative glioma is challenging. On the basis of previous studies of tumor biology, we defined five glioma molecular groups with the use of three alterations: mutations in the TERT promoter, mutations in IDH, and codeletion of chromosome arms 1p and 19q (1p/19q codeletion). We tested the hypothesis that within groups based on these features, tumors would have similar clinical variables, acquired somatic alterations, and germline variants. METHODS: We scored tumors as negative or positive for each of these markers in 1087 gliomas and compared acquired alterations and patient characteristics among the five primary molecular groups. Using 11,590 controls, we assessed associations between these groups and known glioma germline variants. RESULTS: Among 615 grade II or III gliomas, 29% had all three alterations (i.e., were triple-positive), 5% had TERT and IDH mutations, 45% had only IDH mutations, 7% were triple-negative, and 10% had only TERT mutations; 5% had other combinations. Among 472 grade IV gliomas, less than 1% were triple-positive, 2% had TERT and IDH mutations, 7% had only IDH mutations, 17% were triple-negative, and 74% had only TERT mutations. The mean age at diagnosis was lowest (37 years) among patients who had gliomas with only IDH mutations and was highest (59 years) among patients who had gliomas with only TERT mutations. The molecular groups were independently associated with overall survival among patients with grade II or III gliomas but not among patients with grade IV gliomas. The molecular groups were associated with specific germline variants. CONCLUSIONS: Gliomas were classified into five principal groups on the basis of three tumor markers. The groups had different ages at onset, overall survival, and associations with germline variants, which implies that they are characterized by distinct mechanisms of pathogenesis. (Funded by the National Institutes of Health and others.).

Free full text available from PubMed PMID: 26061753

2.4 Gastrointestinal Cancer

This section includes articles on the following topics:

• Gastrointestinal Cancer Review (Section 2.4.1); • Microsatellite Instability (Section 2.4.2); • Targeted Therapy and Predictive Markers (Section 2.4.3); • Short Presentations on Emerging Concepts: Colorectal Cancer (Section 2.4.4); • Upper Gastrointestinal System (Section 2.4.5); • Lower Gastrointestinal System (Section 2.4.6); and • Hepatopancreatobilary (Section 2.4.7).

2.4.1 Gastrointestinal Cancer Review

A) Targeted Therapies and Predictive Markers in Epithelial Malignancies of the Gastrointestinal Tract McIntire M, Redston M. Targeted Therapies and Predictive Markers in Epithelial Malignancies of the Gastrointestinal Tract. Arch Pathol Lab Med. 2012;136(5):496-503.

Summary: In recent years, there has been a tremendous amount of interest in the development of targeted therapies for the treatment of human cancers. Increased understanding of the specific molecular pathways and driver mutations critical to cancer cell growth have allowed the development of these advanced therapeutics. Among these, inhibitors of the epidermal growth factor receptor and HER2/neu pathways now play a major role in the management of gastrointestinal cancers in addition to other solid malignancies. In colorectal and gastric cancers, the use of epidermal growth factor receptor inhibitors and HER2/neu inhibitors has increased the available treatment options for patients with advanced disease. Objective.— To focus on the current targeted therapies and predictors of response in malignancies of the gastrointestinal tract. Data Sources.—Medical literature searchable on PubMed (US National Library of Medicine) as well as older studies revealed by the literature review were used as the source of data. Conclusion.—Gene testing of critical elements of the pathways targeted by these agents (such as KRAS mutational analysis in colorectal tumors and

HER2/neu testing in gastric cancers) allows the ability to predict which patients will respond to these treatments. As the molecular profiling of tumors and our understanding of cancer genomics and epigenetic alterations continues to grow, it is expected that these personalized targeted therapies will form one of the mainstays of gastrointestinal cancer treatment.

Free full article available from the CAP’s Archives PMID: 22229849

B) Gastrointestinal Stromal Tumours: Origin and Molecular Oncology Corless CL, Barnett CM, Heinrich MC. Gastrointestinal stromal tumours: origin and molecular oncology. Nat Rev Cancer. 2011; 11(12): 865-878.

Summary: Gastrointestinal stromal tumours (GISTs) are a paradigm for the development of personalized treatment for cancer patients. The nearly simultaneous discovery of a biomarker that is reflective of their origin and the presence of gain-of-function kinase mutations in these tumours set the stage for more accurate diagnosis and the development of kinase inhibitor therapy. Subsequent studies of genotype and phenotype have led to a molecular classification of GIST and to treatment optimization on the basis of molecular subtype. The study of drug-resistant tumours has advanced our understanding of kinase biology, enabling the development of novel kinase inhibitors. Further improvements in GIST treatment may require targeting GIST stem cell populations and/or additional genomic events.

Full text available from Nature Reviews Cancer (USD 32.00) PMID: 22089421

2.4.2 Microsatellite Instability

A) Relevance, Pathogenesis, and Testing Algorithm for Mismatch Repair- Defective Colorectal Carcinomas: A Report of the Association for Molecular Pathology Funkhouser WK Jr, Lubin IM, Monzon FA, et al. Relevance, pathogenesis, and testing algorithm for mismatch repair-defective colorectal carcinomas: a report of the association for molecular pathology. J Mol Diagn. 2012; 14(2):91-103.

Summary: Loss-of-function defects in DNA mismatch repair (MMR), which manifest as high levels of microsatellite instability (MSI), occur in approximately 15% of all colorectal carcinomas (CRCs). This molecular subset of CRC characterizes patients with better stage-specific prognoses

who experience no benefit from 5-fluorouracil chemotherapy. Most MMR- deficient (dMMR) CRCs are sporadic, but 15% to 20% are due to inherited predisposition (Lynch syndrome). High penetrance of CRCs in germline MMR gene mutation carriers emphasizes the importance of accurate diagnosis of Lynch syndrome carriers. Family-based (Amsterdam), patient/family-based (Bethesda), morphology-based, microsatellite-based, and IHC-based screening criteria do not individually detect all germline mutation carriers. These limitations support the use of multiple concurrent tests and the screening of all patients with newly diagnosed CRC. This approach is resource intensive but would increase detection of inherited and de novo germline mutations to guide family screening. Although CRC prognosis and prediction of 5-fluorouracil response are similar in both the Lynch and sporadic dMMR subgroups, these subgroups differ significantly with regard to the implications for family members. We recommend that new CRCs should be classified into sporadic MMR-proficient, sporadic dMMR, or Lynch dMMR subgroups. The concurrent use of MSI testing, MMR protein IHC, and BRAF c.1799T>A mutation analysis would detect almost all dMMR CRCs, would classify 94% of all new CRCs into these MMR subgroups, and would guide secondary molecular testing of the remainder.

Free full text available from Journal of Molecular Diagnostics PMID: 22260991

B) Coloseq Provides Comprehensive Lynch and Polyposis Syndrome Mutational Analysis Using Massively Parallel Sequencing Pritchard CC, Smith C, Salipante SJ, et al. Coloseq Provides Comprehensive Lynch and Polyposis Syndrome Mutational Analysis Using Massively Parallel Sequencing. J Mol Diagn. 2012; 14(4): 357-366.

Summary: Lynch syndrome (hereditary nonpolyposis colon cancer) and adenomatous polyposis syndromes frequently have overlapping clinical features. Current approaches for molecular genetic testing are often stepwise, taking a best-candidate gene approach with testing of additional genes if initial results are negative. We report a comprehensive assay called ColoSeq that detects all classes of mutations in Lynch and polyposis syndrome genes using targeted capture and massively parallel next- generation sequencing on the Illumina HiSeq2000 instrument. In blinded specimens and colon cancer cell lines with defined mutations, ColoSeq correctly identified 28/28 (100%) pathogenic mutations in MLH1, MSH2, MSH6, PMS2, EPCAM, APC, and MUTYH, including single nucleotide variants (SNVs), small insertions and deletions, and large copy number variants. There was 100% reproducibility of detection mutation between independent runs. The assay correctly identified 222 of 224 heterozygous

SNVs (99.4%) in HapMap samples, demonstrating high sensitivity of calling all variants across each captured gene. Average coverage was greater than 320 reads per base pair when the maximum of 96 index samples with barcodes were pooled. In a specificity study of 19 control patients without cancer from different ethnic backgrounds, we did not find any pathogenic mutations but detected two variants of uncertain significance. ColoSeq offers a powerful, cost-effective means of genetic testing for Lynch and polyposis syndromes that eliminates the need for stepwise testing and multiple follow- up clinical visits.

Free full text available from PubMed PMID: 22658618

2.4.3 Targeted Therapy and Predictive Markers

A) Select Biomarkers for Tumors of the Gastrointestinal Tract: Present and Future Bartley AN, Hamilton SR. Select biomarkers for tumors of the gastrointestinal tract: present and future. Arch Pathol Lab Med. 2015; 139(4): 457-468.

Summary: CONTEXT: Advances in molecular biomarkers of the gastrointestinal tract have contributed to a decline in the incidence of and mortality from diseases of the gastrointestinal tract. The discovery and clinical validation of new biomarkers are important to personalized cancer therapy, and numerous clinical trials are currently ongoing to help identify individualized therapy affecting these biomarkers and molecular mechanisms they represent. Distinct molecular pathways leading to cancers of the colorectum, esophagus, stomach, small bowel, and pancreas have been identified. Using biomarkers in these pathways to direct patient care, including selection of proper molecular testing for identification of actionable mutations and reporting the results of these biomarkers to guide clinicians and genetic counselors, is paramount. OBJECTIVE: To examine and review select clinically actionable biomarkers of the colon, esophagus, stomach, small bowel, and pancreas, including present and future biomarkers with relevant clinical trials. DATA SOURCES: Extensive literature review and practical and consultation experience of the authors. CONCLUSIONS: Although numerous biomarkers have been identified and are currently guiding patient therapy, few have shown evidence of clinical utility in the management of patients with gastrointestinal cancers. Inconsistent results and discordant proposed algorithms for testing were identified throughout the literature; however, the potential for biomarkers to improve outcomes for

patients with gastrointestinal cancer remains high. Continued advances through high-quality studies are needed.

Free full text available from the CAP’s Archives PMID: 25333834

B) Panitumumab-FOLFOX4 Treatment and RAS Mutations in Colorectal Cancer Douillard JY, Oliner KS, Siena S, et al. Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. N Engl J Med. 2013; 369(11): 1023- 1034.

Summary: BACKGROUND: Patients with metastatic colorectal cancer that harbors KRAS mutations in exon 2 do not benefit from anti-epidermal growth factor receptor (EGFR) therapy. Other activating RAS mutations may also be negative predictive biomarkers for anti-EGFR therapy. METHODS: In this prospective-retrospective analysis, we assessed the efficacy and safety of panitumumab plus oxaliplatin, fluorouracil, and leucovorin (FOLFOX4) as compared with FOLFOX4 alone, according to RAS (KRAS or NRAS) or BRAF mutation status. A total of 639 patients who had metastatic colorectal cancer without KRAS mutations in exon 2 had results for at least one of the following: KRAS exon 3 or 4; NRAS exon 2, 3, or 4; or BRAF exon 15. The overall rate of ascertainment of RAS status was 90%. RESULTS: Among 512 patients without RAS mutations, progression-free survival was 10.1 months with panitumumab-FOLFOX4 versus 7.9 months with FOLFOX4 alone (hazard ratio for progression or death with combination therapy, 0.72; 95% confidence interval [CI], 0.58 to 0.90; P=0.004). Overall survival was 26.0 months in the panitumumab-FOLFOX4 group versus 20.2 months in the FOLFOX4-alone group (hazard ratio for death, 0.78; 95% CI, 0.62 to 0.99; P=0.04). A total of 108 patients (17%) with nonmutated KRAS exon 2 had other RAS mutations. These mutations were associated with inferior progression-free survival and overall survival with panitumumab-FOLFOX4 treatment, which was consistent with the findings in patients with KRAS mutations in exon 2. BRAF mutations were a negative prognostic factor. No new safety signals were identified. CONCLUSIONS: Additional RAS mutations predicted a lack of response in patients who received panitumumab-FOLFOX4. In patients who had metastatic colorectal cancer without RAS mutations, improvements in overall survival were observed with panitumumab-FOLFOX4 therapy. (Funded by Amgen and others; PRIME ClinicalTrials.gov number, NCT00364013.).

Free full text available from New England Journal of Medicine PMID: 24024839

NOTE: Also cited in Section 2.1.2

C) Colorectal Cancer Diagnostics: Biomarkers, Cell-Free DNA, Circulating Tumor Cells and Defining Heterogeneous Populations by Single-Cell Analysis Kin C, Kidess E, Poultsides GA, Visser BC, Jeffrey SS. Colorectal cancer diagnostics: biomarkers, cell-free DNA, circulating tumor cells and defining heterogeneous populations by single-cell analysis. Expert Rev Mol Diagn. 2013; 13(6): 581-599.

Summary: Reliable biomarkers are needed to guide treatment of colorectal cancer, as well as for surveillance to detect recurrence and monitor therapeutic response. In this review, the authors discuss the use of various biomarkers in addition to serum carcinoembryonic antigen, the current surveillance method for metastatic recurrence after resection. The clinical relevance of mutations including microsatellite instability, KRAS, BRAF and SMAD4 is addressed. The role of circulating tumor cells and cell-free DNA with regards to their implementation into clinical use is discussed, as well as how single-cell analysis may fit into a monitoring program. The detection and characterization of circulating tumor cells and cell-free DNA in colorectal cancer patients will not only improve the understanding of the development of metastasis, but may also supplant the use of other biomarkers.

Free full text available from Expert Review of Molecular Diagnostics PMID: 23895128

D) Aspirin Use, Tumor PIK3CA Mutation, and Colorectal-Cancer Survival Liao X, Lochhead P, Nishihara R, et al. Aspirin use, tumor PIK3CA mutation, and colorectal-cancer survival. N Engl J Med. 2012; 367(17): 1596-1606.

Summary: BACKGROUND: Regular use of aspirin after a diagnosis of colon cancer has been associated with a superior clinical outcome. Experimental evidence suggests that inhibition of prostaglandin-endoperoxide synthase 2 (PTGS2) (also known as cyclooxygenase-2) by aspirin down-regulates phosphatidylinositol 3-kinase (PI3K) signaling activity. We hypothesized that the effect of aspirin on survival and prognosis in patients with cancers characterized by mutated PIK3CA (the phosphatidylinositol-4,5- bisphosphonate 3-kinase, catalytic subunit alpha polypeptide gene) might differ from the effect among those with wild-type PIK3CA cancers. METHODS: We obtained data on 964 patients with rectal or colon cancer from the Nurses' Health Study and the Health Professionals Follow-up Study, including data on aspirin use after diagnosis and the presence or absence of PIK3CA mutation. We used a Cox proportional-hazards model to compute

the multivariate hazard ratio for death. We examined tumor markers, including PTGS2, phosphorylated AKT, KRAS, BRAF, microsatellite instability, CpG island methylator phenotype, and methylation of long interspersed nucleotide element 1. RESULTS: Among patients with mutated- PIK3CA colorectal cancers, regular use of aspirin after diagnosis was associated with superior colorectal cancer-specific survival (multivariate hazard ratio for cancer-related death, 0.18; 95% confidence interval [CI], 0.06 to 0.61; P<0.001 by the log-rank test) and overall survival (multivariate hazard ratio for death from any cause, 0.54; 95% CI, 0.31 to 0.94; P=0.01 by the log-rank test). In contrast, among patients with wild-type PIK3CA, regular use of aspirin after diagnosis was not associated with colorectal cancer- specific survival (multivariate hazard ratio, 0.96; 95% CI, 0.69 to 1.32; P=0.76 by the log-rank test; P=0.009 for interaction between aspirin and PIK3CA variables) or overall survival (multivariate hazard ratio, 0.94; 95% CI, 0.75 to 1.17; P=0.96 by the log-rank test; P=0.07 for interaction). CONCLUSIONS: Regular use of aspirin after diagnosis was associated with longer survival among patients with mutated-PIK3CA colorectal cancer, but not among patients with wild-type PIK3CA cancer. The findings from this molecular pathological epidemiology study suggest that the PIK3CA mutation in colorectal cancer may serve as a predictive molecular biomarker for adjuvant aspirin therapy. (Funded by The National Institutes of Health and others.).

Free full text available from PubMed PMID: 23094721 NOTE: Also cited in Section 2.1.2

E) Extended RAS Gene Mutation Testing in Metastatic Colorectal Carcinoma to Predict Response to Anti-Epidermal Growth Factor Receptor Monoclonal Antibody Therapy: American Society of Clinical Oncology Provisional Clinical Opinion Update 2015 Allegra CJ, Rumble RB, Hamilton SR, et al. Extended RAS Gene Mutation Testing in Metastatic Colorectal Carcinoma to Predict Response to Anti- Epidermal Growth Factor Receptor Monoclonal Antibody Therapy: American Society of Clinical Oncology Provisional Clinical Opinion Update 2015. J Clin Oncol. 2016; 34(2): 179-185.

Summary: PURPOSE: An American Society of Clinical Oncology Provisional Clinical Opinion (PCO) offers timely clinical direction after publication or presentation of potentially practice-changing data from major studies. This PCO update addresses the utility of extended RAS gene mutation testing in patients with metastatic colorectal cancer (mCRC) to detect resistance to anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MoAb)

therapy. CLINICAL CONTEXT: Recent results from phase II and III clinical trials in mCRC demonstrate that patients whose tumors harbor RAS mutations in exons 2 (codons 12 and 13), 3 (codons 59 and 61), and 4 (codons 117 and 146) are unlikely to benefit from therapy with MoAbs directed against EGFR, when used as monotherapy or combined with chemotherapy. RECENT DATA: In addition to the evidence reviewed in the original PCO, 11 systematic reviews with meta-analyses, two retrospective analyses, and two health technology assessments based on a systematic review were obtained. These evaluated the outcomes for patients with mCRC with no mutation detected or presence of mutation in additional exons in KRAS and NRAS. PCO: All patients with mCRC who are candidates for anti-EGFR antibody therapy should have their tumor tested in a Clinical Laboratory Improvement Amendments-certified laboratory for mutations in both KRAS and NRAS exons 2 (codons 12 and 13), 3 (codons 59 and 61), and 4 (codons 117 and 146). The weight of current evidence indicates that anti-EGFR MoAb therapy should only be considered for treatment of patients whose tumor is determined to not have mutations detected after such extended RAS testing.

Free full text available from Journal of Clinical Oncology PMID: 26438111

2.4.4 Short Presentations on Emerging Concepts: Colorectal Cancer

A) CAP Short Presentations on Emerging Concepts (SPECS): Emerging Concepts in Molecular Testing for Inherited and Sporadic Colorectal Cancer [PowerPoint slides] Anderson MW, Caughron SK, Chandra PK, Crothers J, Misialek MJ, Nowak JA. CAP Short Presentations on Emerging Concepts (SPECS): Emerging Concepts in Molecular Testing for Inherited and Sporadic Colorectal Cancer (v 1.0e rev 7/27/16) [PowerPoint slides]. Foo WC, Witte D, eds. Northfield, IL: College of American Pathologists; 2016.

Access the slides

B) NCCN Clinical Practice Guidelines in Oncology: Colon and Rectal Cancer v 3.2015 National Comprehensive Cancer Network©. NCCN Clinical Practice Guidelines in Oncology: Colon and Rectal Cancer v 3.2015 v 1.2015. http://www.nccn.org/professionals/physician_gls/f_guidelines.asp Accessed June 18, 2015.

Full text available from NCCN website (subscription required)

2.4.5 Upper Gastrointestinal System

A) Comprehensive Molecular Characterization of Gastric Adenocarcinoma Cancer Genome Atlas Research Network. Comprehensive molecular characterization of gastric adenocarcinoma. Nature. 2014; 513(7517): 202- 209.

Summary: Gastric cancer is a leading cause of cancer deaths, but analysis of its molecular and clinical characteristics has been complicated by histological and aetiological heterogeneity. Here we describe a comprehensive molecular evaluation of 295 primary gastric adenocarcinomas as part of The Cancer Genome Atlas (TCGA) project. We propose a molecular classification dividing gastric cancer into four subtypes: tumours positive for Epstein-Barr virus, which display recurrent PIK3CA mutations, extreme DNA hypermethylation, and amplification of JAK2, CD274 (also known as PD-L1) and PDCD1LG2 (also known as PD-L2); microsatellite unstable tumours, which show elevated mutation rates, including mutations of genes encoding targetable oncogenic signalling proteins; genomically stable tumours, which are enriched for the diffuse histological variant and mutations of RHOA or fusions involving RHO-family GTPase-activating proteins; and tumours with chromosomal instability, which show marked aneuploidy and focal amplification of receptor tyrosine kinases. Identification of these subtypes provides a roadmap for patient stratification and trials of targeted therapies.

Free full text available from PubMed PMID: 25079317

B) Molecular Diagnosis for Personalized Target Therapy in Gastric Cancer Cho JY. Molecular Diagnosis for Personalized Target Therapy in Gastric Cancer. J Gastric Cancer. 2013; 13(3): 129-135.

Summary: Gastric cancer is the second leading cause of cancer-related deaths worldwide. In advanced and metastatic gastric cancer, the conventional chemotherapy with limited efficacy shows an overall survival period of about 10 months. Patient specific and effective treatments known as personalized cancer therapy is of significant importance. Advances in high-throughput technologies such as microarray and next generation sequencing for genes, protein expression profiles and oncogenic signaling pathways have reinforced the discovery of treatment targets and personalized treatments. However, there are numerous challenges from cancer target discoveries to practical clinical benefits. Although there is a flood of biomarkers and target agents, only a minority of patients are tested and treated accordingly. Numerous molecular target agents have been under investigation for gastric cancer. Currently, targets for gastric cancer include the epidermal growth factor receptor family, mesenchymal-epithelial transition factor axis, and the phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathways. Deeper insights of molecular characteristics for gastric cancer has enabled the molecular classification of gastric cancer, the diagnosis of gastric cancer, the prediction of prognosis, the recognition of gastric cancer driver genes, and the discovery of potential therapeutic targets. Not only have we deeper insights for the molecular diversity of gastric cancer, but we have also prospected both affirmative potentials and hurdles to molecular diagnostics. New paradigm of transdisciplinary team science, which is composed of innovative explorations and clinical investigations of oncologists, geneticists, pathologists, biologists, and bioinformaticians, is mandatory to recognize personalized target therapy.

Free full text available from PubMed PMID: 24156032

2.4.6 Lower Gastrointestinal System

A) Comprehensive Molecular Characterization of Human Colon and Rectal Cancer The Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012; 487(7407): 330-337.

Summary: To characterize somatic alterations in colorectal carcinoma, we conducted a genome-scale analysis of 276 samples, analysing exome sequence, DNA copy number, promoter methylation and messenger RNA and microRNA expression. A subset of these samples (97) underwent low- depth-of-coverage whole-genome sequencing. In total, 16% of colorectal

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carcinomas were found to be hypermutated: three-quarters of these had the expected high microsatellite instability, usually with hypermethylation and MLH1 silencing, and one-quarter had somatic mismatch-repair gene and polymerase epsilon (POLE) mutations. Excluding the hypermutated cancers, colon and rectum cancers were found to have considerably similar patterns of genomic alteration. Twenty-four genes were significantly mutated, and in addition to the expected APC, TP53, SMAD4, PIK3CA and KRAS mutations, we found frequent mutations in ARID1A, SOX9 and FAM123B. Recurrent copy-number alterations include potentially drug-targetable amplifications of ERBB2 and newly discovered amplification of IGF2. Recurrent chromosomal translocations include the fusion of NAV2 and WNT pathway member TCF7L1. Integrative analyses suggest new markers for aggressive colorectal carcinoma and an important role for MYC-directed transcriptional activation and repression.

Free full text available from PubMed PMID: 22810696

2.4.7 Hepatopancreatobilary

A) Genome-Wide Characterization of Pancreatic Adenocarcinoma Patients Using Next Generation Sequencing Liang WS, Craig DW, Carpten J, et al. Genome-wide characterization of pancreatic adenocarcinoma patients using next generation sequencing. PLoS One. 2012; 7(10):e43192.

Summary: Pancreatic adenocarcinoma (PAC) is among the most lethal malignancies. While research has implicated multiple genes in disease pathogenesis, identification of therapeutic leads has been difficult and the majority of currently available therapies provide only marginal benefit. To address this issue, our goal was to genomically characterize individual PAC patients to understand the range of aberrations that are occurring in each tumor. Because our understanding of PAC tumorigenesis is limited, evaluation of separate cases may reveal aberrations, that are less common but may provide relevant information on the disease, or that may represent viable therapeutic targets for the patient. We used next generation sequencing to assess global somatic events across 3 PAC patients to characterize each patient and to identify potential targets. This study is the first to report whole genome sequencing (WGS) findings in paired tumor/normal samples collected from 3 separate PAC patients. We generated on average 132 billion mappable bases across all patients using WGS, and identified 142 somatic coding events including point mutations,

insertion/deletions, and chromosomal copy number variants. We did not identify any significant somatic translocation events. We also performed RNA sequencing on 2 of these patients' tumors for which tumor RNA was available to evaluate expression changes that may be associated with somatic events, and generated over 100 million mapped reads for each patient. We further performed pathway analysis of all sequencing data to identify processes that may be the most heavily impacted from somatic and expression alterations. As expected, the KRAS signaling pathway was the most heavily impacted pathway (P<0.05), along with tumor-stroma interactions and tumor suppressive pathways. While sequencing of more patients is needed, the high resolution genomic and transcriptomic information we have acquired here provides valuable information on the molecular composition of PAC and helps to establish a foundation for improved therapeutic selection.

Free full text available from PubMed PMID: 23071490

2.5 Lung Cancer

A) Biomarker Testing in Non-Small Cell Lung Cancer: A Clinician's Perspective Bernicker E. Biomarker testing in non-small cell lung cancer: a clinician's perspective. Arch Pathol Lab Med. 2015; 139(4): 448-450.

No summary available

Free full text available from the CAP’s Archives PMID: 25166873

B) Using Multiplexed Assays of Oncogenic Drivers in Lung Cancers to Select Targeted Drugs Kris MG, Johnson BE, Berry LD, et al. Using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs. JAMA. 2014; 311(19): 1998-2006.

Summary: IMPORTANCE: Targeting oncogenic drivers (genomic alterations critical to cancer development and maintenance) has transformed the care of patients with lung adenocarcinomas. The Lung Cancer Mutation Consortium was formed to perform multiplexed assays testing adenocarcinomas of the lung for drivers in 10 genes to enable clinicians to select targeted treatments

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and enroll patients into clinical trials. OBJECTIVES: To determine the frequency of oncogenic drivers in patients with lung adenocarcinomas and to use the data to select treatments targeting the identified driver(s) and measure survival. DESIGN, SETTING, AND PARTICIPANTS: From 2009 through 2012, 14 sites in the United States enrolled patients with metastatic lung adenocarcinomas and a performance status of 0 through 2 and tested their tumors for 10 drivers. Information was collected on patients, therapies, and survival. INTERVENTIONS: Tumors were tested for 10 oncogenic drivers, and results were used to select matched targeted therapies. MAIN OUTCOMES AND MEASURES: Determination of the frequency of oncogenic drivers, the proportion of patients treated with genotype-directed therapy, and survival. RESULTS: From 2009 through 2012, tumors from 1007 patients were tested for at least 1 gene and 733 for 10 genes (patients with full genotyping). An oncogenic driver was found in 466 of 733 patients (64%). Among these 733 tumors, 182 tumors (25%) had the KRAS driver; sensitizing EGFR, 122 (17%); ALK rearrangements, 57 (8%); other EGFR, 29 (4%); 2 or more genes, 24 (3%); ERBB2 (formerly HER2), 19 (3%); BRAF, 16 (2%); PIK3CA, 6 (<1%); MET amplification, 5 (<1%); NRAS, 5 (<1%); MEK1, 1 (<1%); AKT1, 0. Results were used to select a targeted therapy or trial in 275 of 1007 patients (28%). The median survival was 3.5 years (interquartile range [IQR], 1.96-7.70) for the 260 patients with an oncogenic driver and genotype-directed therapy compared with 2.4 years (IQR, 0.88-6.20) for the 318 patients with any oncogenic driver(s) who did not receive genotype-directed therapy (propensity score-adjusted hazard ratio, 0.69 [95% CI, 0.53-0.9], P = .006). CONCLUSIONS AND RELEVANCE: Actionable drivers were detected in 64% of lung adenocarcinomas. Multiplexed testing aided physicians in selecting therapies. Although individuals with drivers receiving a matched targeted agent lived longer, randomized trials are required to determine if targeting therapy based on oncogenic drivers improves survival. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01014286.

Free full text available from PubMed PMID: 24846037

C) Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors: Guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology Lindeman NI, Cagle PT, Beasley MB, et al. Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors: Guideline from the College of American Pathologists, International

Association for the Study of Lung Cancer, and Association for Molecular Pathology. Arch Pathol Lab Med. 2013; 137(6):828-60.

Summary: To establish evidence-based recommendations for the molecular analysis of lung cancers that are required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed. Participants.-Three cochairs without conflicts of interest were selected, one from each of the 3 sponsoring professional societies: College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Writing and advisory panels were constituted from additional experts from these societies. Evidence.-Three unbiased literature searches of electronic databases were performed to capture articles published from January 2004 through February 2012, yielding 1533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for their relevance to the recommendations. Evidence was formally graded for each recommendation. Consensus Process.-Initial recommendations were formulated by the cochairs and panel members at a public meeting. Each guideline section was assigned to at least 2 panelists. Drafts were circulated to the writing panel (version 1), advisory panel (version 2), and the public (version 3) before submission (version 4). Conclusions.- The 37 guideline items address 14 subjects, including 15 recommendations (evidence grade A/B). The major recommendations are to use testing for EGFR mutations and ALK fusions to guide patient selection for therapy with an epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) inhibitor, respectively, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, and to prioritize EGFR and ALK testing over other molecular predictive tests. As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards. Already, new treatments targeting genetic alterations in other, less common driver oncogenes are being evaluated in lung cancer, and testing for these may be addressed in future versions of these guidelines.

Free full text available from the CAP’s Archives PMID: 23551194 NOTE: Also cited in Section 1.1.2

D) Non-small Cell Lung Cancer and Precision Medicine: A Model for the Incorporation of Genomic Features into Clinical Trial Design Pasche B,Grant SC. Non-small cell lung cancer and precision medicine: a model for the incorporation of genomic features into clinical trial design. JAMA. 2014; 311(19): 1975-1976.

Summary: Lung cancer remains the leading cause of cancer death in the United States. It is estimated that 228,190 patients were diagnosed with the disease and 159,480 died of lung cancer in 2013. Historically, lung cancer has been categorized, based on histology, into non–small cell lung cancer (NSCLC) constituting approximately 85% of cases and small cell lung cancer accounting for approximately 15% of the cases. Treatment for advanced NSCLC has been associated with relatively low response rates and significant toxicity, and only in the last 20 years has an agreement been reached that chemotherapy for this disease confers a survival benefit. At the same time, no distinction was made in treatment between the various subtypes of NSCLC until 2008 when a randomized trial demonstrated differences in response rates to specific chemotherapy regimens between squamous and nonsquamous lung carcinomas. Addition of the antiangiogenic agent bevacizumab resulted in a 2-month improvement in median survival in patients with nonsquamous cell lung cancer.4 However, a subsequent study suggested that the benefit of this therapy may be limited to patients 65 years or younger.

Full text available from Journal of American Medical Association (USD 30.00 for 24 hour access) PMID: 24846033 NOTE: Also cited in Section 1.2.1

E) Current and Future Molecular Diagnostics in Non-Small-Cell Lung Cancer Li CM, Chu WY, Wong DL, et al. Current and Future Molecular Diagnostics in Non-Small-Cell Lung Cancer. Expert Rev Mol Diagn. 2015; 15(8): 1061- 1074.

Summary: The molecular investigation of lung cancer has opened up an advanced area for the diagnosis and therapeutic management of lung cancer patients. Gene alterations in cancer initiation and progression provide not only information on molecular changes in lung cancer but also opportunities in advanced therapeutic regime by personalized targeted therapy. EGFR mutations and ALK rearrangement are important predictive biomarkers for the efficiency of tyrosine kinase inhibitor treatment in lung cancer patients. Moreover, epigenetic aberration and microRNA dysregulation are recent

advances in the early detection and monitoring of lung cancer. Although a wide range of molecular tests are available, standardization and validation of assay protocols are essential for the quality of the test outcome. In this review, current and new advancements of molecular biomarkers for non- small-cell lung cancer will be discussed. Recommendations on future development of molecular diagnostic services will also be explored.

Full text available from Expert Review of Molecular Diagnostics (USD 89.00) PMID: 26153330

2.6 Melanoma

This section includes articles on the following topics:

• Articles on Melanoma (Section 2.6.1); and • Short Presentations on Emerging Concepts: Melanoma (Section 2.6.2).

2.6.1 Articles on Melanoma

A) A Landscape of Driver Mutations in Melanoma Hodis E, Watson IR, Kryukov GV, et al. A landscape of driver mutations in melanoma. Cell. 2012; 150(2): 251-263.

Summary: Despite recent insights into melanoma genetics, systematic surveys for driver mutations are challenged by an abundance of passenger mutations caused by carcinogenic UV light exposure. We developed a permutation-based framework to address this challenge, employing mutation data from intronic sequences to control for passenger mutational load on a per gene basis. Analysis of large-scale melanoma exome data by this approach discovered six novel melanoma genes (PPP6C, RAC1, SNX31, TACC1, STK19, and ARID2), three of which-RAC1, PPP6C, and STK19- harbored recurrent and potentially targetable mutations. Integration with chromosomal copy number data contextualized the landscape of driver mutations, providing oncogenic insights in BRAF- and NRAS-driven melanoma as well as those without known NRAS/BRAF mutations. The landscape also clarified a mutational basis for RB and p53 pathway deregulation in this malignancy. Finally, the spectrum of driver mutations provided unequivocal genomic evidence for a direct mutagenic role of UV light in melanoma pathogenesis.

Free full text available from PubMed PMID: 22817889

B) New Strategies in Melanoma: Molecular Testing in Advanced Disease Woodman SE, Lazar AJ, Aldape KD, Davies MA. New strategies in melanoma: molecular testing in advanced disease. Clin Cancer Res. 2012; 18(5): 1195-1200.

Summary: Melanoma is one of the most aggressive forms of skin cancer. The management of melanoma is evolving rapidly due to an improved understanding of the molecular heterogeneity of this disease and the development of effective, personalized, targeted therapy strategies. Although previous classification systems were based predominantly on clinical and histologic criteria, there is now a strong rationale for adding molecular markers to the diagnostic evaluation of these tumors. Research has shown that the types and prevalence of genetic alterations vary among melanoma subtypes. Thus, rational molecular testing should be based on an understanding of the events that are likely to occur in a given tumor and the clinical implications of test results. This review summarizes the existing data that support the rationale for molecular testing in clinically defined melanoma subtypes. Emerging challenges and controversies regarding the use of various molecular testing platforms, and their implications for clinical testing, are also discussed.

Free full text available from Clinical Cancer Research PMID: 22275506

C) The Molecular Pathology of Melanoma: An Integrated Taxonomy of Melanocytic Neoplasia Bastian BC. The Molecular Pathology of Melanoma: An Integrated Taxonomy of Melanocytic Neoplasia. Annu Rev Pathol. 2014; 9:239-271.

Summary: Melanomas comprise multiple biologically distinct categories, which differ in cell of origin, age of onset, clinical and histologic presentation, pattern of metastasis, ethnic distribution, causative role of UV radiation, predisposing germ-line alterations, mutational processes, and patterns of somatic mutations. Neoplasms are initiated by gain-of-function mutations in one of several primary oncogenes, which typically lead to benign melanocytic nevi with characteristic histologic features. The progression of nevi is restrained by multiple tumor-suppressive mechanisms. Secondary genetic alterations override these barriers and promote intermediate or overtly malignant tumors along distinct progression trajectories. The current knowledge about the pathogenesis and clinical, histologic, and genetic

features of primary melanocytic neoplasms is reviewed and integrated into a taxonomic framework.

Free full text available from PubMed PMID: 24460190

2.6.2 Short Presentations on Emerging Concepts: Melanoma

A) CAP Short Presentations on Emerging Concepts (SPEC): Emerging Concepts in Therapeutic Guidance for Metastatic Melanoma [PowerPoint slides] Caughron SK, Wood J. CAP Short Presentations on Emerging Concepts (SPECS): Emerging Concepts in Therapeutic Guidance for Metastatic Melanoma (v 4.0e rev 7/7/15) [PowerPoint slides]. Foo W, Nowak JA, Witte DL, eds. Northfield, IL: College of American Pathologists; 2015.

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The article included is a selected reference from the Metastatic Melanoma SPEC:

A) Improved Survival with Vemurafenib in Melanoma with BRAF V600E Mutation Chapman PB, Hauschild A, Robert C, et al, Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011; 364(26):2507-16.

Summary: Phase 1 and 2 clinical trials of the BRAF kinase inhibitor vemurafenib (PLX4032) have shown response rates of more than 50% in patients with metastatic melanoma with the BRAF V600E mutation. METHODS: We conducted a phase 3 randomized clinical trial comparing vemurafenib with dacarbazine in 675 patients with previously untreated, metastatic melanoma with the BRAF V600E mutation. Patients were randomly assigned to receive either vemurafenib (960 mg orally twice daily) or dacarbazine (1000 mg per square meter of body-surface area

intravenously every 3 weeks). Coprimary end points were rates of overall and progression-free survival. Secondary end points included the response rate, response duration, and safety. A final analysis was planned after 196 deaths and an interim analysis after 98 deaths. RESULTS: At 6 months, overall survival was 84% (95% confidence interval [CI], 78 to 89) in the vemurafenib group and 64% (95% CI, 56 to 73) in the dacarbazine group. In the interim analysis for overall survival and final analysis for progression-free survival, vemurafenib was associated with a relative reduction of 63% in the risk of death and of 74% in the risk of either death or disease progression, as compared with dacarbazine (P<0.001 for both comparisons). After review of the interim analysis by an independent data and safety monitoring board, crossover from dacarbazine to vemurafenib was recommended. Response rates were 48% for vemurafenib and 5% for dacarbazine. Common adverse events associated with vemurafenib were arthralgia, rash, fatigue, alopecia, keratoacanthoma or squamous-cell carcinoma, photosensitivity, nausea, and diarrhea; 38% of patients required dose modification because of toxic effects. CONCLUSIONS: Vemurafenib produced improved rates of overall and progression-free survival in patients with previously untreated melanoma with the BRAF V600E mutation. (Funded by Hoffmann-La Roche; BRIM-3 ClinicalTrials.gov number, NCT01006980.).

Free full text available from New England Journal of Medicine PMID: 21639808

2.7 Thyroid Cancer

This section includes articles on the following topics:

• Articles on Thyroid Cancer (Section 2.7.1); and • Short Presentations on Emerging Concepts: Thyroid (Section 2.7.2).

2.7.1 Articles on Thyroid Cancer

A) Integrated Genomic Characterization of Papillary Thyroid Carcinoma Cancer Genome Atlas Research Network. Integrated Genomic Characterization of Papillary Thyroid Carcinoma. Cell. 2014; 159(3):676-90.

Summary: Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Here, we describe the genomic landscape of 496 PTCs. We observed a low frequency of somatic alterations (relative to other

carcinomas) and extended the set of known PTC driver alterations to include EIF1AX, PPM1D, and CHEK2 and diverse gene fusions. These discoveries reduced the fraction of PTC cases with unknown oncogenic driver from 25% to 3.5%. Combined analyses of genomic variants, gene expression, and methylation demonstrated that different driver groups lead to different pathologies with distinct signaling and differentiation characteristics. Similarly, we identified distinct molecular subgroups of BRAF-mutant tumors, and multidimensional analyses highlighted a potential involvement of oncomiRs in less-differentiated subgroups. Our results propose a reclassification of thyroid cancers into molecular subtypes that better reflect their underlying signaling and differentiation properties, which has the potential to improve their pathological classification and better inform the management of the disease.

Free full text available from Cell PMID: 25417114

B) Molecular Diagnosis for Indeterminate Thyroid Nodules on Fine Needle Aspiration: Advances and Limitations Keutgen XM, Filicori F, Fahey TJ 3rd. Molecular diagnosis for indeterminate thyroid nodules on fine needle aspiration: advances and limitations. Expert Rev Mol Diagn. 2013; 13(6): 613-623.

Summary: Indeterminate thyroid lesions are diagnosed in up to 30% of fine needle aspirations. These nodules harbor malignancy in more than 25% of cases, and hemithyroidectomy or total thyroidectomy has therefore been advocated in order to achieve definitive diagnosis. Recently, many molecular markers have been investigated in an attempt to increase diagnostic accuracy of indeterminate fine needle aspiration cytology and thereby avoid unnecessary complications and costs associated with thyroid surgery. Somatic mutation testing, mRNA gene expression platforms, protein immunocytochemistry and miRNA panels have improved the diagnostic accuracy of indeterminate thyroid nodules, and although no test is perfectly accurate, in the authors' opinion, these methods will most certainly become an important part of the diagnostic tools for clinicians and cytopathologists in the future.

Full text available from Expert Review of Molecular Diagnostics (USD 89.00) PMID: 23895130

C) Association Between BRAF V600E Mutation and Mortality in Patients with Papillary Thyroid Cancer Xing M, Alzahrani AS, Carson KA, et al. Association between BRAF V600E mutation and mortality in patients with papillary thyroid cancer. JAMA. 2013; 309(14): 1493-1501.

Summary: BRAF V600E is a prominent oncogene in papillary thyroid cancer (PTC), but its role in PTC-related patient mortality has not been established. OBJECTIVE: To investigate the relationship between BRAF V600E mutation and PTC-related mortality. DESIGN, SETTING, AND PARTICIPANTS: Retrospective study of 1849 patients (1411 women and 438 men) with a median age of 46 years (interquartile range, 34-58 years) and an overall median follow-up time of 33 months (interquartile range, 13-67 months) after initial treatment at 13 centers in 7 countries between 1978 and 2011. MAIN OUTCOMES AND MEASURES: Patient deaths specifically caused by PTC. RESULTS: Overall, mortality was 5.3% (45/845; 95% CI, 3.9%-7.1%) vs 1.1% (11/1004; 95% CI, 0.5%-2.0%) (P < .001) in BRAF V600E-positive vs mutation-negative patients. Deaths per 1000 person-years in the analysis of all PTC were 12.87 (95% CI, 9.61-17.24) vs 2.52 (95% CI, 1.40-4.55) in BRAF V600E-positive vs mutation-negative patients; the hazard ratio (HR) was 2.66 (95% CI, 1.30-5.43) after adjustment for age at diagnosis, sex, and medical center. Deaths per 1000 person-years in the analysis of the conventional variant of PTC were 11.80 (95% CI, 8.39-16.60) vs 2.25 (95% CI, 1.01-5.00) in BRAF V600E-positive vs mutation-negative patients; the adjusted HR was 3.53 (95% CI, 1.25-9.98). When lymph node metastasis, extrathyroidal invasion, and distant metastasis were also included in the model, the association of BRAF V600E with mortality for all PTC was no longer significant (HR, 1.21; 95% CI, 0.53-2.76). A higher BRAF V600E- associated patient mortality was also observed in several clinicopathological subcategories, but statistical significance was lost with adjustment for patient age, sex, and medical center. For example, in patients with lymph node metastasis, the deaths per 1000 person-years were 26.26 (95% CI, 19.18- 35.94) vs 5.93 (95% CI, 2.96-11.86) in BRAF V600E-positive vs mutation- negative patients (unadjusted HR, 4.43 [95% CI, 2.06-9.51]; adjusted HR, 1.46 [95% CI, 0.62-3.47]). In patients with distant tumor metastasis, deaths per 1000 person-years were 87.72 (95% CI, 62.68-122.77) vs 32.28 (95% CI, 16.14-64.55) in BRAF V600E-positive vs mutation-negative patients (unadjusted HR, 2.63 [95% CI, 1.21-5.72]; adjusted HR, 0.84 [95% CI, 0.27- 2.62]). CONCLUSIONS AND RELEVANCE: In this retrospective multicenter study, the presence of the BRAF V600E mutation was significantly associated with increased cancer-related mortality among patients with PTC. Because overall mortality in PTC is low and the association was not independent of tumor features, how to use BRAF V600E to manage mortality

risk in patients with PTC is unclear. These findings support further investigation of the prognostic and therapeutic implications of BRAF V600E status in PTC.

Full text available from Journal of the American Medical Association (USD 30.00) PMID: 23571588

D) Molecular Diagnostics of Thyroid Tumors Nikiforov YE. Molecular diagnostics of thyroid tumors. Arch Pathol Lab Med. 2011; 135(5):569–577.

Summary: Thyroid cancer is the most common type of endocrine malignancy and its incidence is steadily increasing. Papillary carcinoma and follicular carcinoma are the most common types of thyroid cancer and represent those tumor types for which use of molecular markers for diagnosis and prognostication is of high clinical significance. Objective.—To review the most common molecular alterations in thyroid cancer and their diagnostic and prognostic utility. Data Sources.—PubMed (US National Library of Medicine)–available review articles, peer-reviewed original articles, and experience of the author. Conclusions.—The most common molecular alterations in thyroid cancer include BRAF and RAS point mutations and RET/PTC and PAX8/PPARγ rearrangements. These nonoverlapping genetic alterations are found in more than 70% of papillary and follicular thyroid carcinomas. These molecular alterations can be detected in surgically resected samples and fine-needle aspiration samples from thyroid nodules and can be of significant diagnostic use. The diagnostic role of BRAF mutations has been studied most extensively, and recent studies also demonstrated a significant diagnostic utility of RAS, RET/PTC, and PAX8/PPARγ mutations, particularly in thyroid fine-needle aspiration samples with indeterminate cytology. In addition to the diagnostic use, BRAF V600E mutation can also be used for tumor prognostication, as this mutation is associated with higher rate of tumor recurrence and tumor-related mortality. The use of these and other emerging molecular markers is expected to improve significantly the accuracy of cancer diagnosis in thyroid nodules and allow more individualized surgical and postsurgical management of patients with thyroid cancer.

Free full text available from the CAP’s Archives PMID: 21526955 NOTE: Also cited in Section 2.1.2

E) Association of TERT Promoter Mutation 1,295,228 C>T with BRAF V600E Mutation, Older Patient Age, and Distant Metastasis in Anaplastic Thyroid Cancer Shi X, Liu R, Qu S, et al. Association of TERT promoter mutation 1,295,228 C>T with BRAF V600E mutation, older patient age, and distant metastasis in anaplastic thyroid cancer. J Clin Endocrinol Metab. 2015; 100(4): E632-637.

Summary: CONTEXT: The aggressive role of TERT promoter mutations has been well established in differentiated thyroid cancer but has not been established in anaplastic thyroid cancer (ATC). RESEARCH DESIGN: We tested the mutation status by sequencing genomic tumor DNA and examined its relationship with clinicopathological characteristics of ATC. RESULTS: Among 106 American and Chinese ATC samples, TERT 1,295,228 C>T (termed TERT C228T) mutation was found in 37 (34.9%) cases, TERT promoter mutation 1,295,250 C>T was found in four cases (3.8%), and the two mutations were mutually exclusive and collectively found in 41 cases (38.7%). TERT C228T occurred in 28 of 90 (31.1%) wild-type BRAF cases vs nine of 16 (56.3%) BRAF V600E cases, with an odds ratio of 2.85 (95% confidence interval, 0.96-8.42; P = .05). Patient age was 67.6 +/- 13.6 vs 61.6 +/- 11.4 years in the TERT C228T vs wild-type TERT patients (P = .02), demonstrating an association between TERT C228T and older patient age. This association was also seen within the American cohort. In this cohort, which had more available clinicopathological data, TERT C228T was associated with distant metastasis of the tumor; specifically, distant metastasis occurred in 15 of 18 (83.3%) TERT C228T patients vs eight of 26 (30.8%) wild-type TERT patients, with an odds ratio of 11.25 (95% confidence interval, 2.53-50.08; P = .001). No association was found with patient sex, tumor size, lymph node metastasis, and extrathyroidal invasion of ATC. CONCLUSIONS: This is the largest study on the aggressive role of TERT promoter mutations in ATC, demonstrating an association of TERT C228T with BRAF V600E, older patient age, and tumor distant metastasis in ATC.

Full text available from The Journal of Clinical Endocrinology & Metabolism (subscription required) PMID: 25584719

F) Identification of the Transforming STRN-ALK Fusion as a Potential Therapeutic Target in the Aggressive Forms of Thyroid Cancer Kelly LM, Barila G, Liu P, et al. Identification of the transforming STRN-ALK fusion as a potential therapeutic target in the aggressive forms of thyroid cancer. Proc Natl Acad Sci U S A. 2014; 111(11): 4233-4238.

Summary: Thyroid cancer is a common endocrine malignancy that encompasses well-differentiated as well as dedifferentiated cancer types. The latter tumors have high mortality and lack effective therapies. Using a paired-end RNA-sequencing approach, we report the discovery of rearrangements involving the anaplastic lymphoma kinase (ALK) gene in thyroid cancer. The most common of these involves a fusion between ALK and the striatin (STRN) gene, which is the result of a complex rearrangement involving the short arm of chromosome 2. STRN-ALK leads to constitutive activation of ALK kinase via dimerization mediated by the coiled-coil domain of STRN and to a kinase-dependent, thyroid-stimulating hormone- independent proliferation of thyroid cells. Moreover, expression of STRN-ALK transforms cells in vitro and induces tumor formation in nude mice. The kinase activity of STRN-ALK and the ALK-induced cell growth can be blocked by the ALK inhibitors crizotinib and TAE684. In addition to well- differentiated papillary cancer, STRN-ALK was found with a higher prevalence in poorly differentiated and anaplastic thyroid cancers, and it did not overlap with other known driver mutations in these tumors. Our data demonstrate that STRN-ALK fusion occurs in a subset of patients with highly aggressive types of thyroid cancer and provide initial evidence suggesting that it may represent a therapeutic target for these patients.

Free full text available from PubMed PMID: 24613930

G) Molecular Pathogenesis and Mechanisms of Thyroid Cancer Xing M. Molecular pathogenesis and mechanisms of thyroid cancer. Nat Rev Cancer. 2013; 13(3): 184-199.

Summary: Thyroid cancer is a common endocrine malignancy. There has been exciting progress in understanding its molecular pathogenesis in recent years, as best exemplified by the elucidation of the fundamental role of several major signalling pathways and related molecular derangements. Central to these mechanisms are the genetic and epigenetic alterations in these pathways, such as mutation, gene copy-number gain and aberrant gene methylation. Many of these molecular alterations represent novel diagnostic and prognostic molecular markers and therapeutic targets for thyroid cancer, which provide unprecedented opportunities for further research and clinical development of novel treatment strategies for this cancer.

Free full text available from PubMed PMID: 23429735

H) Targeted Next-Generation Sequencing Panel (ThyroSeq) for Detection of Mutations in Thyroid Cancer Nikiforova MN, Wald AI, Roy S, Durso MB, Nikiforov YE. Targeted Next- Generation Sequencing Panel (ThyroSeq) for Detection of Mutations in Thyroid Cancer. J Clin Endocrinol Metab. 2013; 98(11): E1852-1860.

Summary: Objectives: Next-generation sequencing (NGS) allows for high- throughput sequencing analysis of large regions of the human genome. We explored the use of targeted NGS for simultaneous testing for multiple mutations in thyroid cancer. Design: A custom panel (ThyroSeq) was designed to target 12 cancer genes with 284 mutational hot spots. Sequencing was performed to analyze DNA from 228 thyroid neoplastic and nonneoplastic samples including 105 frozen, 72 formalin-fixed, and 51 fine- needle aspiration samples representing all major types of thyroid cancer. Results: Only 5-10 ng of input DNA was sufficient for successful analysis of 99.6% of samples. The analytical accuracy for mutation detection was 100% with the sensitivity of 3%-5% of mutant allele. ThyroSeq DNA assay identified mutations in 19 of 27 of classic papillary thyroid carcinomas (PTCs) (70%), 25 of 30 follicular variant PTCs (83%), 14 of 18 conventional (78%) and 7 of 18 oncocytic follicular carcinomas (39%), 3 of 10 poorly differentiated carcinomas (30%), 20 of 27 anaplastic (ATCs) (74%), and 11 of 15 medullary thyroid carcinomas (73%). In contrast, 5 of 83 benign nodules (6%) were positive for mutations. Most tumors had a single mutation, whereas several ATCs and PTCs demonstrated two or three mutations. The most common mutations detected were BRAF and RAS followed by PIK3CA, TP53, TSHR, PTEN, GNAS, CTNNB1, and RET. The BRAF mutant allele frequency was 18%-48% in PTCs and was lower in ATCs. Conclusions: The ThyroSeq NGS panel allows simultaneous testing for multiple mutations with high accuracy and sensitivity, requires a small amount of DNA and can be performed in a variety of thyroid tissue and fine-needle aspiration samples, and provides quantitative assessment of mutant alleles. Using this approach, the point mutations were detected in 30%-83% of specific types of thyroid cancer and in only 6% of benign thyroid nodules and were shown to be present in the majority of cells within the cancer nodule.

Free full text available from Journal of Clinical Endocrinology and Metabolism PMID: 23979959

2.7.2 Short Presentations on Emerging Concepts: Thyroid Cancer

CAP’s Pathology Short Presentations in Emerging Concepts (SPECs) are prewritten PowerPoint presentations on selected diseases where molecular tests

play a key role in patient management. To learn more about this resource, view Section 9.1 or the Resources & Publications tab on cap.org.

A) CAP Short Presentations on Emerging Concepts (SPECS): Emerging Concepts in the Diagnosis and Work-up of Thyroid Cancer [PowerPoint slides] Foo WC, Misialek MJ. CAP Short Presentations on Emerging Concepts (SPECS): Emerging Concepts in the Diagnosis and Work-up of Thyroid Cancer (v 4.0e rev 6/30/15) [PowerPoint slides]. Caughron SK, Nowak JA, Witte DL, eds. Northfield, IL: College of American Pathologists; 2015.

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2.8 Soft Tissue

A) Special Issue: Seminars in Surgical Oncology: Multidisciplinary Advances in Soft Tissue Sarcoma Kraybill WG, Pollock RE (eds). Special Issue: Seminars in Surgical Oncology: Multidisciplinary Advances in Soft Tissue Sarcoma. Journal of Surgical Oncology. 2015; 111(5): 489–655.

No summary available.

Full text available from Journal of Surgical Oncology

B) Sarcoma Diagnosis in the Age of Molecular Pathology Demicco EG. Sarcoma diagnosis in the age of molecular pathology. Adv Anat Pathol. 2013; 20(4): 264-274.

Summary: Mesenchymal neoplasia presents numerous challenges to pathologic classification. Histologic features can be deceiving, and traditional immunohistochemical markers of differentiation may be of little use in narrowing the diagnosis. Fortunately, great strides have been made in unraveling the genetic and genomic alterations associated with both sarcomagenesis and benign neoplasia. In turn, these advances have led to an expansion of the available diagnostic toolkit for sarcoma pathology. In order to assist the practicing pathologist in integrating these tools into their repertoire, this article will discuss some of the latest advances in sarcoma diagnosis, including an update on translocation-associated sarcomas, and will review a number of sarcoma-specific immunohistochemical studies

developed over the past decade. Some of the potential uses and pitfalls of commonly used tests will be addressed. Finally, the discussion will briefly touch upon the impact that advances in molecular technologies, particularly targeted gene expression analysis, may have on altering the face of diagnostic pathology.

Full text available from Advances in Anatomic Pathology (subscription required) PMID: 23752088

C) Molecular Diagnostics of Soft Tissue Tumors Bridge JA, Cushman-Vokoun AM. Molecular diagnostics of soft tissue tumors. Arch Pathol Lab Med. 2011; 135(5):588–601.

Summary: Soft tissue pathology encompasses a remarkably diverse assortment of benign and malignant soft tissue tumors. Rendering a definitive diagnosis is complicated not only by the large volume of existing histologic subtypes (>100) but also frequently by the presence of overlapping clinical, histologic, immunohistochemical, and/or radiographic features. During the past 3 decades, mesenchymal tumor–specific, cytogenetic and molecular genetic abnormalities have demonstrated an increasingly important, ancillary role in mesenchymal tumor diagnostics. Objectives.—To review molecular diagnostic tools available to the pathologist to further classify specific soft tissue tumor types and recurrent aberrations frequently examined. Advantages and limitations of individual approaches will also be highlighted. Data Sources.—Previously published review articles, peer- reviewed research publications, and the extensive cytogenetic and molecular diagnostic experience of the authors to include case files of The University of Nebraska Medical Center. Conclusions.—Cytogenetic and molecular genetic assays are used routinely for diagnostic purposes in soft tissue pathology and represent a powerful adjunct to complement conventional microscopy and clinicoradiographic evaluation in the formulation of an accurate diagnosis. Care should be taken, however, to recognize the limitations of these approaches. Ideally, more than one technical approach should be available to a diagnostic laboratory to compensate for the shortcomings of each approach in the assessment of individual specimens.

Free full text available from CAP’s Archives PMID: 21526957

2.9 Head & Neck

A) An Introduction to Molecular Pathology of the Head and Neck Hunt JL. An introduction to molecular pathology of the head and neck. Arch Pathol Lab Med. 2014; 138(6): 730.

No summary available

Free full text available from the CAP’s Archives PMID: 24878012

B) Molecular Pathology of Skin Neoplasms of the Head and Neck Kraft S, Granter SR. Molecular pathology of skin neoplasms of the head and neck. Arch Pathol Lab Med. 2014; 138(6): 759-787.

Summary: CONTEXT: Skin neoplasms include the most common malignancies affecting humans. Many show an ultraviolet (UV)-induced pathogenesis and often affect the head and neck region. OBJECTIVE: To review literature on cutaneous neoplasms that show a predilection for the head and neck region and that are associated with molecular alterations. DATA SOURCES: Literature review. CONCLUSIONS: Common nonmelanoma skin cancers, such as basal and squamous cell carcinomas, show a UV-induced pathogenesis. Basal cell carcinomas are characterized by molecular alterations of the Hedgehog pathway, affecting patched and smoothened genes. While squamous cell carcinomas show UV-induced mutations in several genes, driver mutations are only beginning to be identified. In addition, certain adnexal neoplasms also predominantly affect the head and neck region and show interesting, recently discovered molecular abnormalities, or are associated with hereditary conditions whose molecular genetic pathogenesis is well understood. Furthermore, recent advances have led to an increased understanding of the molecular pathogenesis of melanoma. Certain melanoma subtypes, such as lentigo maligna melanoma and desmoplastic melanoma, which are more often seen on the chronically sun-damaged skin of the head and neck, show differences in their molecular signature when compared to the other more common subtypes, such as superficial spreading melanoma, which are more prone to occur at sites with acute intermittent sun damage. In summary, molecular alterations in cutaneous neoplasms of the head and neck are often related to UV exposure. Their molecular footprint often reflects the histologic tumor type, and familiarity with these changes will be increasingly necessary for diagnostic and therapeutic considerations.

Free full text available from the CAP’s Archives

PMID: 24878016

C) Comprehensive Genomic Characterization of Head and Neck Squamous Cell Carcinomas Cancer Genome Atlas Network. Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature. 2015; 517(7536):576-82.

Summary: The Cancer Genome Atlas profiled 279 head and neck squamous cell carcinomas (HNSCCs) to provide a comprehensive landscape of somatic genomic alterations. Here we show that human-papillomavirus- associated tumours are dominated by helical domain mutations of the oncogene PIK3CA, novel alterations involving loss of TRAF3, and amplification of the cell cycle gene E2F1. Smoking-related HNSCCs demonstrate near universal loss-of-function TP53 mutations and CDKN2A inactivation with frequent copy number alterations including amplification of 3q26/28 and 11q13/22. A subgroup of oral cavity tumours with favourable clinical outcomes displayed infrequent copy number alterations in conjunction with activating mutations of HRAS or PIK3CA, coupled with inactivating mutations of CASP8, NOTCH1 and TP53. Other distinct subgroups contained loss-of-function alterations of the chromatin modifier NSD1, WNT pathway genes AJUBA and FAT1, and activation of oxidative stress factor NFE2L2, mainly in laryngeal tumours. Therapeutic candidate alterations were identified in most HNSCCs.

Free full text available from Nature PMID: 25631445

D) Nasopharyngeal Cancer: Molecular Landscape Bruce JP, Yip K, Bratman SV, Ito E, Liu FF. Nasopharyngeal Cancer: Molecular Landscape. J Clin Oncol. 2015; 33(29): 3346-3355.

Summary: Nasopharyngeal carcinoma (NPC) is a unique epithelial malignancy arising from the superior aspect of the pharyngeal mucosal space, associated with latent Epstein-Barr virus infection in most cases. The capacity to characterize cancer genomes in unprecedented detail is now providing insights into the genesis and molecular underpinnings of this disease. Herein, we provide an overview of the molecular aberrations that likely drive nasopharyngeal tumor development and progression. The contributions of major Epstein-Barr virus-encoded factors, including proteins, small RNAs, and microRNAs, along with their interactions with pathways regulating cell proliferation and survival are highlighted. We review recent analyses that clearly define the role of genetic and epigenetic variations affecting the human genome in NPC. These findings point to the impact of DNA methylation and histone modifications on gene expression programs

that promote this malignancy. The molecular interactions that allow NPC cells to evade immune recognition and elimination, which is crucial for the survival of cells expressing potentially immunogenic viral proteins, are also described. Finally, the potential utility of detecting host and viral factors for the diagnosis and prognosis of NPC is discussed. Altogether, the studies summarized herein have greatly expanded our knowledge of the molecular biology of NPC, yet much remains to be uncovered. Emerging techniques for using and analyzing well-annotated biospecimens from patients with NPC will ultimately lead to a greater level of understanding, and enable improvements in precision therapies and clinical outcomes.

Full text available from Journal of Clinical Oncology (USD 30.00) PMID: 26351340

E) The Tip of the Iceberg: Clinical Implications of Genomic Sequencing Projects in Head and Neck Cancer Birkeland AC, Ludwig ML, Meraj TS, Brenner JC,Prince ME. The Tip of the Iceberg: Clinical Implications of Genomic Sequencing Projects in Head and Neck Cancer. Cancers (Basel). 2015; 7(4): 2094-2109.

Summary: Recent genomic sequencing studies have provided valuable insight into genetic aberrations in head and neck squamous cell carcinoma. Despite these great advances, certain hurdles exist in translating genomic findings to clinical care. Further correlation of genetic findings to clinical outcomes, additional analyses of subgroups of head and neck cancers and follow-up investigation into genetic heterogeneity are needed. While the development of targeted therapy trials is of key importance, numerous challenges exist in establishing and optimizing such programs. This review discusses potential upcoming steps for further genetic evaluation of head and neck cancers and implementation of genetic findings into precision medicine trials.

Free full text available from PubMed PMID: 26506389

F) Emerging Biomarkers in Head and Neck Cancer in the Era of Genomics Kang H, Kiess A,Chung CH. Emerging Biomarkers in Head and Neck Cancer in the Era of Genomics. Nat Rev Clin Oncol. 2015; 12(1): 11-26.

Summary: Head and neck cancer (HNC) broadly includes carcinomas arising from the mucosal epithelia of the head and neck region as well as various cell types of salivary glands and the thyroid. As reflected by the multiple sites and histologies of HNC, the molecular characteristics and clinical outcomes of this disease vary widely. In this Review, we focus on established and emerging biomarkers that are most relevant to

nasopharyngeal carcinoma and head and neck squamous-cell carcinoma (HNSCC), which includes primary sites in the oral cavity, oropharynx, hypopharynx and larynx. Applications and limitations of currently established biomarkers are discussed along with examples of successful biomarker development. For emerging biomarkers, preclinical or retrospective data are also described in the context of recently completed comprehensive molecular analyses of HNSCC, which provide a broad genetic landscape and molecular classification beyond histology and clinical characteristics. We will highlight the ongoing effort that will see a shift from prognostic to predictive biomarker development in HNC with the goal of delivering individualized cancer therapy.

Full text available from Nature Reviews Clinical Oncology (USD 9.99) PMID: 25403939

G) The Head and Neck Cancer Cell Oncogenome: A Platform for the Development of Precision Molecular Therapies Martin D, Abba MC, Molinolo AA, et al. The Head and Neck Cancer Cell Oncogenome: A Platform for the Development of Precision Molecular Therapies. Oncotarget. 2014; 5(19): 8906-8923.

Summary: The recent elucidation of the genomic landscape of head and neck squamous cell carcinoma (HNSCC) has provided a unique opportunity to develop selective cancer treatment options. These efforts will require the establishment of relevant HNSCC models for preclinical testing. Here, we performed full exome and transcriptome sequencing of a large panel of HNSCC-derived cells from different anatomical locations and human papillomavirus (HPV) infection status. These cells exhibit typical mutations in TP53, FAT1, CDK2NA, CASP8, and NOTCH1, and copy number variations (CNVs) and mutations in PIK3CA, HRAS, and PTEN that reflect the widespread activation of the PI3K-mTOR pathway. SMAD4 alterations were observed that may explain the decreased tumor suppressive effect of TGF- beta in HNSCC. Surprisingly, we identified HPV+ HNSCC cells harboring TP53 mutations, and documented aberrant TP53 expression in a subset of HPV+ HNSCC cases. This analysis also revealed that most HNSCC cells harbor multiple mutations and CNVs in epigenetic modifiers (e.g., EP300, CREBP, MLL1, MLL2, MLL3, KDM6A, and KDM6B) that may contribute to HNSCC initiation and progression. These genetically-defined experimental HNSCC cellular systems, together with the identification of novel actionable molecular targets, may now facilitate the pre-clinical evaluation of emerging therapeutic agents in tumors exhibiting each precise genomic alteration.

Free full text available from PubMed PMID: 25275298

H) Translational Genomics and Head and Neck Cancer: Toward Precision Medicine Razzouk S. Translational Genomics and Head and Neck Cancer: Toward Precision Medicine. Clin Genet. 2014; 86(5): 412-421.

Summary: Head and neck squamous cell carcinoma (HNSCC) comprise a wide spectrum of neoplasms with different tumor biologies, prognosis and response to therapies. Current tumor classification and traditional diagnostic methods (e.g. clinical assessment, histopathology) are limited in their capacity to determine prognosis and clinical decision-making. Despite recent improvements in treatment, the outcome for patients with HNSCC remains poor. Similar to most tumors, several patient-related factors, (e.g. genetics and environment) and disease-related factors (e.g. tumor location, TMN staging) play a significant role on survival. Thus, the problem in defining the prognosis is that the clinical course and response to treatment differ considerably among patients. Such interindividual variability is related to the heterogeneity of the tumor, genetic and epigenetic variations, thus reflecting the interaction of multiple biological components that result in a unique phenotype. Integrative genomics are developed to identify the molecular pathways leading to cancer at the individual level and find novel prognostic markers for HNSCC, hence tailoring a treatment accordingly. Such genetic- based personalized diagnosis allows tumor stratification and implementation of targeted therapy. Modern medicine includes new drugs that disrupt the implicated molecules and their signaling pathways. Here, we summarize the current state of knowledge that elucidates the translation of genetic data into clinical benefit.

Full text available from Clinical Genetics (USD 6.00-38.00) PMID: 25143247

I) An Overview of Molecular and Genetic Alterations in Selected Benign Odontogenic Disorders Cabay RJ. An overview of molecular and genetic alterations in selected benign odontogenic disorders. Arch Pathol Lab Med. 2014; 138(6): 754-758.

Summary: CONTEXT: Some dental abnormalities have environmental causes. Other odontogenic alterations are idiopathic and may have hereditary etiologies. Investigations of these conditions are ongoing. OBJECTIVE: To provide a discussion of developmental odontogenic abnormalities and benign odontogenic overgrowths and neoplasms for which genetic alterations have been well demonstrated and well documented. DATA SOURCES: Relevant peer-reviewed literature. CONCLUSIONS: The understanding of benign odontogenic lesions at a molecular level is rather well developed for some lesions and at the initial stages for many others.

Further characterization of the molecular underpinnings of these and other odontogenic lesions would result in an enhanced comprehension of odontogenesis and the pathogenesis of a variety of odontogenic aberrations. These advancements may lead to better prevention and treatment paradigms and improved patient outcomes.

Free full text available from the CAP’s Archives PMID: 24878015

2.10 Genitourinary Cancer

This section includes articles on the following topics:

• Urothelial and Renal (Section 2.10.1); and • Female Genitourinary (Section 2.10.2); and • Male Genitourinary (Section 2.10.3).

2.10.1 Urothelial and Renal

A) Intratumour Heterogeneity in Urologic Cancers: From Molecular Evidence to Clinical Implications Gerlinger M, Catto JW, Orntoft TF, Real FX, Zwarthoff EC, Swanton C. Intratumour Heterogeneity in Urologic Cancers: From Molecular Evidence to Clinical Implications. Eur Urol. 2015; 67(4): 729-737.

Summary: CONTEXT: Intratumour heterogeneity (ITH) can impair the precise molecular analysis of tumours and may contribute to difficulties encountered in cancer biomarker qualification and treatment personalisation. OBJECTIVE: This review summarises the evidence for genetic ITH in renal, bladder, and prostate carcinomas and potential strategies to address the clinical and translational research challenges arising from ITH. EVIDENCE ACQUISITION: Publications that assessed ITH in the relevant urologic cancers were identified in a literature review. EVIDENCE SYNTHESIS: ITH with functionally distinct tumour subclones has been identified in all three tumour types. Heterogeneity of actionable genetic changes and of prognostic biomarkers between different tumour regions in the same patient suggests limitations of single biopsy-based molecular analyses for precision medicine approaches. Evolutionary constraints may differ between patients and may allow the prediction of specific evolutionary trajectories. CONCLUSIONS: Assessment of multiple tumour regions for precision medicine purposes, monitoring of subclonal dynamics over time, and the preferential targeting of

genetic alterations located on the trunk of the phylogenetic tree of individual cancers may accelerate the development of personalised medicine strategies and improve our understanding of treatment failure. PATIENT SUMMARY: Genetic alterations can be heterogeneous within urologic tumours, complicating their use as biomarkers for treatment personalisation. We present novel strategies to address these challenges.

Full text available from European Urology (USD 35.95) PMID: 24836153

B) Precision Medicine for Metastatic Renal Cell Carcinoma Sonpavde G, Choueiri TK. Precision Medicine for Metastatic Renal Cell Carcinoma. Urol Oncol. 2014; 32(1): 5-15.

Summary: OBJECTIVES: This review provides a broad overview of emerging data that provide hope that rational precision medicine for metastatic renal cell carcinoma (RCC) may be possible. METHODS: PubMed and major conferences were searched for studies reporting potential predictive biomarkers for the therapy of metastatic RCC. RESULTS: The availability of multiple new agents for the therapy of advanced RCC poses new challenges in terms of optimal selection of patients for the appropriate drug. Prognostic stratification based on routine histopathologic, clinical and laboratory factors have been utilized to broadly select individuals based, i.e. high-dose interleukin (IL)-2 or vascular endothelial growth factor (VEGF) inhibitors for good and intermediate risk patients and temsirolimus for poor risk patients. While multiple candidate predictive molecular biomarkers suggest that rational selection of patients for high-dose interleukin (IL)-2, and VEGF and mammalian target of rapamycin (mTOR) inhibitors may be possible, none have been validated for use in the clinic. Tumor heterogeneity and standardization of tissue collection and analysis are massive challenges that need to be addressed. Predictive molecules derived from tumor tissue, plasma and host tissue may all be predictive for therapeutic benefit. Moreover, gene expression may be modulated by multiple factors including epigenetics, transcription factors and post-transcriptional and post- translational modifications. Indeed, study of the interaction of molecular factors from all of these sources with environmental and clinical factors may be necessary to develop a unified profile composed of a panel of factors predictive of benefit from specific agents (i.e. sustained response, limited toxicity and overall a positive benefit/risk ratio). CONCLUSIONS: Conducting clinical trials with 1) prospective incorporation of promising candidate predictive molecular biomarkers, 2) novel biomarkers endpoints, and 3) mandatory biopsies of metastatic sites at different time points on therapy, are potential important steps in developing the concept of "the right medication for the right patient".

Full text available from Urologic Oncology (USD 31.50) PMID: 24239472

C) Comprehensive Molecular Characterization of Clear Cell Renal Cell Carcinoma The Cancer Genome Atlas Research Network. Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature. 2013; 499(7456): 43-49.

Summary: Genetic changes underlying clear cell renal cell carcinoma (ccRCC) include alterations in genes controlling cellular oxygen sensing (for example, VHL) and the maintenance of chromatin states (for example, PBRM1). We surveyed more than 400 tumours using different genomic platforms and identified 19 significantly mutated genes. The PI(3)K/AKT pathway was recurrently mutated, suggesting this pathway as a potential therapeutic target. Widespread DNA hypomethylation was associated with mutation of the H3K36 methyltransferase SETD2, and integrative analysis suggested that mutations involving the SWI/SNF chromatin remodelling complex (PBRM1, ARID1A, SMARCA4) could have far-reaching effects on other pathways. Aggressive cancers demonstrated evidence of a metabolic shift, involving downregulation of genes involved in the TCA cycle, decreased AMPK and PTEN protein levels, upregulation of the pentose phosphate pathway and the glutamine transporter genes, increased acetyl-CoA carboxylase protein, and altered promoter methylation of miR-21 (also known as MIR21) and GRB10. Remodelling cellular metabolism thus constitutes a recurrent pattern in ccRCC that correlates with tumour stage and severity and offers new views on the opportunities for disease treatment.

Free full text available from PubMed PMID: 23792563

2.10.2 Female Genitourinary

A) Distinct Evolutionary Trajectories of Primary High-Grade Serous Ovarian Cancers Revealed Through Spatial Mutational Profiling Bashashati A, Ha G, Tone A, et al. Distinct evolutionary trajectories of primary high-grade serous ovarian cancers revealed through spatial mutational profiling. J Pathol. 2013; 231(1): 21-34.

Summary: High-grade serous ovarian cancer (HGSC) is characterized by poor outcome, often attributed to the emergence of treatment-resistant subclones. We sought to measure the degree of genomic diversity within

primary, untreated HGSCs to examine the natural state of tumour evolution prior to therapy. We performed exome sequencing, copy number analysis, targeted amplicon deep sequencing and gene expression profiling on 31 spatially and temporally separated HGSC tumour specimens (six patients), including ovarian masses, distant metastases and fallopian tube lesions. We found widespread intratumoural variation in mutation, copy number and gene expression profiles, with key driver alterations in genes present in only a subset of samples (eg PIK3CA, CTNNB1, NF1). On average, only 51.5% of mutations were present in every sample of a given case (range 10.2-91.4%), with TP53 as the only somatic mutation consistently present in all samples. Complex segmental aneuploidies, such as whole-genome doubling, were present in a subset of samples from the same individual, with divergent copy number changes segregating independently of point mutation acquisition. Reconstruction of evolutionary histories showed one patient with mixed HGSC and endometrioid histology, with common aetiologic origin in the fallopian tube and subsequent selection of different driver mutations in the histologically distinct samples. In this patient, we observed mixed cell populations in the early fallopian tube lesion, indicating that diversity arises at early stages of tumourigenesis. Our results revealed that HGSCs exhibit highly individual evolutionary trajectories and diverse genomic tapestries prior to therapy, exposing an essential biological characteristic to inform future design of personalized therapeutic solutions and investigation of drug- resistance mechanisms.

Free full text available from PubMed PMID: 23780408

B) Comprehensive Genomic Profiling of Epithelial Ovarian Cancer by Next Generation Sequencing-Based Diagnostic Assay Reveals New Routes to Targeted Therapies Ross JS, Ali SM, Wang K, et al. Comprehensive genomic profiling of epithelial ovarian cancer by next generation sequencing-based diagnostic assay reveals new routes to targeted therapies. Gynecol Oncol. 2013; 130(3): 554-559.

Summary: OBJECTIVE: Targeted next generation sequencing (NGS) was evaluated for its ability to identify unanticipated targetable genomic alterations (GA) for patients with relapsed ovarian epithelial carcinoma (OC). METHODS: DNA sequencing was performed for 3320 exons of 182 cancer- related genes and 37 introns of 14 genes frequently rearranged in cancer on indexed, adaptor ligated, hybridization-captured libraries using DNA isolated from FFPE sections from 48 histologically verified relapsed OC specimens. The original primary tumor was sequenced in 26 (54%) of the cases and

recurrent/metastatic tumor site biopsies were sequenced in 22 (46%) of the cases. Actionability was defined as: GA that predict sensitivity or resistance to approved or standard therapies or are inclusion or exclusion criteria for specific experimental therapies in NCI registered clinical trials. RESULTS: There were 38 (80%) serous, 5 (10%) endometrioid, 3 (6%) clear cell, 1 mucinous (2%) and 1 (2%) undifferentiated carcinomas. 141 GA were identified with an average of 2.9 GA (range 0-8) per tumor, of which 67 were actionable for an average of 1.4 actionable GA per patient (range 0-5). 33/48 (69%) of OC patient samples harbored at least one actionable GA. Most common GA were TP53 (79%); MYC (25%); BRCA1/2 (23%); KRAS (16.6%) and NF1 (14.5%). One tumor featured an ERBB2 point mutation. One of 3 (33%) of clear cell tumors featured cMET amplification validated by both FISH and IHC. CONCLUSIONS: NGS assessment of therapy resistant OC identifies an unexpectedly high frequency of GA that could influence targeted therapy selection for the disease.

Free full text available from Gynecologic Oncology PMID: 23791828

C) Mutations in 12 Genes for Inherited Ovarian, Fallopian Tube, and Peritoneal Carcinoma Identified by Massively Parallel Sequencing Walsh T, Casadei S, Lee MK, et al. Mutations in 12 Genes for Inherited Ovarian, Fallopian Tube, and Peritoneal Carcinoma Identified by Massively Parallel Sequencing. Proc Natl Acad Sci U S A. 2011; 108(44): 18032-18037.

Summary: Inherited loss-of-function mutations in BRCA1 and BRCA2 and other tumor suppressor genes predispose to ovarian carcinomas, but the overall burden of disease due to inherited mutations is not known. Using targeted capture and massively parallel genomic sequencing, we screened for germ-line mutations in 21 tumor suppressor genes in genomic DNA from women with primary ovarian, peritoneal, or fallopian tube carcinoma. Subjects were consecutively enrolled at diagnosis and not selected for age or family history. All classes of mutations, including point mutations and large genomic deletions and insertions, were detected. Of 360 subjects, 24% carried germ-line loss-of-function mutations: 18% in BRCA1 or BRCA2 and 6% in BARD1, BRIP1, CHEK2, MRE11A, MSH6, NBN, PALB2, RAD50, RAD51C, or TP53. Six of these genes were not previously implicated in inherited ovarian carcinoma. Primary carcinomas were generally characterized by genomic loss of normal alleles of the mutant genes. Of women with inherited mutations, >30% had no family history of breast or ovarian carcinoma, and >35% were 60 y or older at diagnosis. More patients with ovarian carcinoma carry cancer-predisposing mutations and in more genes than previously appreciated. Comprehensive genetic testing for inherited carcinoma is warranted for all women with ovarian, peritoneal, or

fallopian tube carcinoma, regardless of age or family history. Clinical genetic testing is currently done gene by gene, with each test costing thousands of dollars. In contrast, massively parallel sequencing allows such testing for many genes simultaneously at low cost.

Free full text available from PubMed PMID: 22006311

D) Characterization of Uterine Leiomyomas by Whole-Genome Sequencing Mehine M, Kaasinen E, Makinen N, et al. Characterization of uterine leiomyomas by whole-genome sequencing. N Engl J Med. 2013; 369(1): 43- 53.

Summary: BACKGROUND: Uterine leiomyomas are benign but affect the health of millions of women. A better understanding of the molecular mechanisms involved may provide clues to the prevention and treatment of these lesions. METHODS: We performed whole-genome sequencing and gene-expression profiling of 38 uterine leiomyomas and the corresponding myometrium from 30 women. RESULTS: Identical variants observed in some separate tumor nodules suggested that these nodules have a common origin. Complex chromosomal rearrangements resembling chromothripsis were a common feature of leiomyomas. These rearrangements are best explained by a single event of multiple chromosomal breaks and random reassembly. The rearrangements created tissue-specific changes consistent with a role in the initiation of leiomyoma, such as translocations of the HMGA2 and RAD51B loci and aberrations at the COL4A5-COL4A6 locus, and occurred in the presence of normal TP53 alleles. In some cases, separate events had occurred more than once in single tumor-cell lineages. CONCLUSIONS: Chromosome shattering and reassembly resembling chromothripsis (a single genomic event that results in focal losses and rearrangements in multiple genomic regions) is a major cause of chromosomal abnormalities in uterine leiomyomas; we propose that tumorigenesis occurs when tissue-specific tumor-promoting changes are formed through these events. Chromothripsis has previously been associated with aggressive cancer; its common occurrence in leiomyomas suggests that it also has a role in the genesis and progression of benign tumors. We observed that multiple separate tumors could be seeded from a single lineage of uterine leiomyoma cells. (Funded by the Academy of Finland Center of Excellence program and others.).

Free full text available from New England Journal of Medicine PMID: 23738515

E) Integrated Genomic Characterization of Endometrial Carcinoma The Cancer Genome Atlas Research Network, Kandoth C, Schultz N, et al. Integrated genomic characterization of endometrial carcinoma. Nature. 2013; 497(7447): 67-73.

Summary: We performed an integrated genomic, transcriptomic and proteomic characterization of 373 endometrial carcinomas using array- and sequencing-based technologies. Uterine serous tumours and approximately 25% of high-grade endometrioid tumours had extensive copy number alterations, few DNA methylation changes, low oestrogen receptor/progesterone receptor levels, and frequent TP53 mutations. Most endometrioid tumours had few copy number alterations or TP53 mutations, but frequent mutations in PTEN, CTNNB1, PIK3CA, ARID1A and KRAS and novel mutations in the SWI/SNF chromatin remodelling complex gene ARID5B. A subset of endometrioid tumours that we identified had a markedly increased transversion mutation frequency and newly identified hotspot mutations in POLE. Our results classified endometrial cancers into four categories: POLE ultramutated, microsatellite instability hypermutated, copy- number low, and copy-number high. Uterine serous carcinomas share genomic features with ovarian serous and basal-like breast carcinomas. We demonstrated that the genomic features of endometrial carcinomas permit a reclassification that may affect post-surgical adjuvant treatment for women with aggressive tumours.

Free full text available from PubMed PMID: 23636398

2.10.3 Male Genitourinary

A) A Prospective Investigation of PTEN Loss and Erg Expression in Lethal Prostate Cancer Ahearn TU, Pettersson A, Ebot EM, et al. A Prospective Investigation of PTEN Loss and Erg Expression in Lethal Prostate Cancer. J Natl Cancer Inst. 2016; 108(2):

Summary: BACKGROUND: PTEN is a tumor suppressor frequently deleted in prostate cancer that may be a useful prognostic biomarker. However, the association of PTEN loss with lethal disease has not been tested in a large, predominantly surgically treated cohort. METHODS: In the Health Professionals Follow-up Study and Physicians' Health Study, we followed 1044 incident prostate cancer cases diagnosed between 1986 and 2009 for cancer-specific and all-cause mortality. A genetically validated PTEN

immunohistochemistry (IHC) assay was performed on tissue microarrays (TMAs). TMPRSS2:ERG status was previously assessed in a subset of cases by a genetically validated IHC assay for ERG. Cox proportional hazards models adjusting for age and body mass index at diagnosis, Gleason grade, and clinical or pathologic TNM stage were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for the association with lethal disease. All statistical tests were two-sided. RESULTS: On average, men were followed 11.7 years, during which there were 81 lethal events. Sixteen percent of cases had complete PTEN loss in all TMA cores and 9% had heterogeneous PTEN loss across cores. After adjustment for clinical-pathologic variables, complete PTEN loss was associated with lethal progression (HR = 1.8, 95% CI = 1.2 to 2.9). The association of PTEN loss (complete or heterogeneous) with lethal progression was only among men with ERG-negative (HR = 3.1, 95% CI = 1.7 to 5.7) but not ERG-positive (HR = 1.2, 95% CI = 0.7 to 2.2) tumors. CONCLUSIONS: PTEN loss is independently associated with increased risk of lethal progression, particularly in the ERG fusion-negative subgroup. These validated and inexpensive IHC assays may be useful for risk stratification in prostate cancer.

Full text available from Journal of the National Cancer Institute (USD 40.00 for 24 hour access) PMID: 26615022

B) Advancing Precision Medicine for Prostate Cancer Through Genomics Roychowdhury S, Chinnaiyan AM. Advancing precision medicine for prostate cancer through genomics. J Clin Oncol. 2013; 31(15): 1866-1873.

Summary: Prostate cancer is the most common type of cancer in men and the second leading cause of cancer death in men in the United States. The recent surge of high-throughput sequencing of cancer genomes has supported an expanding molecular classification of prostate cancer. Translation of these basic science studies into clinically valuable biomarkers for diagnosis and prognosis and biomarkers that are predictive for therapy is critical to the development of precision medicine in prostate cancer. We review potential applications aimed at improving screening specificity in prostate cancer and differentiating aggressive versus indolent prostate cancers. Furthermore, we review predictive biomarker candidates involving ETS gene rearrangements, PTEN inactivation, and androgen receptor signaling. These and other putative biomarkers may signify aberrant oncogene pathway activation and provide a rationale for matching patients with molecularly targeted therapies in clinical trials. Lastly, we advocate innovations for clinical trial design to incorporate tumor biopsy and molecular

characterization to develop biomarkers and understand mechanisms of resistance.

Full text available from Journal of Clinical Oncology (subscription required) PMID: 23589550

2.11 Cancer of Unknown Primary Origin

A) Cancer of Unknown Primary Origin in the Genomic Era: Elucidating the Dark Box of Cancer Economopoulou P, Mountzios G, Pavlidis N, Pentheroudakis G. Cancer of Unknown Primary Origin in the Genomic Era: Elucidating the Dark Box of Cancer. Cancer Treat Rev. 2015; 41(7): 598-604.

Summary: Cancer of Unknown Primary (CUP) comprises a heterogeneous disease group with diagnosis of metastatic malignancy in the absence of an identifiable primary site after diagnostic work up. CUP may either resemble a specific primary tumor site sharing common clinicopathological characteristics and prognosis, or present as a distinct disease entity with undifferentiated pathological features, usually bearing dismal prognosis. Diagnosis and management have traditionally been based on clinicopathological characteristics and therapeutic strategies have been mainly empirical. In the last decade, the advent of massive gene sequencing and the advances in genomic technologies have shed light on the genomic landscape of CUP. Several gene panel tests are currently commercially available and are used in an effort to correlate the genomic characteristics of a specific CUP tumor to those of a known primary tumor, guiding thus therapeutic management. Nevertheless, these efforts are hampered by the rarity of CUP and the inability to validate the results of such tests due to the paucity of randomized clinical trials. In the current work, we provide an overview of CUP with emphasis on the impact of the genome sequencing technologies on diagnosis and management of these tumors. We also discuss potential implications of genomics for the future treatment of CUP and address the challenges of the implementation of these therapeutic strategies in routine clinical practice.

Full text available from Cancer Treatment Reviews (USD 31.50) PMID: 26033502

B) Comprehensive Tumor Profiling Identifies Numerous Biomarkers of Drug Response in Cancers of Unknown Primary Site: Analysis of 1806 Cases Gatalica Z, Millis SZ, Vranic S, et al. Comprehensive Tumor Profiling Identifies Numerous Biomarkers of Drug Response in Cancers of Unknown Primary Site: Analysis of 1806 Cases. Oncotarget. 2014; 5(23): 12440- 12447.

Summary: BACKGROUND: Cancer of unknown primary (CUP) accounts for approximately 3% of all malignancies. Despite extensive laboratory and imaging efforts, the primary site usually cannot be unequivocally confirmed, and the treatment for the most part remains empirical. Recently, identification of common cancer pathway alterations in diverse cancer lineages has offered an opportunity to provide targeted therapies for patients with CUP, irrespective of the primary site. PATIENTS AND METHODS: 1806 cancers of unknown primary were identified among more than 63,000 cases profiled at Caris Life Sciences. Multiplatform profiling of the tumor samples included immunohistochemistry, gene sequencing and in situ hybridization methods in an effort to identify changes in biomarkers that are predictive of drug responses. RESULTS: Biomarkers associated with a potential drug benefit were identified in 96% of cases. Biomarkers identified included those associated with potential benefit in nearly all classes of approved cancer drugs (cytotoxic, hormonal, targeted biological drugs). Additionally, biomarkers associated with a potential lack of benefit were identified in numerous cases, which could further refine the management of patients with CUP. CONCLUSION: Comprehensive biomarker profiling of CUP may provide additional choices in treatment of patients with these difficult to treat malignancies.

Free full text available from PubMed PMID: 25415047

C) Gene Expression Profiling in Patients with Carcinoma of Unknown Primary Site: From Translational Research to Standard of Care Hainsworth JD,Greco FA. Gene Expression Profiling in Patients with Carcinoma of Unknown Primary Site: From Translational Research to Standard of Care. Virchows Arch. 2014; 464(4): 393-402.

Summary: Carcinoma of unknown primary site (CUP) is diagnosed in approximately 3 % of patients with advanced cancer, and most patients have traditionally been treated with empiric chemotherapy. As treatments improve and become more specific for individual solid tumor types, therapy with a single empiric combination chemotherapy regimen becomes increasingly inadequate. Gene expression profiling (GEP) is a new diagnostic method that allows prediction of the site of tumor origin based on gene expression

patterns retained from the normal tissues of origin. In blinded studies in tumors of known origin, GEP assays correctly identified the site of origin in 85 % of cases and compares favorably with immunohistochemical (IHC) staining. In patients with CUP, GEP is able to predict a site of origin in >95 % of patients versus 35-55 % for IHC staining. Although confirmation of the accuracy of these predictions is difficult, the diagnoses made by IHC staining and GEP are identical in 77 % of cases when IHC staining predicts a single primary site. GEP diagnoses appear to be most useful when IHC staining is inconclusive. Site-specific treatment of CUP patients based on GEP and/or IHC predictions appears to improve overall outcomes; patients predicted to have treatment-sensitive tumor types derived the most benefit. GEP adds to the diagnostic evaluation of patients with CUP and should be included when IHC staining is unable to predict a single site of origin. Site-specific treatment, based on tissue of origin diagnosis, should replace empiric chemotherapy in patients with CUP.

Full text available from Virchows Archiv (USD 39.95) PMID: 24487792

D) Massively-Parallel Sequencing Assists the Diagnosis and Guided Treatment of Cancers of Unknown Primary Tothill RW, Li J, Mileshkin L, et al. Massively-Parallel Sequencing Assists the Diagnosis and Guided Treatment of Cancers of Unknown Primary. J Pathol. 2013; 231(4): 413-423.

Summary: The clinical management of patients with cancer of unknown primary (CUP) is hampered by the absence of a definitive site of origin. We explored the utility of massively-parallel (next-generation) sequencing for the diagnosis of a primary site of origin and for the identification of novel treatment options. DNA enrichment by hybridization capture of 701 genes of clinical and/or biological importance, followed by massively-parallel sequencing, was performed on 16 CUP patients who had defied attempts to identify a likely site of origin. We obtained high quality data from both fresh- frozen and formalin-fixed, paraffin-embedded samples, demonstrating accessibility to routine diagnostic material. DNA copy-number obtained by massively-parallel sequencing was comparable to that obtained using oligonucleotide microarrays or quantitatively hybridized fluorescently tagged oligonucleotides. Sequencing to an average depth of 458-fold enabled detection of somatically acquired single nucleotide mutations, insertions, deletions and copy-number changes, and measurement of allelic frequency. Common cancer-causing mutations were found in all cancers. Mutation profiling revealed therapeutic gene targets and pathways in 12/16 cases, providing novel treatment options. The presence of driver mutations that are enriched in certain known tumour types, together with mutational signatures indicative of exposure to sunlight or smoking, added to clinical, pathological,

and molecular indicators of likely tissue of origin. Massively-parallel DNA sequencing can therefore provide comprehensive mutation, DNA copy- number, and mutational signature data that are of significant clinical value for a majority of CUP patients, providing both cumulative evidence for the diagnosis of primary site and options for future treatment.

Full text available from Journal of Pathology (USD 6.00-38.00) PMID: 24037760

2.12 Hematopoietic Neoplasms

This section includes articles on the following topics:

• Hematopoietic Neoplasms - Quick Reference Tables (Section 2.12.1); • Myeloid Neoplasia (Section 2.12.2); and • Lymphoid Neoplasia (Section 2.12.3).

A) Massively Parallel Sequencing: The New Frontier of Hematologic Genomics Johnsen JM, Nickerson DA, Reiner AP. Massively parallel sequencing: the new frontier of hematologic genomics. Blood. 2013; 122(19): 3268-3275.

Summary: Genomic technologies are becoming a routine part of human genetic analysis. The exponential growth in DNA sequencing capability has brought an unprecedented understanding of human genetic variation and the identification of thousands of variants that impact human health. In this review, we describe the different types of DNA variation and provide an overview of existing DNA sequencing technologies and their applications. As genomic technologies and knowledge continue to advance, they will become integral in clinical practice. To accomplish the goal of personalized genomic medicine for patients, close collaborations between researchers and clinicians will be essential to develop and curate deep databases of genetic variation and their associated phenotypes.

Free full text available from Blood PMID: 24021669

B) Leveraging Cancer Genome Information in Hematologic Malignancies Rampal R, Levine RL. Leveraging cancer genome information in hematologic malignancies. J Clin Oncol. 2013; 31(15): 1885-1892.

Summary: The use of candidate gene and genome-wide discovery studies in the last several years has led to an expansion of our knowledge of the spectrum of recurrent, somatic disease alleles, which contribute to the pathogenesis of hematologic malignancies. Notably, these studies have also begun to fundamentally change our ability to develop informative prognostic schema that inform outcome and therapeutic response, yielding substantive insights into mechanisms of hematopoietic transformation in different tissue compartments. Although these studies have already had important biologic and translational impact, significant challenges remain in systematically applying these findings to clinical decision making and in implementing new technologies for genetic analysis into clinical practice to inform real-time decision making. Here, we review recent major genetic advances in myeloid and lymphoid malignancies, the impact of these findings on prognostic models, our understanding of disease initiation and evolution, and the implication of genomic discoveries on clinical decision making. Finally, we discuss general concepts in genetic modeling and the current state-of-the-art technology used in genetic investigation.

Full text available from Journal of Clinical Oncology (subscription required) PMID: 23589554

2.12.1 Hematopoietic Neoplasms – Quick Reference Table: Mutated Genes of Potential Clinical Significance

The genes involved in the pathogenesis and progression of hematopoietic neoplasms is continually growing. A comprehensive list is difficult to assemble and quickly falls out-of-date. The following tables provide a selection of genes for which mutational analysis may be clinically relevant, either for diagnosis or prognostication, even if not routinely available. The decision of whether or not to perform a test for a mutation should include consideration of not only its prognostic or diagnostic significance, but also, the frequency at which the mutation is observed. It may not be effective to perform a test for a mutation that is rarely seen, even if it has large prognostic or diagnostic significance when present.

NOTE: cytogenetic abnormalities are critically important in both diagnostic and prognostic assessment of hematopoietic neoplasms. They are, however, beyond the scope of this resource guide and do not appear below.

Quick Reference Table: Mutated Genes of Potential Clinical Significance

Disease Gene Clinical Significance Ref Mature lymphoid CLL ATM Adverse prognostic risk in 7, 42 neoplasms patients with del(11q) IGHV Somatic hypermutation 7, 42 confers improved prognostic risk, while unmutated IGHV is associated with poor prognosis NOTCH1 Adverse prognostic risk, 5, 7, 42 even when only present in a minor clone. Portends higher risk of Richter transformation. TP53 Adverse prognostic risk, 5, 7, 42 even when only present in a minor clone. ZAP70 Adverse prognostic risk 7, 42

BIRC3 Adverse prognostic risk, 5, 7, 42 observed in fludarabine- refractory disease SF3B1 Adverse prognostic risk, 5, 7, 42 observed in fludarabine- refractory disease FAT1 Associated with fludarabine- 5 refractory disease SAMHD1 Associated with 5 relapsed/therapy-refractory disease MYD88 Improved prognosis 5

Mature lymphoid DLBCL FOXO1 Adverse prognostic risk in 50 neoplasms patients treated with R- CHOP, irrespective of cell-of- origin BCL2 Associated with germinal 26, 36 center type EZH2 Associated with germinal 26, 36 center type GNA13 Associated with germinal 26, 36 center type MEF2B Associated with germinal 26, 36 center type SGK1 Associated with germinal 26, 36 center type CD79A Associated with activated B- 36 cell type CD79B Associated with activated B- 36 cell type MYD88 Associated with activated B- 36 cell type HCL BRAF Detected in all cases of 47 (V600E) classical hairy cell leukemia, with rare exceptions, aiding in the distinction from variant hairy cell leukemia and splenic marginal zone lymphoma SMZL KLF2 Frequently observed in 9 SMZL, less often in other B- cell neoplasms. Though not specific, the detection of this mutation may aid in confirmation of a diagnosis of SMZL in the proper clinicopathologic context

Mature lymphoid SMZL NOTCH2 Frequently observed in 20 neoplasms SMZL, less often in other B- cell neoplasms. Though not specific, the detection of this mutation may aid in confirmation of a diagnosis of SMZL in the proper clinicopathologic context MYD88 Occasionally present in 10- 9 (L265P) 17% of SMZL, particularly with plasmacytic differentiation and IgM paraproteinemia LPL MYD88 Recurrently observed in LPL 49 (L265P) but infrequently in other B- cell neoplasms. Occasionally observed in splenic marginal zone lymphoma. Also present in upwards of 54% of IgM MGUS, in which a rising mutation level heralds progression to Waldenstrom macroglobulinemia. CXCR4 C1013G activating mutation 5 reported to promote tumor progression and drug resistance CXCR4 The absence of both MYD88 5 and CXCR4 mutations portends a lesser prognosis

MM BRAF 32 Adverse prognostic risk (V600E) KRAS May herald progression from 40 MGUS to myeloma, or from medullary to extramedullary disease IRF4 Improved prognosis 52 EGR1 Improved prognosis 52 TP53 Adverse prognostic risk, 48, 52 found at increased incidence in plasma cell leukemia

Mature lymphoid MM ATM Adverse prognostic risk 52 neoplasms ATR Adverse prognostic risk 52 ZFHX4 Adverse prognostic risk 52 Mature T-cell AITL IDH2 Predominantly detected in 6 neoplasms AITL but not other peripheral T-cell lymphomas RHOA Predominantly detected in 43 (G17V) AITL, along with a subset of PTCL with T-follicular-helper phenotype, but not observed in other PTCL. Precursor B-ALL ERG Improved prognosis 27 lymphoid IKZF1 Adverse prognostic risk, 16, 27 neoplasms commonly observed in Ph+ and Ph-like ALL TP53 Adverse prognostic risk 27 CREBBP Associated with resistance to 27 glucocorticoid therapy NT5C2 Activating mutations may 51 confer nucleoside analog chemotherapy resistance T-ALL FBXW7 Has been reported as a 8, 19, predictor of favorable risk in 53 conjunction with NOTCH1 mutations, but not all trials have shown an effect on survival NOTCH1 Has been reported as a 8, 19, favorable indicator in some 53 pediatric trials but may be protocol-specific. Others, including a meta-analysis, have shown no effect on survival, however. RUNX1 Adverse prognostic indicator 14 Myeloid neoplasia AML ASXL1 Adverse prognostic risk, 24, 31, including in cases otherwise 38 characterized as intermediate-risk (normal cytogenetics, FLT3-ITD wild- type)

Myeloid neoplasia AML PHF6 Adverse prognostic risk 37 BRAF Adverse prognostic risk 34 CEBPA Double mutations 33, 41 demonstrate favorable risk in the context of AML with normal karyotype DNMT3A 24, 31, Adverse prognostic risk 38

FLT3 Intermediate risk in the 24, 31, context of AML with normal 38 karyotype IDH1 Effect on risk varies by study, 24, 31, gene, and specific mutation. 38 Some reports of adverse outcome, though targeted inhibitors may show promising results. Concomitant IDH/NPM1- mutant patients show a very favorable outcome in the context of wild-type FLT3 IDH2 Effect on risk varies by study, 24, 31, gene, and specific mutation. 38 Some reports of adverse outcome, though targeted inhibitors may show promising results. Concomitant IDH/NPM1- mutant patients show a very favorable outcome in the context of wild-type FLT3 JAK2 Adverse prognostic risk in 24, 31, the context of core-binding 38 factor AML KIT Associated with increased 24, 29, risk of relapse in AML with 31, 38 t(8;21) and inv(16) KRAS Adverse prognostic risk has 24, 31, been reported but may also 38 predict sensitivity to cytarabine therapy

Myeloid neoplasia AML MLL Adverse prognostic risk, 24, 31, including in cases otherwise 38 characterized as intermediate-risk (normal cytogenetics, FLT3-ITD wild- type) NF1 Associated with unfavorable 24, 31, cytogenetic risk, including 38 monosomal karyotype. NPM1 Favorable risk in the context 24, 31, of AML with normal 38 karyotype and wild-type FTL3 NRAS 24, 31, Adverse prognostic risk 38

RUNX1 Adverse prognostic risk in 13, 25 the context of normal karyotype AML SRSF2 24, 31, Adverse prognostic risk 38

TET2 Adverse prognostic risk, 24, 31, including in cases otherwise 38 characterized as intermediate-risk (normal cytogenetics, FLT3-ITD wild- type) TP53 Adverse prognostic risk. 24, 31, Associated with complex 38 karyotype and monosomy 17 ZRSR2 Adverse prognostic risk 34 Myeloid neoplasia MDS ASXL1 Adverse prognostic risk 1 DNMT3A Possibly associated with 1, 30 adverse prognostic risk ETV6 Adverse prognostic risk 3, 4 IDH1 Adverse prognostic risk 1, 30 IDH2 Possibly associated with 1 adverse prognostic risk SF3B1 Possibly improved prognostic 1, 30 risk in MDS with ring sideroblasts

Myeloid neoplasia MDS SRSF2 Adverse prognostic risk; 1, 30 associated with RAEB patients RUNX1 Adverse prognostic risk 30 ZRSR2 Adverse prognosis and a 10 higher rate of transformation in patients with wild-type TET2 TP53 Adverse prognostic risk 30 FLT3 Associated with complex 2 cytogenetics and independently associated with progression to AML TET2 Mutations predict response 30 to hypomethylating agents. Unclear prognostic import though possibly improved EZH2 Adverse prognostic risk 30 SETBP1 Adverse prognostic risk 23, 30 KMT2A Adverse prognostic risk 30 STAG2 Adverse prognostic risk 46 MPN JAK2 Supports the diagnosis of a myeloproliferative neoplasm in the appropriate clinicopathologic context. Mutations are observed in 90% of polycythemia vera and 50% of ET and PMF MPL Supports the diagnosis of essential thrombocythemia in the appropriate clinicopathologic context CALR Supports the diagnosis of a 21, 28 myeloproliferative neoplasm in the appropriate clinicopathologic context. Found in 2/3rds of ET or PMF without JAK2 or MPL mutations

Myeloid neoplasia CNL ASXL1 Adverse prognostic risk 11 CSF3R Mutations of CSF3R, 11 especially T618I, are highly associated with the diagnosis of chronic neutrophilic leukemia SETBP1 Adverse prognostic risk 11 CMML ASXL1 Adverse prognostic risk 17 CBL Combination of CBL and 23 SETBP1 mutations independently predicts poor prognosis SETBP1 Combination of CBL and 23 SETBP1 mutations independently predicts poor prognosis SRSF2 Adverse prognostic risk 17 Atypical SETBP1 39 Adverse prognostic risk CML JMML NF1 Mutations in NF1, NRAS, 22, 44 KRAS KRAS, PTPN11 and CBL 22, 44 NRAS occur in 85% of patients. 22, 44 PTPN11 PTPN11 mutations may 22, 44 CBL portend inferior prognosis. 22, 44 Additionally, two or more somatic alterations within the RAS pathway portend an inferior prognosis. RRAS2 Observed in several JMML 22, 44 patients without other driver mutation, potentially representing a further RAS pathway diagnostic molecular marker SETBP1 Adverse prognostic risk 45 SM KIT Potentially valuable for 12, 15 (D816V) disease monitoring and may have prognostic significance

Myeloid neoplasia SM SRSF2 Adverse prognostic risk; 18 associated with RAEB patients (now referred to as "MDS with excess blasts" in the 2016 revision of the World Health Organization Classification of myeloid neoplasms) ASXL1 Adverse prognostic risk 18, 35 RUNX1 Associated with inferior 35 prognosis in advanced SM Source: Joshua Coleman, MD CLL=chronic lymphocytic leukemia, DLBCL=diffuse large B-cell lymphoma, HCL=hairy cell leukemia, SMZL=splenic marginal zone lymphoma, LPL=lymphoplasmacytic lymphoma, MM=multiple myeloma, MGUS=monoclonal gammopathy of undetermined significance, AITL=angioimmunoblastic T-cell lymphoma, PTCL=peripheral T-cell lymphoma, B-ALL=B-cell acute lymphoblastic leukemia, T-ALL=T-cell acute lymphoblastic leukemia, AML=acute myeloid leukemia, MDS=myelodysplastic syndrome, RAEB=refractory anemia with excess blasts, MPN=myeloproliferative neoplasm, ET=essential thrombocythemia, PMF=primary myelofibrosis, CMML=chronic myelomonocytic leukemia, JMML=juvenile myelomonocytic leukemia, SM=systemic mastocytosis

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2.12.2 Myeloid Neoplasia

This section includes articles on the following topics:

• Chronic Myelogenous Leukemia (Section 2.12.2.1); • BCR-ABL1-Negative Myeloproliferative Neoplasms (Section 2.12.2.2); • Acute Myelongenous Leukemia (Section 2.12.2.3);

• Myelodysplastic Syndrome (Section 2.12.2.4); and • Short Presentations on Emerging Concepts: Polycythemia and Thrombocythemia (Section 2.12.2.5).

2.12.2.1 Chronic Myelogenous Leukemia

A) BCR-ABL1 Compound Mutations in Tyrosine Kinase Inhibitor-Resistant CML: Frequency and Clonal Relationships Khorashad JS, Kelley TW, Szankasi P, et al. BCR-ABL1 compound mutations in tyrosine kinase inhibitor-resistant CML: frequency and clonal relationships. Blood. 2013; 121(3): 489-498.

Summary: BCR-ABL1 compound mutations can confer high-level resistance to imatinib and other ABL1 tyrosine kinase inhibitors (TKIs). The third- generation ABL1 TKI ponatinib is effective against BCR-ABL1 point mutants individually, but remains vulnerable to certain BCR-ABL1 compound mutants. To determine the frequency of compound mutations among chronic myeloid leukemia patients on ABL1 TKI therapy, in the present study, we examined a collection of patient samples (N = 47) with clear evidence of 2 BCR-ABL1 kinase domain mutations by direct sequencing. Using a cloning and sequencing method, we found that 70% (33/47) of double mutations detected by direct sequencing were compound mutations. Sequential, branching, and parallel routes to compound mutations were common. In addition, our approach revealed individual and compound mutations not detectable by direct sequencing. The frequency of clones harboring compound mutations with more than 2 missense mutations was low (10%), whereas the likelihood of silent mutations increased disproportionately with the total number of mutations per clone, suggesting a limited tolerance for BCR-ABL1 kinase domain missense mutations. We conclude that compound mutations are common in patients with sequencing evidence for 2 BCR-ABL1 mutations and frequently reflect a highly complex clonal network, the evolution of which may be limited by the negative impact of missense mutations on kinase function.

Free full text available from Blood PMID: 23223358

B) Laboratory Recommendations for Scoring Deep Molecular Responses Following Treatment for Chronic Myeloid Leukemia Cross NC, White HE, Colomer D, et al. Laboratory Recommendations for Scoring Deep Molecular Responses Following Treatment for Chronic Myeloid Leukemia. Leukemia. 2015; 29(5): 999-1003.

Summary: Treatment of chronic myeloid leukemia (CML) with tyrosine kinase inhibitors has advanced to a stage where many patients achieve very low or undetectable levels of disease. Remarkably, some of these patients remain in sustained remission when treatment is withdrawn, suggesting that they may be at least operationally cured of their disease. Accurate definition of deep molecular responses (MRs) is therefore increasingly important for optimal patient management and comparison of independent data sets. We previously published proposals for broad standardized definitions of MR at different levels of sensitivity. Here we present detailed laboratory recommendations, developed as part of the European Treatment and Outcome Study for CML (EUTOS), to enable testing laboratories to score MR in a reproducible manner for CML patients expressing the most common BCR-ABL1 variants.

Free full text available from PubMed PMID: 25652737

2.12.2.2 BCR-ABL1-Negative Myeloproliferative Neoplasms

A) Calreticulin Mutations in Myeloproliferative Neoplasms and New Methodology for Their Detection and Monitoring Chi J, Manoloukos M, Pierides C, et al. Calreticulin mutations in myeloproliferative neoplasms and new methodology for their detection and monitoring. Ann Hematol. 2015; 94(3):399-408.

Summary: The diagnosis of the BCR-ABL-negative myeloproliferative neoplasms (MPN), namely polycythemia vera, essential thombocythemia and primary myelofibrosis has relied significantly on the detection of known causative mutations in the JAK2 or MPL genes, which account for the majority of MPN patients. However, around 30 % of patients with MPN, primarily essential thombocythemia and primary myelofibrosis, lack mutations in these two genes making it difficult to reach a confident diagnosis in these cases. The recent discovery of frameshift mutations in CALR in approximately 70 % of MPN patients lacking the JAK2 and MPL mutations offers a reliable diagnostic marker for the latter group. A review of the current literature, plus unpublished data from our laboratory, shows that 55 different CALR insertion/deletion mutations have been identified so far in MPN patients. Among these 55 variants reported to date, a 52-base pair deletion and a 5-base pair insertion are by far the most prominent representing 50 and 35 %, respectively, of all cases with CALR mutations. In this paper, we describe a high-resolution melting (HRM) analysis and a Taqman(R) Real- Time PCR (RQ-PCR) assay and we propose a new clinical laboratory

diagnostic algorithm for CALR mutation analysis. According to this algorithm, samples can go through front-line screening with HMR or fragment analysis, followed by the newly developed RQ-PCR to both discriminate and quantify the two most common mutations in CALR gene.

Full text available from Annals of Hematology (USD 39.95) PMID: 25349114

B) BCR-ABL Negative Myeloproliferative Neoplasia: A Review of Involved Molecular Mechanisms Koopmans SM, Schouten HC,van Marion AM. BCR-ABL negative myeloproliferative neoplasia: A review of involved molecular mechanisms. Histol Histopathol. 2015; 30(2):151-61..

Summary: The clonal bone marrow stem cell disorders essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) belong to the group of Philadelphia chromosome negative myeloproliferative neoplasia (Ph- MPN). In 2005 the JAK2V617F mutation was discovered which has generated more insight in the pathogenetic mechanism of the MPNs. More mutations have been detected in MPN patients since. However, the underlying cause of MPN has not been discovered so far. The mechanism of increased angiogenesis in MPNs and the development of fibrosis in the bone marrow in PMF patients and in some ET and PV patients is still not known. This review will focus on the most important molecular pathogenetic mechanisms in MPN patients.

Free full text available from Histology and Histopathology PMID: 25196073

C) An Overview on CALR and CSF3R Mutations and a Proposal for Revision of WHO Diagnostic Criteria for Myeloproliferative Neoplasms Tefferi A, Thiele J, Vannucchi AM, Barbui T. An overview on CALR and CSF3R mutations and a proposal for revision of WHO diagnostic criteria for myeloproliferative neoplasms. Leukemia. 2014; 28(7): 1407-1413.

Summary: Disease-specific mutations facilitate diagnostic precision and drug target discovery. In myeloproliferative neoplasms (MPN), this is best exemplified by the chronic myeloid leukemia-associated BCR-ABL1. No other mutation in MPN has thus far shown a similar degree of diagnostic accuracy or therapeutic relevance. However, JAK2 and KIT mutations are detected in more than 90% of patients with polycythemia vera and systemic mastocytosis, respectively, and are therefore used as highly sensitive clonal markers in these diseases. JAK2 and MPL mutations also occur in essential

thrombocythemia (ET) and primary myelofibrosis (PMF), but their diagnostic value is limited by suboptimal sensitivity and specificity. The molecular diagnostic gap in JAK2/MPL-unmutated ET/PMF is now partially addressed by the recent discovery of calreticulin (CALR) mutations in the majority of such cases. However, bone marrow morphology remains the central diagnostic platform and is essential for distinguishing ET from prefibrotic PMF and diagnosing patients those do not express JAK2, MPL or CALR (triple-negative). The year 2013 was also marked by the description of CSF3R mutations in the majority of patients with chronic neutrophilic leukemia (CNL). Herein, we argue for the inclusion of CALR and CSF3R mutations in the World Health Organization classification system for ET/PMF and CNL, respectively.

Full text available from Leukemia (USD 32.00) PMID: 24441292

2.12.2.3 Acute Myeloid Leukemia

A) Prospective Evaluation of Gene Binding Factor Acute Myeloid Leukemia Jourdan E, Boissel N, Chevret S. Prospective evaluation of gene mutations and minimal residual disease in patients with core binding factor acute myeloid leukemia. Blood. 2013; 121(12): 2213-2223.

Summary: Not all patients with core binding factor acute myeloid leukemia (CBF-AML) display a good outcome. Modern risk factors include KIT and/or FLT3 gene mutations and minimal residual disease (MRD) levels, but their respective values have never been prospectively assessed. A total of 198 CBF-AML patients were randomized between a reinforced and a standard induction course, followed by 3 high-dose cytarabine consolidation courses. MRD levels were monitored prospectively. Gene mutations were screened at diagnosis. Despite a more rapid MRD decrease after reinforced induction, induction arm did not influence relapse-free survival (RFS) (64% in both arms; P = .91). Higher WBC, KIT, and/or FLT3-ITD/TKD gene mutations, and a less than 3-log MRD reduction after first consolidation, were associated with a higher specific hazard of relapse, but MRD remained the sole prognostic factor in multivariate analysis. At 36 months, cumulative incidence of relapse and RFS were 22% vs 54% (P < .001) and 73% vs 44% (P < .001) in patients who achieved 3-log MRD reduction vs the others. These results suggest that MRD, rather than gene mutations, should be used for future treatment stratifications in CBF-AML patients. This trial was registered at

EudraCT as #2006-005163-26 and at www.clinicaltrials.gov as #NCT 00428558.

Free full text available from Blood PMID: 23321257

B) Genetic Profiling in Acute Myeloid Leukaemia--Where Are We and What is Its Role in Patient Management Ofran Y, Rowe JM. Genetic profiling in acute myeloid leukaemia--where are we and what is its role in patient management. Br J Haematol. 2013; 160(3): 303-320.

Summary: Genetic profiling in acute myeloid leukaemia (AML) is a moving target. Only 4 years ago, AML was re-classified, based on karyotypic abnormalities. However, numerous important new mutations and other genetic abnormalities that were not considered in this classification have been identified. Current cytogenetic-based classification is limited by the substantial number of intermediate-risk patients in whom the preferred therapy is debatable. In addition, the majority of AML patients co-express multiple mutations and cannot be easily categorized into predefined homogenous groups. The tremendous progress in mass sequencing allows parallel identification of multiple genetic aberrations in large cohorts. Thus, a new concept of genetic profiling has arisen. Genes and proteins biologically interact with each other; therefore, it should not be surprising that mutations in different genes interact. Prognosis is determined by the composition of mutations and aberrations in leukaemic stem cells. As a consequence, clinical decisions no longer rely on scant genetic data and require comprehensive genetic evaluation. Some non-genetic parameters are also important and should be incorporated into the clinical decision algorithm. Genetic interaction-based profiles are challenging and recent studies demonstrate an improvement in prognostic predictions with this model. Thus, genetic profiling is likely to have a major therapeutic impact, at least for intermediate-risk cytogenetics.

Free full text available from British Journal of Haematology PMID: 23240632 NOTE: Also cited in Section 2.12.1

C) Mutational Landscape of AML with Normal Cytogenetics: Biological and Clinical Implications Martelli MP, Sportoletti P, Tiacci E, Martelli MF, Falini B. Mutational landscape of AML with normal cytogenetics: biological and clinical implications. Blood Rev. 2013; 27(1): 13-22.

Summary: Acute myeloid leukemia (AML) is a molecularly heterogeneous disease. Based on cytogenetics and FISH, AML patients are stratified into three major risk categories: favourable, intermediate and unfavourable. However, prognostic stratification and treatment decision for the intermediate risk category, that mostly comprises AML patients with normal cytogenetics (CN-AML), has been difficult due to the clinical heterogeneity and scarce knowledge of the molecular alterations underlying this large AML subgroup. During the past decade, the identification of several mutations associated with CN-AML has resulted into important advances in the AML field. In this review, we address the biological features of the main mutations associated with CN-AML and the impact of next generation sequencing studies in expanding our knowledge of the molecular landscape of CN-AML. In addition, we outline the prognostic value of mutations for risk stratification of CN-AML patients and discuss the potential of mutations discovery process for developing new molecular targeted therapies.

Free full text available from Blood Reviews PMID: 23261068 NOTE: Also cited in Section 2.12.1

2.12.2.4 Myelodysplastic Syndrome

A) Clonal Hematopoiesis of Indeterminate Potential and Its Distinction from Myelodysplastic Syndromes Steensma DP, Bejar R, Jaiswal S, et al. Clonal Hematopoiesis of Indeterminate Potential and Its Distinction from Myelodysplastic Syndromes. Blood. 2015; 126(1): 9-16.

Summary: Recent genetic analyses of large populations have revealed that somatic mutations in hematopoietic cells leading to clonal expansion are commonly acquired during human aging. Clonally restricted hematopoiesis is associated with an increased risk of subsequent diagnosis of myeloid or lymphoid neoplasia and increased all-cause mortality. Although myelodysplastic syndromes (MDS) are defined by cytopenias, dysplastic morphology of blood and marrow cells, and clonal hematopoiesis, most individuals who acquire clonal hematopoiesis during aging will never develop MDS. Therefore, acquisition of somatic mutations that drive clonal expansion in the absence of cytopenias and dysplastic hematopoiesis can be considered clonal hematopoiesis of indeterminate potential (CHIP), analogous to monoclonal gammopathy of undetermined significance and monoclonal B-cell lymphocytosis, which are precursor states for hematologic neoplasms but are usually benign and do not progress. Because mutations

are frequently observed in healthy older persons, detection of an MDS- associated somatic mutation in a cytopenic patient without other evidence of MDS may cause diagnostic uncertainty. Here we discuss the nature and prevalence of CHIP, distinction of this state from MDS, and current areas of uncertainty regarding diagnostic criteria for myeloid malignancies.

Free full text available from Blood PMID: 25931582

B) Recent Developments in Myelodysplastic Syndromes Bejar R, Steensma DP. Recent Developments in Myelodysplastic Syndromes. Blood. 2014; 124(18): 2793-2803.

Summary: Once thought to be rare disorders, the myelodysplastic syndromes (MDS) are now recognized as among the most common hematological neoplasms, probably affecting >30 000 patients per year in the United States. US regulatory approval of azacitidine, decitabine, and lenalidomide between 2004 and 2006 seemed to herald a new era in the development of disease-modifying therapies for MDS, but there have been no further drug approvals for MDS indications in the United States in the last 8 years. The available drugs are not curative, and few of the compounds that are currently in development are likely to be approved in the near future. As a result, MDS diagnoses continue to place a heavy burden on both patients and health care systems. Incomplete understanding of disease pathology, the inherent biological complexity of MDS, and the presence of comorbid conditions and poor performance status in the typical older patient with MDS have been major impediments to development of effective novel therapies. Here we discuss new insights from genomic discoveries that are illuminating MDS pathogenesis, increasing diagnostic accuracy, and refining prognostic assessment, and which will one day contribute to more effective treatments and improved patient outcomes.

Free full text available from Blood PMID: 25237199

2.12.2.5 Short Presentations on Emerging Concepts: Polycythemia and Thrombocythemia

A) CAP Short Presentations on Emerging Concepts (SPECs): Emerging Concepts on Molecular Testing in the Workup of Polycythemia and Thrombocythemia [PowerPoint slides] Anderson MW, Bagg A, Witte DL. CAP Short Presentations on Emerging Concepts (SPECs): Emerging Concepts on Molecular Testing in the Workup of Polycythemia and Thrombocythemia (v 2.0e rev 7/7/15) [PowerPoint slides]. Caughron SK, Chandra PK, Foo WC, Misialek MJ, Nowak JA, Wood J, eds. Northfield, IL: College of American Pathologists; 2015.

Access slides here

2.12.3 Lymphoid Neoplasia

This section includes articles on the following topics:

• Precursor Lymphoid Neoplasms (Section 2.12.3.1); • Mature B-Cell Neoplasms (Section 2.12.3.2); and • Mature T-Cell Neoplasms (Section 2.12.3.3).

A) EuroClonality/BIOMED-2 Guidelines for Interpretation and Reporting of Ig/TCR Clonality Testing in Suspected Lymphoproliferations Langerak AW, Groenen PJ, Brüggemann M, et al. EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations. Leukemia. 2012; 26(10): 2159-2171.

Summary: PCR-based immunoglobulin (Ig)/T-cell receptor (TCR) clonality testing in suspected lymphoproliferations has largely been standardized and has consequently become technically feasible in a routine diagnostic setting. Standardization of the pre-analytical and post-analytical phases is now essential to prevent misinterpretation and incorrect conclusions derived from clonality data. As clonality testing is not a quantitative assay, but rather concerns recognition of molecular patterns, guidelines for reliable interpretation and reporting are mandatory. Here, the EuroClonality (BIOMED-2) consortium summarizes important pre- and post-analytical aspects of clonality testing, provides guidelines for interpretation of clonality testing results, and presents a uniform way to report the results of the Ig/TCR assays. Starting from an immunobiological concept, two levels to report Ig/TCR profiles are discerned: the technical description of individual (multiplex) PCR reactions and the overall molecular conclusion for B and T cells. Collectively, the EuroClonality (BIOMED-2) guidelines and consensus reporting system should help to improve the general performance level of

clonality assessment and interpretation, which will directly impact on routine clinical management (standardized best-practice) in patients with suspected lymphoproliferations.

Free full text available from PubMed PMID: 22918122

2.12.3.1 Precursor Lymphoid Neoplasms

A) Childhood Acute Lymphoblastic Leukemia: Integrating Genomics into Therapy Tasian SK, Loh ML,Hunger SP. Childhood Acute Lymphoblastic Leukemia: Integrating Genomics into Therapy. Cancer. 2015; 121(20): 3577-3590.

Summary: Acute lymphoblastic leukemia (ALL), the most common malignancy of childhood, is a genetically complex entity that remains a major cause of childhood cancer-related mortality. Major advances in genomic and epigenomic profiling during the past decade have appreciably enhanced knowledge of the biology of de novo and relapsed ALL and have facilitated more precise risk stratification of patients. These achievements have also provided critical insights regarding potentially targetable lesions for the development of new therapeutic approaches in the era of precision medicine. In this review, the authors delineate the current genetic landscape of childhood ALL, emphasizing patient outcomes with contemporary treatment regimens as well as therapeutic implications of newly identified genomic alterations in specific subsets of ALL.

Full text available from Cancer (USD 6.00-38.00) PMID: 26194091

2.12.3.2 Mature B-Cell Neoplasms

A) The Genetic Landscape of Diffuse Large B-Cell Lymphoma Pasqualucci L, Dalla-Favera R. The Genetic Landscape of Diffuse Large B- Cell Lymphoma. Semin Hematol. 2015; 52(2): 67-76.

Summary: Diffuse large B-cell lymphoma (DLBCL), the most common lymphoid malignancy in the western world, is an aggressive disease that remains incurable in approximately 30% of patients. Over the past decade, the rapid expansion of sequencing technologies allowing the genome-wide assessment of genomic and transcriptional changes has revolutionized our understanding of the genetic basis of DLBCL by providing a comprehensive

and unbiased view of the genes/pathways that are disrupted by genetic alterations in this disease, and may contribute to tumor initiation and expansion. These studies uncovered the existence of several previously unappreciated alterations in key cellular pathways that may also influence treatment outcome. Indeed, a number of newly identified genetic lesions are currently being explored as markers for improved diagnosis and risk stratification, or are entering clinical trials as promising therapeutic targets. This review focuses on recent advances in the genomic characterization of DLBCL and discusses how information gained from these efforts has provided new insights into its biology, uncovering potential targets of prognostic and therapeutic relevance.

Free full text available from PubMed PMID: 25805586 NOTE: Also cited in Section 2.12.1

B) The Genomic Landscape of Chronic Lymphocytic Leukaemia: Biological and Clinical Implications Strefford JC. The Genomic Landscape of Chronic Lymphocytic Leukaemia: Biological and Clinical Implications. Br J Haematol. 2015; 169(1): 14-31.

Summary: Chronic lymphocytic leukaemia (CLL) remains at the forefront of the genetic analysis of human tumours, principally due its prevalence, protracted natural history and accessibility to suitable material for analysis. With the application of high-throughput genetic technologies, we have an unbridled view of the architecture of the CLL genome, including a comprehensive description of the copy number and mutational landscape of the disease, a detailed picture of clonal evolution during pathogenesis, and the molecular mechanisms that drive genomic instability and therapeutic resistance. This work has nuanced the prognostic importance of established copy number alterations, and identified novel prognostically relevant gene mutations that function within biological pathways that are attractive treatment targets. Herein, an overview of recent genomic discoveries will be reviewed, with associated biological and clinical implications, and a view into how clinical implementation may be facilitated.

Full text available from British Journal of Haematology (USD 6.00-38.00) PMID: 25496136 NOTE: Also cited in Section 2.12.3

C) Targeted Therapies in CLL: Mechanisms of Resistance and Strategies for Management Woyach JA, Johnson AJ. Targeted Therapies in CLL: Mechanisms of Resistance and Strategies for Management. Blood. 2015; 126(4): 471-477.

Summary: The therapy of relapsed chronic lymphocytic leukemia (CLL) has changed dramatically in the past year with the regulatory approval of idelalisib and ibrutinib, with other therapeutic small molecules likely to become widely available in the next few years. Although durable remissions are being seen in many patients with these agents, it is becoming apparent that some patients with high genomic risk disease will relapse. Next- generation sequencing in patients as well as in vitro models is affording us the opportunity to understand the biology behind these relapses, which is the first step to designing rational therapies to prevent and treat targeted therapy-resistant CLL. These strategies are critical, as these relapses can be very difficult to manage, and a coordinated effort to put these patients on clinical trials will be required to efficiently determine the optimal therapies for these patients. In this review, we will describe mechanisms of resistance, both proven and hypothesized, for idelalisib, ibrutinib, and venetoclax, describe patterns of resistance that have been described with ibrutinib, and discuss potential strategies for management of disease resistant to these drugs as well as potential strategies to prevent resistance.

Free full text available from PubMed PMID: 26065659

D) The Basis and Rational Use of Molecular Genetic Testing in Mature B- Cell Lymphomas Roullet M, Bagg A. The basis and rational use of molecular genetic testing in mature B-cell lymphomas. Adv Anat Pathol. 2010; 17(5): 333-358.

Summary: An increasing number of neoplasms are associated with variably specific genetic abnormalities. This is best exemplified by hematological malignancies, in which there is a growing list of entities that are defined by their genetic lesion(s); this is not (yet) the case in mature B-cell lymphomas. However, enhanced insights into the pathogenesis of this large and diverse group of lymphomas have emerged with the ongoing unraveling of a plethora of fascinating genetic abnormalities. The purpose of this review is to synthesize well-recognized data and nascent discoveries in our understanding of the genetic basis of a spectrum of mature B-cell lymphomas, and how this may be applied to contemporary clinical practice. Despite the explosion of new and exciting knowledge in this arena, with the potential for enhanced diagnostic and prognostic strategies, it is essential to remain cognizant of the limitations (and complexity) of genetic investigations, so that assays can be developed and used both judiciously and rationally.

Full text available from Advances in Anatomic Pathology (subscription required) PMID: 20733353

E) Myeloma Genetics and Genomics: Practice Implications and Future Directions Faiman B. Myeloma Genetics and Genomics: Practice Implications and Future Directions. Clin Lymphoma Myeloma Leuk. 2014; 14(6): 436-440.

Summary: Multiple myeloma (MM) is a heterogeneous, clonal disorder of the plasma cells originating from the B-cell line. The diagnosis and monitoring of MM requires routine measurement of biomarkers such as serum protein electrophoresis, urine protein electrophoresis, serum free light chains, among others. Prognostic models such as the Durie-Salmon staging system and International Staging System are available and account for the disease burden. Advanced biomarker and genetic testing includes cytogenetics, fluorescent in situ hybridization, and gene expression profiling to estimate the aggressiveness of the disease and personalize the patient's treatment. Future goals of therapy will be to achieve minimal residual disease (MRD), which incorporates biomarkers and genomic data. MRD testing might provide a better estimate of the depth of response to therapy and overall survival. A robust genomic program of research is still needed to provide additional information for the best MM care practices and to gain new strategies to treat the disease, in particular, in the relapsed and/or refractory setting.

Full text available from Clinical Lymphoma, Myeloma & Leukemia (USD 31.50 for 24 hour access) PMID: 25127058

2.12.3.3 Mature T-Cell Neoplasms

A) Molecular Diagnostics of T-Cell Lymphoproliferative Disorders Bailey NG, Elenitoba-Johnson KS. Molecular Diagnostics of T-Cell Lymphoproliferative Disorders. Cancer J. 2014; 20(1): 48-60.

Summary: T-cell neoplasms include both mature T-cell leukemias and lymphomas and immature proliferations of precursor T cells. Molecular laboratories routinely assay suspected T-cell proliferations for evidence of clonality. In addition, some T-cell neoplasms are characterized by recurrent structural abnormalities that can be readily identified by such techniques as fluorescence in situ hybridization. New massively parallel sequencing technologies have led to the identification of numerous recurrent gene mutations in T-cell neoplasms. These findings are reviewed. As new technologies become implemented in molecular diagnostic laboratories and as targeted therapies are developed, it is anticipated that more extensive genomic characterization of T-cell neoplasms will be routinely performed in the future.

Full text available from Cancer Journal (subscription required) PMID: 24445765

This section includes the following topics:

• The Basics - Quick Reference Table: Commonly Tested Genes (Section 3.1); • Cardiovascular Disorders (Section 3.2); • Cystic Fibrosis (Section 3.3); • Overgrowth Syndromes (Section 3.4); • Inflammatory Bowel Disorders (Section 3.5); • Eye Disorders (Section 3.6); • Renal Disorders (Section 3.7); • Neurologic, Neuromuscular, and Neuropsychiatric Disorders (Section 3.8) • Ethical Issues (Section 3.9); • Prenatal (Section 3.10); • Mitochondrial Disorders (Section 3.11); • Hereditary Cancer Syndromes (Section 3.12); and • Post Mortem (Section 3.13).

3.1 The Basics - Quick Reference Table: Commonly Tested Genes for Hereditary Disease

There is a wide range of uses for genomics in the arena of hereditary diseases. Genomic studies have proven invaluable in the process of identifying new genetic associations for not only single gene diseases, but also multifactorial diseases such as autism and schizophrenia. As genomics enters the clinical realm, the technologies can be utilized in a variety of ways. From a technical standpoint, the different technologies can be employed to reveal specific types of genetic aberrations. For example, microarrays traditionally identify copy number variations in regions much smaller than conventional cytogenetics and FISH, while next-generation sequencing is very useful in identifying point mutations over large areas of the genome.

Clinically, in patients presenting with non-specific signs and symptoms (e.g. developmental delay or failure to thrive) genomics can help elucidate the

underlying genetic cause and hopefully provide useful information to guide clinical management. At the same time, the increased number of variants of uncertain significance and incidental findings that can be detected via genomic methods raise new ethical and counseling challenges. Early studies of sequencing-based methods for noninvasive prenatal aneuploidy screening and of microarrays in the pediatric and prenatal settings have highlighted the need for careful evaluation of the relationship between older and newer methods as the newest methods are considered for incorporation into routine clinical practice.

Factors considered in selecting a genetic test Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews:Genetics, Katsanis et al151, Molecular genetic testing and the future of clinical genomics; 14(6), copyright 2013.

A) A Prospective Evaluation of Whole-Exome Sequencing as a First-Tier Molecular Test in Infants with Suspected Monogenic Disorders Stark Z, Tan TY, Chong B, et al. A Prospective Evaluation of Whole-Exome Sequencing as a First-Tier Molecular Test in Infants with Suspected Monogenic Disorders. Genet Med. 2016. doi: 10.1038/gim.2016.1. [Epub ahead of print]

Summary: PURPOSE: To prospectively evaluate the diagnostic and clinical utility of singleton whole-exome sequencing (WES) as a first-tier test in

infants with suspected monogenic disease. METHODS: Singleton WES was performed as a first-tier sequencing test in infants recruited from a single pediatric tertiary center. This occurred in parallel with standard investigations, including single- or multigene panel sequencing when clinically indicated. The diagnosis rate, clinical utility, and impact on management of singleton WES were evaluated. RESULTS: Of 80 enrolled infants, 46 received a molecular genetic diagnosis through singleton WES (57.5%) compared with 11 (13.75%) who underwent standard investigations in the same patient group. Clinical management changed following exome diagnosis in 15 of 46 diagnosed participants (32.6%). Twelve relatives received a genetic diagnosis following cascade testing, and 28 couples were identified as being at high risk of recurrence in future pregnancies. CONCLUSIONS: This prospective study provides strong evidence for increased diagnostic and clinical utility of singleton WES as a first-tier sequencing test for infants with a suspected monogenic disorder. Singleton WES outperformed standard care in terms of diagnosis rate and the benefits of a diagnosis, namely, impact on management of the child and clarification of reproductive risks for the extended family in a timely manner.

Full text available from Genetics in Medicine (USD 32.00) PMID: 26938784

B) Diagnostic Clinical Genome and Exome Sequencing Biesecker LG, Green RC. Diagnostic clinical genome and exome sequencing. N Engl J Med. 2014; 371(12): 1170.

No summary available.

Free full text available from New England Journal of Medicine PMID: 25229935

C) Clinical Interpretation and Implications of Whole-Genome Sequencing Dewey FE, Grove ME, Pan C, et al. Clinical interpretation and implications of whole-genome sequencing. JAMA. 2014; 311(10): 1035-1045.

Summary: IMPORTANCE: Whole-genome sequencing (WGS) is increasingly applied in clinical medicine and is expected to uncover clinically significant findings regardless of sequencing indication. OBJECTIVES: To examine coverage and concordance of clinically relevant genetic variation provided by WGS technologies; to quantitate inherited disease risk and pharmacogenomic findings in WGS data and resources required for their discovery and interpretation; and to evaluate clinical action prompted by WGS findings. DESIGN, SETTING, AND PARTICIPANTS: An exploratory study of 12 adult participants recruited at Stanford University Medical Center

who underwent WGS between November 2011 and March 2012. A multidisciplinary team reviewed all potentially reportable genetic findings. Five physicians proposed initial clinical follow-up based on the genetic findings. MAIN OUTCOMES AND MEASURES: Genome coverage and sequencing platform concordance in different categories of genetic disease risk, person-hours spent curating candidate disease-risk variants, interpretation agreement between trained curators and disease genetics databases, burden of inherited disease risk and pharmacogenomic findings, and burden and interrater agreement of proposed clinical follow-up. RESULTS: Depending on sequencing platform, 10% to 19% of inherited disease genes were not covered to accepted standards for single nucleotide variant discovery. Genotype concordance was high for previously described single nucleotide genetic variants (99%-100%) but low for small insertion/deletion variants (53%-59%). Curation of 90 to 127 genetic variants in each participant required a median of 54 minutes (range, 5-223 minutes) per genetic variant, resulted in moderate classification agreement between professionals (Gross kappa, 0.52; 95% CI, 0.40-0.64), and reclassified 69% of genetic variants cataloged as disease causing in mutation databases to variants of uncertain or lesser significance. Two to 6 personal disease-risk findings were discovered in each participant, including 1 frameshift deletion in the BRCA1 gene implicated in hereditary breast and ovarian cancer. Physician review of sequencing findings prompted consideration of a median of 1 to 3 initial diagnostic tests and referrals per participant, with fair interrater agreement about the suitability of WGS findings for clinical follow-up (Fleiss kappa, 0.24; P < 001). CONCLUSIONS AND RELEVANCE: In this exploratory study of 12 volunteer adults, the use of WGS was associated with incomplete coverage of inherited disease genes, low reproducibility of detection of genetic variation with the highest potential clinical effects, and uncertainty about clinically reportable findings. In certain cases, WGS will identify clinically actionable genetic variants warranting early medical intervention. These issues should be considered when determining the role of WGS in clinical medicine.

Free full text available from PubMed PMID: 24618965

D) Sequencing Studies in Human Genetics: Design and Interpretation Goldstein DB, Allen A, Keebler J, et al. Sequencing studies in human genetics: design and interpretation. Nat Rev Genet. 2013; 14(7): 460-470.

Summary: Next-generation sequencing is becoming the primary discovery tool in human genetics. There have been many clear successes in identifying genes that are responsible for Mendelian diseases, and sequencing

approaches are now poised to identify the mutations that cause undiagnosed childhood genetic diseases and those that predispose individuals to more common complex diseases. There are, however, growing concerns that the complexity and magnitude of complete sequence data could lead to an explosion of weakly justified claims of association between genetic variants and disease. Here, we provide an overview of the basic workflow in next- generation sequencing studies and emphasize, where possible, measures and considerations that facilitate accurate inferences from human sequencing studies.

Free full text available from PubMed PMID: 23752795

E) Developing Genome and Exome Sequencing for Candidate Gene Identification in Inherited Disorders: An Integrated Technical and Bioinformatics Approach Coonrod EM, Durtschi JD, Margraf RL, Voelkerding KV. Developing genome and exome sequencing for candidate gene identification in inherited disorders: an integrated technical and bioinformatics approach. Arch Pathol Lab Med. 2013; 137(3): 415-433.

Return to Table of Contents Section 3

Summary: CONTEXT: Advances in sequencing technology with the commercialization of next-generation sequencing (NGS) has substantially increased the feasibility of sequencing human genomes and exomes. Next- generation sequencing has been successfully applied to the discovery of disease-causing genes in rare, inherited disorders. By necessity, the advent of NGS has fostered the concurrent development of bioinformatics approaches to expeditiously analyze the large data sets generated. Next- generation sequencing has been used for important discoveries in the research setting and is now being implemented into the clinical diagnostic arena. OBJECTIVE: To review the current literature on technical and bioinformatics approaches for exome and genome sequencing and highlight examples of successful disease gene discovery in inherited disorders. To discuss the challenges for implementing NGS in the clinical research and diagnostic arenas. DATA SOURCES: Literature review and authors' experience. CONCLUSIONS: Next-generation sequencing approaches are powerful and require an investment in infrastructure and personnel expertise for effective use; however, the potential for improvement of patient care through faster and more accurate molecular diagnoses is high.

Free full text available from the CAP’s Archives PMID: 22770468 NOTE: Also cited in Section 6.1

F) Molecular Genetic Testing and the Future of Clinical Genomics Katsanis SH, Katsanis N. Molecular genetic testing and the future of clinical genomics. Nat Rev Genet. 2013; 14(6): 415-426.

Summary: Genomic technologies are reaching the point of being able to detect genetic variation in patients at high accuracy and reduced cost, offering the promise of fundamentally altering medicine. Still, although scientists and policy advisers grapple with how to interpret and how to handle the onslaught and ambiguity of genome-wide data, established and well- validated molecular technologies continue to have an important role, especially in regions of the world that have more limited access to next- generation sequencing capabilities. Here we review the range of methods currently available in a clinical setting as well as emerging approaches in clinical molecular diagnostics. In parallel, we outline implementation challenges that will be necessary to address to ensure the future of genetic medicine.

Free full text available from PubMed PMID: 23681062

G) Disease-Targeted Sequencing: A Cornerstone in the Clinic Rehm HL. Disease-Targeted Sequencing: A Cornerstone in the Clinic. Nat Rev Genet. 2013; 14(4): 295-300.

Summary: With the declining cost of sequencing and the ongoing discovery of disease genes, it is now possible to examine hundreds of genes in a single disease-targeted test. Although exome- and genome-sequencing approaches are beginning to compete, disease-targeted testing retains certain advantages and still holds a firm place in the diagnostic evaluation. Here I examine the current state of clinical disease-targeted sequencing and evaluate the benefits and challenges of incorporating sequencing tests into patient care.

Free full text available from PubMed PMID: 23478348

Quick Reference Table: Commonly Tested Genes for Hereditary Disease

This table is for quick reference only. Clinical decision making should be not be based solely on this information Present in the CAP Disease ACMG PT Disease Name MIM Gene Incidental Avail- Number Findings able Recomm endations Achrondoplasia 100800 FGFR3 610474 Hypochrondroplasia 146000 FGFR3 (MIM LADD Syndrome 149730 134934) Muenke Syndrome 602849 187600 Thanatophoric Dysplasia and Types I & II 187601 Adenomatous Polyposis APC (MIM 175100 x Coli 611731) ABCD1 (MIM Adrenoleukodystrophy 300100 300371) Afibrinogenemia, 202400 Congenital FGA (MIM Dysfibrinogenemia/Hypody 134820) 616004 sfibrinogenemia, Congenital Alpha-1-Antitrypsin SERPINA1 613490 x Deficiency (MIM 107400) Amyotrophic Lateral C9orf72 (MIM Sclerosis and/or 105550 614260) Frontotemporal Dementia Aortic Aneurysm, Familial MYH11 (MIM 132900 x Thoracic 4 160745) Aortic Aneurysm, Familial ACTA2 (MIM 611788 x Thoracic 6 102620) Aortic Aneurysm, Familial MYLK (MIM 613780 x Thoracic 7 600922) Arrhythmogenic Right TMEM43 (MIM Ventricular 604400 x 612048) Cardiomyopathy, Type 5 Arrhythmogenic Right DSP (MIM Ventricular 607450 x 125647) Cardiomyopathy, Type 8 Arrhythmogenic Right PKP2 (MIM Ventricular 609040 x 602861) Cardiomyopathy, Type 9

Arrhythmogenic Right DSG2 (MIM Ventricular 610193 x 125671) Cardiomyopathy, Type 10 Arrhythmogenic Right DSC2 (MIM Ventricular 610476 x 125645) Cardiomyopathy, Type 11 Chr 11p15 (ICR1, H19, Beckwith-Wiedemann KCNQ1OT1, 130650 Syndrome CDKN1C) NSD1 (MIM 606681) FLCN (MIM Birt-Hogg-Dube Syndrome 135150 607273) BLM (MIM Bloom Syndrome 210900 x 604610) Breast-Ovarian Cancer, BRCA1 (MIM 604370 x x Familial 1 113705) Breast-Ovarian Cancer, BRCA2 (MIM 612555 x x Familial 2 600185) Breast Cancer, Familial 114480 CDH1 (MIM Hereditary Diffuse Gastric 137215 192090) Cancer SCN5A (MIM Brugada Syndrome 1 601144 x 600163) Catecholaminergic RYR2 (MIM Polymorphic Ventricular 604772 180902) x Tachycardia 218800 Crigle-Najjar Syndrome and Types I & II UGT1A1 606785 (MIM191740) Familial Transient Neonatal 237900 Hyperbilirubinemia CFTR (MIM Cystic Fibrosis 219700 x 602421) LMNA (MIM 115200 x 150330) Dilated Cardiomyopathy 1A MYBPC3 (MIM 115200 x 600958) Ehlers-Danlos Syndrome, COL3A1 (MIM 130050 x Type 4 120180) GLA (MIM Fabry’s Disease 301500 x 300644) Familial Amyloidosis, TTR (MIM 105210 Transthyretin-Related 176300) APOA1 (MIM 107680) Familial Amyloidosis, 105200 FGA (MIM Visceral 134820) LYZ (MIM 153450)

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IKBKAP (MIM Familial Dysautonomia 223900 x 603722) APOB x Familial (MIM107730) 143890 Hypercholesterolemia LDLR (MIM x 606945) Familial APOE (MIM Hyperlipoproteinemia, Type 104310 107741) x x III, Alzheimer Disease Familial Hypercalciuric 145980 Hypercalcemia CASR (MIM Neonatal Severe Primary 239200 601199) Hyperparathyroidism Familial Hypertrophic MYH7 (MIM 192600 x Cardiomyopathy 1 160760) Familial Hypertrophic TPM1 (MIM 115196 x Cardiomyopathy 3 191010) Familial Hypertrophic MYBPC3 (MIM 115197 x Cardiomyopathy 4 600958) Familial Hypertrophic PRKAG2 (MIM 600858 x Cardiomyopathy 6 602743) Familial Hypertrophic TNNI3 (MIM 613690 x Cardiomyopathy 7 191044) Familial Hypertrophic MYL3 (MIM 608751 x Cardiomyopathy 8 160790) Familial Hypertrophic MYL2 (MIM 608758 x Cardiomyopathy 10 160781) Familial Hypertrophic ACTC1 (MIM 612098 x Cardiomyopathy 11 102540) Familial Medullary Thyroid RET (MIM 155240 x Carcinoma 164761) Glucose-6-Phosphate G6PD (MIM 300908 Dehydrogenase Deficiency 305900) HFE (MIM x 613609) Hemochromatosis 235200 BMP2 (MIM x 112261) Alpha globins: HBA1 (MIM x 141800) HBA2 (MIM x 141850) Beta globin: Hemoglobinopathies HBB (MIM x 141900) Gamma: HBG1 x (MIM 142200) HBG2 (MIM x 142250) F8 (MIM Hemophilia A 306700 300841) F9 (MIM Hemophilia B 306900 300746)

HTT (MIM Huntington Disease 143100 x 613004) Hypercholesterolemia, PCSK9 (MIM 603776 x Autosomal Dominant, 3 607786) GBA (MIM x 606463) HEXA (MIM x Lysosomal Storage 606869) Diseases (Gaucher, Tay- SMPD1 (MIM x Sachs,Niemann-Pick, 607608) Fabry, etc.) GLA (MIM x 300644) others x ACADM (MCAD; MIM x Fatty Acid Oxidation 607008), Disorders ACADVL (VLCAD; MIM x 609575) GALT (MIM Galactosemia 230400 606999) BTD (MIM Biotinidase Deficiency 253260 609019) PAH (MIM Phenylketonuria 261600 612349) OTC (MIM 300461) ASS1 (MIM 603470) CPS1 (MIM 608307) Urea Cycle and Related ASL (MIM Disorders 608310) ARG1 (MIM 608313) NAGS (MIM 608300) SLC25A13 (MIM 603859) 105830 Angelman Syndrome Chr 15q11.2 x Deletion/Abnor 176270 Prader-Willi Syndrome mal Methylation x FMR1 Repeat 300624 Fragile X Syndrome Expansion(MIM x 309550)

PTPN11 (MIM 176876) SOS1 (MIM 182530) RAF1 (MIM 163950 164760) Noonan Syndrome KRAS (MIM 190070) CBL (MIM 165360) RIT1(MIM 609591) 312750 MECP2 (MIM Rett Syndrome x 300005) PTEN (MIM PTEN-Related Disorders x 601728) BMPR1A (MIM 174900 601299) Juvenile Polyposis SMAD4 (MIM 600993) Left Ventricular 601494 TNNT2 (MIM x Noncompaction 6 191045) 151623 TP53 (MIM Li-Fraumeni Syndrome 1 x 191170) Loeys-Dietz Syndrome 609192 TGFBR1 (MIM x Type 1A 190181) Loeys-Dietz Syndrome 610168 TGFBR2 (MIM x Type 1B 190182) Loeys-Dietz Syndrome 608967 TGFBR1 (MIM x Type 2A 190181) Loeys-Dietz Syndrome 610380 TGFBR2 (MIM x Type 2B 190182) Loeys-Dietz Syndrome 613795 SMAD3 (MIM x Type 3 603109) 192500 KCNQ1 (MIM Long QT Syndrome 1 x 607542) KCNH2 (MIM Long QT Syndrome 2 613688 x 152427) SCN5A (MIM Long QT Syndrome 3 603830 x 600163) MLH1 (MIM x 120436) MSH2 (MIM x 609309) Lynch Syndrome 120435 MSH6 (MIM x 600678) PMS2 (MIM x 600259) RYR1 (MIM x 180901) Malignant Hyperthermia 145600 CACNA1S x (MIM 114208)

FBN1 (MIM x 134797) Marfan’s Syndrome 154700 TGFBR1 (MIM x 190181) Multiple Endocrine MEN1 (MIM 131100 x Neoplasia, Type 1 613733) Multiple Endocrine 171400 x x Neoplasia, Type 2A RET (MIM Multiple Endocrine 162300 164761) x x Neoplasia, Type 2B Muscular Dystrophy, 310200 x Duchenne Type Muscular Dystrophy, 300376 DMD (MIM x Becker Type 300377) Muscular Dystrophy, 302045 Cardiomyopathy Dilated, x 3B DMPK Myotonic Dystrophy 1 160900 x (MIM605377) ZNF9 (MIM Myotonic Dystrophy 2 602668 x 116955) 162200 Neurofibromatosis, Type 1 NF1 (MIM Neurofibromatosis-Noonan 601321 613113) Syndrome NF2 (MIM Neurofibromatosis, Type 2 101000 x 607379) Niemann Pick Disease, 257200 x Type A SMPD1 (MIM Niemann Pick Disease, 607616 607608) x Type B GJB2 (connexin 26; x Nonsyndromic Hearing MIM 121011), GJB6 (MIM Loss x 604418) many others x Oligodontia-Colorectal AXIN2 (MIM 608615 Cancer Syndrome 604025) PRSS1 (MIM 276000) PRSS2 (MIM 601564) SPINK1 (MIM Pancreatitis, Hereditary 167800 167790) CFTR (MIM 602421) CTRC (MIM 601405) SDHD (MIM Paragangliomas 1 168000 x 602690) SDHAF2 (MIM Paragangliomas 2 601650 x 613019)

SDHC (MIM Paragangliomas 3 605373 x 602413) SDHB (MIM Paragangliomas 4 115310 x 185470) STK11 (MIM Peutz-Jeghers Syndrome 175200 x 602216) SDHB (MIM x 185470) SDHD (MIM x 602690) VHL (MIM x 608537) RET (MIM x 164761) Pheochromocytoma 171300 MAX (MIM x 154950) TMEM127 x (MIM 613403) KIF1B (MIM x 605995) GDNF (MIM x 600837) 132600 Pilomatrixoma 608456 MUTYH (MIM MYH-Associated Polyposis 604933) 613659 Neoplasms of Stomach Polycystic Kidney Disease, PKD1 (MIM 173900 Adult Type I 601313) Polycystic Kidney Disease, PKD2 (MIM 613095 Adult Type II 173910) Polycystic Kidney Disease, PKHD1 (MIM Autosmal Recessive and 263200 606702) Hepatic Disease MET (MIM Renal Cell Carcinoma, 164860) 605074 Papillary 1, Familial PRCC (MIM 179755) Hereditary Leiomyomatosis FH (MIM 150800 and Renal Cell Cancer 136850) RB1 (MIM Retinoblastoma 180200 x 614041) RHD (MIM x Rh Gehnotyping (Maternal 111680) Alloimmunization) RHCE (MIM x 111700) Spinal and Bulbar Muscular AR (MIM 313200 Atrophy, X-Linked 313700) SMN1 (MIM x 600354) Spinal Muscular Atrophy SMN2 (MIM x 601627)

F2 (prothrombin; x MIM 176930) F5 (Factor V Leiden; MIM x 612309) MTHFR (MIM x 607093) Thrombophilia SERPINC1 x (MIM 107300) SERPINE1 x (MIM 173360) PROC (MIM x 612283) PROS1 (MIM x 176880) TSC1 (MIM Tuberous Sclerosis 1 191100 x 605284) TSC2 (MIM Tuberous Sclerosis 2 613254 x 191092) Von Hippel-Lindau VHL (MIM 193300 x Syndrome 608537) VWF (MIM Von Willebrand Disease 613160) WT1 (MIM Wilms’ Tumor 194070 x 607102) ATP7B (MIM Wilson Disease 277900 606882) ASPA (MIM Canavan Disease 271900 x 608034) FANCA (MIM x 607139) FANCC (MIM Fanconi Anemia x 613899) FANCG (MIM x 602956) FXN (MIM Friedreich Ataxia 229300 x 606829) MCOLN1 (MIM Mucolipidosis IV 252650 x 605248) Mitochondrial Cytopathies (Leber Hereditary Optic MT-ND4, MT- Neuropathy, Myoclonus ND6, MT-ND1, x Epilepsy with Ragged Red MT-TK Fibers, etc.) Source: Ann M. Moyer, MD, PhD

Additional References:  GeneReviews. Pagon RA, Adam MP, Ardinger HH, et al (eds). Seattle (WA): University of Washington, Seattle; 1993-2016.  Online Mendelian Inheritance in Man. McKusic-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine. www.omin.org. Access July 29, 2016.  Green RC, Berg JS, Grody WW, et al. Genet Med. 2013; 15(7):565-74.

3.2 Cardiovascular Disorders

Quick Reference Table: Inherited Cardiovascular Disorders and Associated Genes This table is for quick reference only. Clinical decision making should be not based solely on this information.

Disease name and MIM number Gene ACTA2 (MIM 102620) Aortic aneurysm, familial thoracic MYH11 (MIM 160745) MYLK (MIM 600922) DSC2 (MIM 125645) DSG2 (MIM 125671) Arrhythmogenic right ventricular DSP (MIM 125647) cardiomyopathy PKP2 (MIM 602861) TMEM43 (MIM 612048) Brugada syndrome SCN5A (MIM 600163) Catecholaminergic polymorphic ventricular RYR2 (MIM 180902) tachycardia LMNA (MIM 150330) Dilated cardiomyopathy 1A MYBPC3 (MIM 600958) APOB (MIM 107730) Familial hypercholesterolemia LDLR (MIM 606945) PCSK9 (MIM 607786) Familial hyperlipoproteinemia, type III APOE (107741) ACTC1 (MIM 102540) MYBPC3 (MIM 600958) MYH7 (MIM 160760) MYL2 (MIM 160781) Familial hypertrophic cardiomyopathy MYL3 (MIM 160790) PRKAG2 (MIM 602743) TPM1 (MIM 191010) TNNI3 (MIM 191044) Left ventricular noncompaction TNNT2 (MIM 191045) KCNH2 (MIM 152427) Long QT syndrome KCNQ1 (MIM 607542) SCN5A (MIM 600163) Source: Ann M Moyer, MD, PhD

 Green RC, Berg JS, Grody WW, et al. Genet Med. 2013; 15(7):565-74.

A) The Spectrum of FBN1, TGFβR1, TGFβR2 and ACTA2 Variants in 594 Individuals with Suspected Marfan Syndrome, Loeys-Dietz Syndrome or Thoracic Aortic Aneurysms and Dissections (TAAD) Lerner-Ellis JP, Aldubayan SH, Hernandez AL, et al. The spectrum of FBN1, TGFβR1, TGFβR2 and ACTA2 variants in 594 individuals with suspected Marfan Syndrome, Loeys-Dietz Syndrome or Thoracic Aortic Aneurysms and Dissections (TAAD). Mol Genet Metab. 2014; 112(2): 171-176.

Summary: INTRODUCTION: In this study, patients suspected of having a clinical diagnosis of Marfan Syndrome (MFS), Loeys-Dietz Syndrome (LDS) and Thoracic Aortic Aneurysms and Dissections (TAAD) were referred for genetic testing and examined for mutations in the FBN1, TGFβR1, TGFβR2 and ACTA2 genes. METHODS: We examined 594 samples from unrelated individuals and different combinations of genes were sequenced, including one or more of the following: FBN1, TGFβR1, TGFβR2, ACTA2, and, in some cases, FBN1 was analyzed by MLPA to detect large deletions. RESULTS: A total of 112 patients had a positive result. Of those, 61 had a clinical diagnosis of MFS, eight had LDS, three had TAAD and 40 patients had clinical features with no specific diagnosis provided. A total of 44 patients had an inconclusive result; of these, 12 patients were referred with a clinical diagnosis of MFS, 4 with LDS and 9 with TAAD and 19 had no clinical diagnosis. A total of 89 mutations were novel. CONCLUSION: This study reveals the rate of detection of variants in several genes associated with MFS, LDS and TAAD. The evaluation of patients by individuals with expertise in the field may decrease the likelihood of ordering unnecessary molecular testing. Nevertheless, genetic testing supports the diagnosis of MFS, LDS and TAAD.

Full text available from Molecular Genetics and Metabolism (USD 31.50) PMID: 24793577 NOTE: Also cited in Section 4.2.11

B) DNA Sequence Capture and Next-Generation Sequencing for the Molecular Diagnosis of Genetic Cardiomyopathies D'Argenio V, Frisso G, Precone V, et al. DNA sequence capture and next- generation sequencing for the molecular diagnosis of genetic cardiomyopathies. J Mol Diagn. 2014; 16(1): 32-44.

Summary: Hypertrophic cardiomyopathy is a relatively frequent disease with a prevalence of 0.2% worldwide and a remarkable genetic heterogeneity,

with more than 30 causative genes reported to date. Current PCR-based strategies are inadequate for genomic investigations involving many candidate genes. Here, we report a next-generation sequencing procedure associated with DNA sequence capture that is able to sequence 202 cardiomyopathy-related genes simultaneously. We developed a complementary data analysis pipeline to select and prioritize genetic variants. The overall procedure can screen a large number of target genes simultaneously, thereby potentially revealing new disease-causing and modifier genes. By using this procedure, we analyzed hypertrophic cardiomyopathy patients in a shorter time and at a lower cost than with current procedures. The specificity of the next-generation sequencing-based procedure is at least as good as other techniques routinely used for mutation searching, and the sensitivity is much better. Analysis of the results showed some novel variants potentially involved in the pathogenesis of hypertrophic cardiomyopathy: a missense mutation in MYH7 and a nonsense variant in INS-IGF2 (patient 1), a splicing variant in MYBPC3 and an indel/frameshift variant in KCNQ1 (patient 2), and two concomitant variations in CACNA1C (patient 3). Sequencing of DNA from the three patients within a pool allowed detection of most variants identified in each individual patient, indicating that this approach is a feasible and cost-effective procedure.

Free full text available from Journal of Molecular Diagnostics PMID: 24183960

3.3 Cystic Fibrosis

A) Population-Based Carrier Screening for Cystic Fibrosis: A Systematic Review of 23 Years of Research Ioannou L, McClaren BJ, Massie J, et al. Population-based carrier screening for cystic fibrosis: a systematic review of 23 years of research. Genet Med. 2014; 16(3): 207-216.

Summary: Cystic fibrosis is the most common severe autosomal recessive disease, with a prevalence of 1 in 2,500-3,500 live births and a carrier frequency of 1 in 25 among Northern Europeans. Population-based carrier screening for cystic fibrosis has been possible since CFTR, the disease- causing gene, was identified in 1989. This review provides a systematic evaluation of the literature from the past 23 years on population-based carrier screening for cystic fibrosis, focusing on the following: uptake of testing; how to offer screening; attitudes, opinions, and knowledge; factors influencing

decision making; and follow-up after screening. Recommendations are given for the implementation and evaluation of future carrier-screening programs.

Free full text available from Genetics in Medicine PMID: 24030436

B) Next Generation Diagnostics of Cystic Fibrosis and CFTR-Related Disorders by Targeted Multiplex High-Coverage Resequencing of CFTR Trujillano D, Ramos MD, Gonzalez J, et al. Next generation diagnostics of cystic fibrosis and CFTR-related disorders by targeted multiplex high- coverage resequencing of CFTR. J Med Genet. 2013; 50(7): 455-462.

Summary: BACKGROUND: Here we have developed a novel and much more efficient strategy for the complete molecular characterisation of the cystic fibrosis (CF) transmembrane regulator (CFTR) gene, based on multiplexed targeted resequencing. We have tested this approach in a cohort of 92 samples with previously characterised CFTR mutations and polymorphisms. METHODS: After enrichment of the pooled barcoded DNA libraries with a custom NimbleGen SeqCap EZ Choice array (Roche) and sequencing with a HiSeq2000 (Illumina) sequencer, we applied several bioinformatics tools to call mutations and polymorphisms in CFTR. RESULTS: The combination of several bioinformatics tools allowed us to detect all known pathogenic variants (point mutations, short insertions/deletions, and large genomic rearrangements) and polymorphisms (including the poly-T and poly-thymidine-guanine polymorphic tracts) in the 92 samples. In addition, we report the precise characterisation of the breakpoints of seven genomic rearrangements in CFTR, including those of a novel deletion of exon 22 and a complex 85 kb inversion which includes two large deletions affecting exons 4-8 and 12-21, respectively. CONCLUSIONS: This work is a proof-of-principle that targeted resequencing is an accurate and cost-effective approach for the genetic testing of CF and CFTR-related disorders (ie, male infertility) amenable to the routine clinical practice, and ready to substitute classical molecular methods in medical genetics.

Free full text available from Journal of Medical Genetics PMID: 23687349

3.4 Overgrowth Syndromes

A) PIK3CA-Related Segmental Overgrowth Mirzaa G, Conway R, Graham JM, Jr., Dobyns WB. PIK3CA-Related Segmental Overgrowth. Genereviews(R). 1993.

Summary: PIK3CA-associated segmental overgrowth includes disorders of brain (e.g., MCAP [megalencephaly-capillary malformation] syndrome, hemimegalencephaly); and segmental body overgrowth (e.g., CLOVES [congenital lipomatous asymmetric overgrowth of the trunk, lymphatic, capillary, venous, and combined-type vascular malformations, epidermal nevi, skeletal and spinal anomalies] syndrome, fibroadipose hyperplasia [FH]). Heterozygous (usually somatic mosaic) mutations of PIK3CA are causative. MCAP syndrome is characterized by the major findings of (1) megalencephaly (MEG) or hemimegalencephaly (HMEG) associated with neurologic findings of hypotonia, seizures, and mild to severe intellectual disability; and (2) cutaneous capillary malformations with focal or generalized somatic overgrowth. Additional findings can include digital anomalies (syndactyly, polydactyly), cortical malformations - most distinctively polymicrogyria (PMG); and variable connective tissue dysplasia. CLOVES (or CLOVE) syndrome and fibroadipose hyperplasia (FH) may be associated with (1) MEG or HMEG; and (2) patchy segmental overgrowth associated with skeletal anomalies, lipomatosis, vascular malformations, and epidermal nevi. PIK3CA-associated segmental overgrowth is confirmed in an individual with a mutation in one PIK3CA allele, typically in affected tissues. Because the vast majority of PIK3CA mutations arise postzygotic (and are thus mosaic), more than one tissue may need to be tested. Failure to detect a PIK3CA mutation does not exclude a clinical diagnosis of the PIK3CA- associated segmental overgrowth disorders in individuals with suggestive features. Treatment of manifestations: Significant or lipomatous segmental overgrowth may require debulking; scoliosis and leg-length discrepancy may require orthopedic care and surgical intervention. Neurologic complications (e.g., obstructive hydrocephalus, increased intracranial pressure, progressive and/or symptomatic cerebellar tonsillar ectopia or Chiari malformation; epilepsy in those with HMEG) may warrant neurosurgical intervention. Routine treatment of the following, when present, is indicated: cardiac and renal abnormalities; intellectual disabilities and behavior problems; motor difficulties; speech, swallowing, and feeding difficulties. Surveillance: MCAP syndrome: Follow up no less than every six months until age six years and at least yearly thereafter to monitor for neurosurgical complications, breathing or sleep disorders, seizures and orthopedic complications. Provisionally recommended imaging in early childhood includes brain MRI every six months for the first two years, then yearly until age eight years for neurologic complications (e.g., hydrocephalus, cerebellar tonsillar ectopia). Consider

screening for Wilms tumor following the protocol suggested for Beckwith- Wiedemann syndrome (BWS) (by ultrasound examination every 3 months until age 8 years); however, tumor risk in PIK3CA-related segmental overgrowth is undetermined and appears to be lower than in BWS. CLOVES syndrome and FH: Monitoring for severe scoliosis, infiltrative lipomatous overgrowth, paraspinal high-flow lesions with spinal cord ischemia, lymphatic malformations, cutaneous vesicles, orthopedic problems, central phlebectasias, and thromboembolism. PIK3CA-associated segmental overgrowth is not typically inherited. Most affected individuals with MCAP reported to date (21/24) had somatic mosaicism for mutations in PIK3CA, suggesting that the mutation occurred post-fertilization in one cell of the multicellular embryo. Two of 24 affected individuals had a de novo germline mutation in PIK3CA. All reported individuals with CLOVES and FH had somatic mosaicism for mutations in PIK3CA. No confirmed instances of vertical transmission or sib recurrence have been reported. Because family members are not known to have an increased risk, prenatal diagnosis is usually not indicated for family members.

Free full text available from PubMed PMID: 23946963

B) PIK3CA-Related Overgrowth Spectrum (PROS): Diagnostic and Testing Eligibility Criteria, Differential Diagnosis, and Evaluation Keppler-Noreuil KM, Rios JJ, Parker VE, et al. PIK3CA-Related Overgrowth Spectrum (PROS): Diagnostic and Testing Eligibility Criteria, Differential Diagnosis, and Evaluation. Am J Med Genet A. 2015; 167A(2): 287-295.

Summary: Somatic activating mutations in the phosphatidylinositol-3- kinase/AKT/mTOR pathway underlie heterogeneous segmental overgrowth phenotypes. Because of the extreme differences among patients, we sought to characterize the phenotypic spectrum associated with different genotypes and mutation burdens, including a better understanding of associated complications and natural history. Historically, the clinical diagnoses in patients with PIK3CA activating mutations have included Fibroadipose hyperplasia or Overgrowth (FAO), Hemihyperplasia Multiple Lipomatosis (HHML), Congenital Lipomatous Overgrowth, Vascular Malformations, Epidermal Nevi, Scoliosis/Skeletal and Spinal (CLOVES) syndrome, macrodactyly, Fibroadipose Infiltrating Lipomatosis, and the related megalencephaly syndromes, Megalencephaly-Capillary Malformation (MCAP or M-CM) and Dysplastic Megalencephaly (DMEG). A workshop was convened at the National Institutes of Health (NIH) to discuss and develop a consensus document regarding diagnosis and treatment of patients with PIK3CA-associated somatic overgrowth disorders. Participants in the workshop included a group of researchers from several institutions who have been studying these disorders and have published their findings, as well as

representatives from patient-advocacy and support groups. The umbrella term of "PIK3CA-Related Overgrowth Spectrum (PROS)" was agreed upon to encompass both the known and emerging clinical entities associated with somatic PIK3CA mutations including, macrodactyly, FAO, HHML, CLOVES, and related megalencephaly conditions. Key clinical diagnostic features and criteria for testing were proposed, and testing approaches summarized. Preliminary recommendations for a uniform approach to assessment of overgrowth and molecular diagnostic testing were determined. Future areas to address include the surgical management of overgrowth tissue and vascular anomalies, the optimal approach to thrombosis risk, and the testing of potential pharmacologic therapies.

Free full text available from PubMed PMID: 25557259

3.5 Inflammatory Bowel Disorders

A) Molecular Diagnosis of Infantile Onset Inflammatory Bowel Disease by Exome Sequencing Dinwiddie DL, Bracken JM, Bass JA, et al. Molecular diagnosis of infantile onset inflammatory bowel disease by exome sequencing. Genomics. 2013; 102(5-6): 442-7.

Summary: Pediatric-onset inflammatory bowel disease (IBD) is known to be associated with severe disease, poor response to therapy, and increased morbidity and mortality. We conducted exome sequencing of two brothers from a non-consanguineous relationship who presented before the age of one with severe infantile-onset IBD, failure to thrive, skin rash, and perirectal abscesses refractory to medical management. We examined the variants discovered in all known IBD-associated and primary immunodeficiency genes in both siblings. The siblings were identified to harbor compound heterozygous mutations in IL10RA (c.784C>T, p.Arg262Cys; c.349C>T, p.Arg117Cys). Upon molecular diagnosis, the proband underwent successful hematopoietic stem cell transplantation and demonstrated marked clinical improvement of all IBD-associated clinical symptoms. Exome sequencing can be an effective tool to aid in the molecular diagnosis of pediatric-onset IBD. We provide additional evidence of the safety and benefit of HSCT for patients with IBD due to mutations in the IL10RA gene.

Free full text available from Genomics PMID: 24001973 NOTE: Also cited in Section 3.11

B) Making a Definitive Diagnosis: Successful Clinical Application of Whole Exome Sequencing in a Child with Intractable Inflammatory Bowel Disease Worthey EA, Mayer AN, Syverson GD, et al. Making a Definitive Diagnosis: Successful Clinical Application of Whole Exome Sequencing in a Child with Intractable Inflammatory Bowel Disease. Journal/Genet Med. 2011; 13(3): 255-262.

Summary: PURPOSE: We report a male child who presented at 15 months with perianal abscesses and proctitis, progressing to transmural pancolitis with colocutaneous fistulae, consistent with a Crohn disease-like illness. The age and severity of the presentation suggested an underlying immune defect; however, despite comprehensive clinical evaluation, we were unable to arrive at a definitive diagnosis, thereby restricting clinical management. METHODS: We sought to identify the causative mutation(s) through exome sequencing to provide the necessary additional information required for clinical management. RESULTS: After sequencing, we identified 16,124 variants. Subsequent analysis identified a novel, hemizygous missense mutation in the X-linked inhibitor of apoptosis gene, substituting a tyrosine for a highly conserved and functionally important cysteine. X-linked inhibitor of apoptosis was not previously associated with Crohn disease but has a central role in the proinflammatory response and bacterial sensing through the NOD signaling pathway. The mutation was confirmed by Sanger sequencing in a licensed clinical laboratory. Functional assays demonstrated an increased susceptibility to activation-induced cell death and defective responsiveness to NOD2 ligands, consistent with loss of normal X-linked inhibitor of apoptosis protein function in apoptosis and NOD2 signaling. CONCLUSIONS: Based on this medical history, genetic and functional data, the child was diagnosed as having an X-linked inhibitor of apoptosis deficiency. Based on this finding, an allogeneic hematopoietic progenitor cell transplant was performed to prevent the development of life-threatening hemophagocytic lymphohistiocytosis, in concordance with the recommended treatment for X-linked inhibitor of apoptosis deficiency. At >42 days posttransplant, the child was able to eat and drink, and there has been no recurrence of gastrointestinal disease, suggesting this mutation also drove the gastrointestinal disease. This report describes the identification of a novel cause of inflammatory bowel disease. Equally importantly, it demonstrates the power of exome sequencing to render a molecular diagnosis in an individual patient in the setting of a novel disease, after all standard diagnoses were exhausted, and illustrates how this technology can be used in a clinical setting.

Free full text available from Genetics in Medicine PMID: 21173700

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3.6 Eye Disorders

A) Panel-Based Genetic Diagnostic Testing for Inherited Eye Diseases Is Highly Accurate and Reproducible, and More Sensitive for Variant Detection, Than Exome Sequencing Consugar MB, Navarro-Gomez D, Place EM, et al. Panel-Based Genetic Diagnostic Testing for Inherited Eye Diseases Is Highly Accurate and Reproducible, and More Sensitive for Variant Detection, Than Exome Sequencing. Genet Med. 2015; 17(4): 253-261.

Summary: PURPOSE: Next-generation sequencing-based methods are being adopted broadly for genetic diagnostic testing, but the performance characteristics of these techniques with regard to test accuracy and reproducibility have not been fully defined. METHODS: We developed a targeted enrichment and next-generation sequencing approach for genetic diagnostic testing of patients with inherited eye disorders, including inherited retinal degenerations, optic atrophy, and glaucoma. In preparation for providing this genetic eye disease (GEDi) test on a CLIA-certified basis, we performed experiments to measure the sensitivity, specificity, and reproducibility, as well as the clinical sensitivity, of the test. RESULTS: The GEDi test is highly reproducible and accurate, with sensitivity and specificity of 97.9 and 100%, respectively, for single-nucleotide variant detection. The sensitivity for variant detection was notably better than the 88.3% achieved by whole-exome sequencing using the same metrics, because of better coverage of targeted genes in the GEDi test as compared with a commercially available exome capture set. Prospective testing of 192 patients with inherited retinal degenerations indicated that the clinical sensitivity of the GEDi test is high, with a diagnostic rate of 51%. CONCLUSION: Based on quantified performance metrics, the data suggest that selective targeted enrichment is preferable to whole-exome sequencing for genetic diagnostic testing.

Free full text available from PubMed PMID: 25412400

3.7 Renal Disorders

Genes associated with steroid-resistant nephritic syndrome (SRNS) Republished with permission of American Society of Nephrology from Clin J Am Soc Nephrol, Simultaneous sequencing of 24 genes with steroid-resistant nephrotic syndrome, McCarthy HJ, et al330, 8(4), copyright 2013; permission conveyed through Copyright Clearance Center, Inc.

A) Molecular Diagnosis of Autosomal Dominant Polycystic Kidney Disease Using Next-Generation Sequencing Tan AY, Michaeel A, Liu G, et al. Molecular diagnosis of autosomal dominant polycystic kidney disease using next-generation sequencing. J Mol Diagn. 2014; 16(2): 216-228.

Summary: Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 and PKD2. However, genetic analysis is complicated by six PKD1 pseudogenes, large gene sizes, and allelic heterogeneity. We developed a new clinical assay for PKD gene analysis using paired-end next-generation sequencing (NGS) by multiplexing individually bar-coded long-range PCR libraries and analyzing them in one Illumina MiSeq flow cell. The data analysis pipeline has been optimized and automated with Unix shell scripts to accommodate variant calls. This approach was validated using a cohort of 25 patients with ADPKD previously analyzed by Sanger sequencing. A total of 250 genetic variants were identified by NGS, spanning the entire exonic and adjacent intronic regions of

PKD1 and PKD2, including all 16 pathogenic mutations. In addition, we identified three novel mutations in a mutation-negative cohort of 24 patients with ADPKD previously analyzed by Sanger sequencing. This NGS method achieved sensitivity of 99.2% (95% CI, 96.8%-99.9%) and specificity of 99.9% (95% CI, 99.7%-100.0%), with cost and turnaround time reduced by as much as 70%. Prospective NGS analysis of 25 patients with ADPKD demonstrated a detection rate comparable with Sanger standards. In conclusion, the NGS method was superior to Sanger sequencing for detecting PKD gene mutations, achieving high sensitivity and improved gene coverage. These characteristics suggest that NGS would be an appropriate new standard for clinical genetic testing of ADPKD.

Free full text available from PubMed PMID: 24374109

B) Simultaneous Sequencing of 24 Genes Associated with Steroid- Resistant Nephrotic Syndrome McCarthy HJ, Bierzynska A, Wherlock M, et al. Simultaneous sequencing of 24 genes associated with steroid-resistant nephrotic syndrome. Clin J Am Soc Nephrol. 2013; 8(4): 637-648.

Summary: BACKGROUND AND OBJECTIVES: Up to 95% of children presenting with steroid-resistant nephrotic syndrome in early life will have a pathogenic single-gene mutation in 1 of 24 genes currently associated with this disease. Others may be affected by polymorphic variants. There is currently no accepted diagnostic algorithm for clinical genetic testing. The hypothesis was that the increasing reliability of next generation sequencing allows comprehensive one-step genetic investigation of this group and similar patient groups. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This study used next generation sequencing to screen 446 genes, including the 24 genes known to be associated with hereditary steroid-resistant nephrotic syndrome. The first 36 pediatric patients collected through a national United Kingdom Renal Registry were chosen with comprehensive phenotypic detail. Significant variants detected by next generation sequencing were confirmed by conventional Sanger sequencing. RESULTS: Analysis revealed known and novel disease-associated variations in expected genes such as NPHS1, NPHS2, and PLCe1 in 19% of patients. Phenotypically unexpected mutations were also detected in COQ2 and COL4A4 in two patients with isolated nephropathy and associated sensorineural deafness, respectively. The presence of an additional heterozygous polymorphism in WT1 in a patient with NPHS1 mutation was associated with earlier-onset disease, supporting modification of phenotype through genetic epistasis. CONCLUSIONS: This study shows that next

generation sequencing analysis of pediatric steroid-resistant nephrotic syndrome patients is accurate and revealing. This analysis should be considered part of the routine genetic workup of diseases such as childhood steroid-resistant nephrotic syndrome, where the chance of genetic mutation is high but requires sequencing of multiple genes.

Free full text available from PubMed PMID: 23349334

3.8 Neurologic, Neuromuscular and Neuropsychiatric Disorders

A) A Comprehensive Genomic Approach for Neuromuscular Diseases Gives a High Diagnostic Yield Ankala A, da Silva C, Gualandi F, et al. A Comprehensive Genomic Approach for Neuromuscular Diseases Gives a High Diagnostic Yield. Ann Neurol. 2015; 77(2): 206-214.

Summary: OBJECTIVE: Neuromuscular diseases (NMDs) are a group of >200 highly genetically as well as clinically heterogeneous inherited genetic disorders that affect the peripheral nervous and muscular systems, resulting in gross motor disability. The clinical and genetic heterogeneities of NMDs make disease diagnosis complicated and expensive, often involving multiple tests. METHODS: To expedite the molecular diagnosis of NMDs, we designed and validated several next generation sequencing (NGS)-based comprehensive gene panel tests that include complementary deletion and duplication testing through comparative genomic hybridization arrays. Our validation established the targeted gene panel test to have 100% sensitivity and specificity for single nucleotide variant detection. To compare the clinical diagnostic yields of single gene (NMD-associated) tests with the various NMD NGS panel tests, we analyzed data from all clinical tests performed at the Emory Genetics Laboratory from October 2009 through May 2014. We further compared the clinical utility of the targeted NGS panel test with that of exome sequencing (ES). RESULTS: We found that NMD comprehensive panel testing has a 3-fold greater diagnostic yield (46%) than single gene testing (15-19%). Sanger fill-in of low-coverage exons, copy number variation analysis, and thorough in-house validation of the assay all complement panel testing and allow the detection of all types of causative pathogenic variants, some of which (about 18%) may be missed by ES. INTERPRETATION: Our results strongly indicate that for molecular diagnosis of heterogeneous disorders such as NMDs, targeted panel testing has the highest clinical yield and should therefore be the preferred first-tier approach.

Full text available from Annals of Neurology (USD 6.00-38.00) PMID: 25380242

B) A Polygenic Burden of Rare Disruptive Mutations in Schizophrenia Purcell SM, Moran JL, Fromer M, et al. A polygenic burden of rare disruptive mutations in schizophrenia. Nature. 2014; 506(7487): 185-190.

Summary: Schizophrenia is a common disease with a complex aetiology, probably involving multiple and heterogeneous genetic factors. Here, by analysing the exome sequences of 2,536 schizophrenia cases and 2,543 controls, we demonstrate a polygenic burden primarily arising from rare (less than 1 in 10,000), disruptive mutations distributed across many genes. Particularly enriched gene sets include the voltage-gated calcium ion channel and the signalling complex formed by the activity-regulated cytoskeleton- associated scaffold protein (ARC) of the postsynaptic density, sets previously implicated by genome-wide association and copy-number variation studies. Similar to reports in autism, targets of the fragile X mental retardation protein (FMRP, product of FMR1) are enriched for case mutations. No individual gene-based test achieves significance after correction for multiple testing and we do not detect any alleles of moderately low frequency (approximately 0.5 to 1 per cent) and moderately large effect. Taken together, these data suggest that population-based exome sequencing can discover risk alleles and complements established gene-mapping paradigms in neuropsychiatric disease.

Free full text available from PubMed PMID: 24463508

C) Exome Sequencing as a Diagnostic Tool for Pediatric-Onset Ataxia Sawyer SL, Schwartzentruber J, Beaulieu CL, et al. Exome sequencing as a diagnostic tool for pediatric-onset ataxia. Hum Mutat. 2014; 35(1): 45-49.

Summary: Ataxia demonstrates substantial phenotypic and genetic heterogeneity. We set out to determine the diagnostic yield of exome sequencing in pediatric patients with ataxia without a molecular diagnosis after standard-of-care assessment in Canada. FORGE (Finding Of Rare disease GEnes) Canada is a nation-wide project focused on identifying novel disease genes for rare pediatric diseases using whole-exome sequencing. We retrospectively selected all FORGE Canada projects that included cerebellar ataxia as a feature. We identified 28 such families and a molecular diagnosis was made in 13; a success rate of 46%. In 11 families, we identified mutations in genes associated with known neurological syndromes

and in two we identified novel disease genes. Exome analysis of sib pairs and/or patients born to consanguineous parents was more likely to be successful (9/13) than simplex cases (4/15). Our data suggest that exome sequencing is an effective first line test for pediatric patients with ataxia where a specific single gene is not immediately suspected to be causative.

Free full text available from Human Mutation PMID: 24108619

D) Evaluating Rare Variants in Complex Disorders Using Next-Generation Sequencing Ezewudo M, Zwick ME. Evaluating rare variants in complex disorders using next-generation sequencing. Curr Psychiatry Rep. 2013; 15(4): 349.

Summary: Determining the genetic architecture of liability for complex neuropsychiatric disorders like autism spectrum disorders and schizophrenia poses a tremendous challenge for contemporary biomedical research. Here we discuss how genetic studies first tested, and rejected, the hypothesis that common variants with large effects account for the prevalence of these disorders. We then explore how the discovery of structural variation has contributed to our understanding of the etiology of these disorders. The rise of fast and inexpensive oligonucleotide sequencing and methods of targeted enrichment and their influence on the search for rare genetic variation contributing to complex neuropsychiatric disorders is the next focus of our article. Finally, we consider the technical challenges and future prospects for the use of next-generation sequencing to reveal the complex genetic architecture of complex neuropsychiatric disorders in both research and the clinical settings.

Free full text available from PubMed PMID: 23435969

3.9 Ethical Issues

This section includes the following topics:

• Genetic Counseling (Section 3.9.1); and • Secondary Findings (Section 3.9.2).

3.9.1 Genetic Counseling

A) A Practice Guideline from the American College of Medical Genetics and Genomics and the National Society of Genetic Counselors: Referral Indications for Cancer Predisposition Assessment Hampel H, Bennett RL, Buchanan A, Pearlman R, Wiesner GL. A practice guideline from the American College of Medical Genetics and Genomics and the National Society of Genetic Counselors: referral indications for cancer predisposition assessment. Genet Med. 2015;17(1):70-87.

Summary: Cancer genetic consultation is an important aspect of the care of individuals at increased risk of a hereditary cancer syndrome. Yet several patient, clinician, and system-level barriers hinder identification of individuals appropriate for cancer genetics referral. Thus, the purpose of this practice guideline is to present a single set of comprehensive personal and family history criteria to facilitate identification and maximize appropriate referral of at-risk individuals for cancer genetic consultation. To develop this guideline, a literature search for hereditary cancer susceptibility syndromes was conducted using PubMed. In addition, GeneReviews and the National Comprehensive Cancer Network guidelines were reviewed when applicable. When conflicting guidelines were identified, the evidence was ranked as follows: position papers from national and professional organizations ranked highest, followed by consortium guidelines, and then peer-reviewed publications from single institutions. The criteria for cancer genetic consultation referral are provided in two formats: (i) tables that list the tumor type along with the criteria that, if met, would warrant a referral for a cancer genetic consultation and (ii) an alphabetical list of the syndromes, including a brief summary of each and the rationale for the referral criteria that were selected. Consider referral for a cancer genetic consultation if your patient or any of their first-degree relatives meet any of these referral criteria.

Free full text available from Genetics in Medicine PMID: 25394175 NOTE: Also cited in Section 3.12

B) Reporting Incidental Findings in Clinical Whole Exome Sequencing: Incorporation of the 2013 ACMG Recommendations into Current Practices of Genetic Counseling Smith LA, Douglas J, Braxton AA, Kramer K. Reporting Incidental Findings in Clinical Whole Exome Sequencing: Incorporation of the 2013 ACMG Recommendations into Current Practices of Genetic Counseling. J Genet Couns. 2015;24(4):654-62.

Summary: The purpose of this study was to investigate how the American College of Medical Genetics and Genomics (ACMG) March 2013 recommendations for reporting incidental findings (IFs) have influenced current practices of genetic counselors involved in utilizing whole exome sequencing (WES) for clinical diagnosis. An online survey was sent to all members of the National Society of Genetic Counselors; members were eligible to participate if they currently offered WES for clinical diagnosis. Forty-six respondents completed the survey of whom 34 were in practice prior to the March 2013 ACMG recommendations. Half of respondents (N = 19, 54.9 %) in practice prior to March 2013 reported that the ACMG recommendations have had a significant impact on the content of their counseling sessions. Approximately half of respondents (N = 21, 45.5 %) report all IFs, regardless of patient age, while one third (N = 14, 30.4 %) consider factors such as age and parent preference in reporting IFs. Approximately 40 % (N = 18) of respondents reported that the testing laboratory's policy for returning IFs has an influence on their choice of laboratory; of those, 72.2 % (N = 13) reported that the option to opt out of receiving reports of IFs has a significant influence on their choice of laboratory. A majority of respondents (N = 43, 93.5 %) found that most patients want to receive reports of IFs. However, respondents report there are patients who wish to decline receiving this information. This study querying genetic counselors identified benefits and challenges that the 2013 ACMG recommendations elicited. Some challenges, such as not having the option to opt out of IFs, have been addressed by the ACMG's most recent updates to their recommendations. Further investigation into larger and more inclusive provider populations as well as patient populations will be valuable for the ongoing discussion surrounding IFs in WES.

Full text available from Journal of Genetic Counseling (USD 39.95) PMID: 25403901

3.9.2 Secondary Findings

A) ACMG Recommendations for Reporting of Incidental Findings in Clinical Exome and Genome Sequencing Green RC, Berg JS, Grody WW, et al. ACMG recommendations for reporting of incidental findings in clinical exome and genome sequencing. Genet Med. 2013; 15(7): 565-574.

care. The American College of Medical Genetics and Genomics (ACMG) recently published a policy statement on clinical sequencing that emphasized the importance of alerting the patient to the possibility of such results in pretest patient discussions, clinical testing, and reporting of results. The ACMG appointed a Working Group on Incidental Findings in Clinical Exome and Genome Sequencing to make recommendations about responsible management of incidental findings when patients undergo exome or genome sequencing. This Working Group conducted a year-long consensus process, including an open forum at the 2012 Annual Meeting and review by outside experts, and produced recommendations that have been approved by the ACMG Board. Specific and detailed recommendations, and the background and rationale for these recommendations, are described herein. The ACMG recommends that laboratories performing clinical sequencing seek and report mutations of the specified classes or types in the genes listed here. This evaluation and reporting should be performed for all clinical germline (constitutional) exome and genome sequencing, including the "normal" of tumor-normal subtractive analyses in all subjects, irrespective of age but excluding fetal samples. We recognize that there are insufficient data on penetrance and clinical utility to fully support these recommendations, and we encourage the creation of an ongoing process for updating these recommendations at least annually as further data are collected.

Free full text available from PubMed PMID: 23788249 NOTE: Also cited in Section 7.2

B) Clinical Exome Performance for Reporting Secondary Genetic Findings Park JY, Clark P, Londin E, Sponziello M, Kricka LJ, Fortina P. Clinical exome performance for reporting secondary genetic findings. Clin Chem. 2015; 61(1): 213-220.

Summary: BACKGROUND: Reporting clinically actionable incidental genetic findings in the course of clinical exome testing is recommended by the American College of Medical Genetics and Genomics (ACMG). However, the performance of clinical exome methods for reporting small subsets of genes has not been previously reported. METHODS: In this study, 57 exome data sets performed as clinical (n = 12) or research (n = 45) tests were retrospectively analyzed. Exome sequencing data was examined for adequacy in the detection of potentially pathogenic variant locations in the 56 genes described in the ACMG incidental findings recommendation. All exons of the 56 genes were examined for adequacy of sequencing coverage. In addition, nucleotide positions annotated in HGMD (Human Gene Mutation Database) were examined. RESULTS: The 56 ACMG genes have 18,336

nucleotide variants annotated in HGMD. None of the 57 exome data sets possessed a HGMD variant. The clinical exome test had inadequate coverage for >50% of HGMD variant locations in 7 genes. Six exons from 6 different genes had consistent failure across all 3 test methods; these exons had high GC content (76%-84%). CONCLUSIONS: The use of clinical exome sequencing for the interpretation and reporting of subsets of genes requires recognition of the substantial possibility of inadequate depth and breadth of sequencing coverage at clinically relevant locations. Inadequate depth of coverage may contribute to false-negative clinical exome results.

Free full text available from PubMed PMID: 25414276

C) Finding Fault? Exploring Legal Duties to Return Incidental Findings in Genomic Research Pike ER, Rothenberg KH, Berkman BE. Finding Fault? Exploring Legal Duties to Return Incidental Findings in Genomic Research. Georgetown Law J. 2014;102:795-843.

Summary: The use of whole-genome sequencing in biomedical research is expected to produce dramatic advances in human health. The increasing use of this powerful, data-rich new technology in research, however, will inevitably give rise to incidental findings (IFs)-findings with individual health or reproductive significance that are beyond the aims of the particular research-and the related questions of whether and to what extent researchers have an ethical obligation to return IFs. Many have concluded that researchers have an ethical obligation to return some findings in some circumstances but have provided vague or context-dependent approaches to determining which IFs must be returned and when. As a result, researchers have started returning IFs inconsistently, giving rise to concerns about legal liability in circumstances in which notification could have potentially prevented injury. Although it is clear that ethical guidance should not be automatically codified as law and that crafting ethical obligations around legal duties can be inappropriate, the ethical debate should not proceed unaware of the potential legal ramifications of advancing and implementing an ethical obligation to return IFs. This Article assesses the legal claims that could be brought for a researcher's failure to return IFs. The potential for researchers to be held liable in tort is still uncertain and turns largely on a number of factors-including customary practice and guidance documents-that are still in flux. Unlike medical care, which has a well-defined duty into which evolving scientific knowledge about genetics and genomics can readily be incorporated, a researcher's duty to return IFs is less well defined, making it difficult to determine at the outset whether and when legal liability will attach.

This Article advocates for a clearer, ethically sound standard of requiring that researchers disclose in the informed consent document which approach to offering IFs will be taken. This approach enables participants to know at the outset which findings, if any, will be returned, allows researchers to ascertain when their failure to appropriately return incidental findings will give rise to liability, and enables courts to make determinations that will produce more consistent legal guidance.

Free full text available from PubMed PMID: 25346543 NOTE: Also cited in Section 7.3

D) Disclosure of Incidental Findings from Next-Generation Sequencing in Pediatric Genomic Research Abdul-Karim R, Berkman BE, Wendler D, et al. Disclosure of incidental findings from next-generation sequencing in pediatric genomic research. Pediatrics. 2013; 131(3): 564-571.

Summary: Next-generation sequencing technologies will likely be used with increasing frequency in pediatric research. One consequence will be the increased identification of individual genomic research findings that are incidental to the aims of the research. Although researchers and ethicists have raised theoretical concerns about incidental findings in the context of genetic research, next-generation sequencing will make this once largely hypothetical concern an increasing reality. Most commentators have begun to accept the notion that there is some duty to disclose individual genetic research results to research subjects; however, the scope of that duty remains unclear. These issues are especially complicated in the pediatric setting, where subjects cannot currently but typically will eventually be able to make their own medical decisions at the age of adulthood. This article discusses the management of incidental findings in the context of pediatric genomic research. We provide an overview of the current literature and propose a framework to manage incidental findings in this unique context, based on what we believe is a limited responsibility to disclose. We hope this will be a useful source of guidance for investigators, institutional review boards, and bioethicists that anticipates the complicated ethical issues raised by advances in genomic technology.

Free full text available from PubMed PMID: 23400601

3.10 Prenatal

This section includes the following topics:

• Articles on Prenatal Testing (Section 3.10.1); and • Short Presentations on Emerging Concepts: Prenatal Screening (Section 3.10.2).

3.10.1 Articles on Prenatal Testing

A) Ethical and Legal Aspects of Noninvasive Prenatal Genetic Diagnosis Dickens BM. Ethical and legal aspects of noninvasive prenatal genetic diagnosis. Int J Gynaecol Obstet. 2014; 124(2): 181-184.

Summary: The new technology that will allow genetic testing of a fetus within the first trimester of pregnancy by isolating cell-free fetal DNA (cffDNA) in the mother's blood raises a range of ethical and legal issues. Considered noninvasive, this test is safe and reliable, and may avoid alternative genetic testing by amniocentesis or chorionic villus sampling, which risks causing spontaneous abortion. Ethical and legal issues of cffDNA testing will become more acute if testing expands to fetal whole-genome sequencing. Critical issues include the state of the science or diagnostic art; the appropriateness of offering the test; the implications of denying the test when it is available and appropriate; disclosure and counseling following test results; and management of patients' choices on acquiring test results. A challenge will be providing patients with appropriate counseling based on up-to-date genetic knowledge, and accommodating informed patients' legal choices.

Full text available from International Journal of Gynecology & Obstetrics (USD 31.50) PMID: 24299974 NOTE: Also cited in Section 7.3

B) Obstetricians' and Gynecologists' Practice and Opinions of Expanded Carrier Testing and Non-invasive Prenatal Testing Benn P, Chapman AR, Erickson K, et al. Obstetricians' and gynecologists' practice and opinions of expanded carrier testing and non-invasive prenatal testing. Prenat Diagn. 2014; 34(2): 145-52.

Summary: OBJECTIVE: Assess opinions of Fellows of the American College of Obstetricians and Gynecologists (ACOG) on expanded carrier testing (molecular detection of > 100 genetic diseases of variable severity)

and non-invasive prenatal testing (NIPT). METHODS: A survey conducted in between March and August, 2012, assessed current use of testing, provision of genetic counseling, types of disorders that should be identified, preferences for future use, ethical aspects and views on regulatory oversight. RESULTS: Expanded carrier testing was offered to all patients by 15% of the responders and 52.1% upon patient request. Most (67.3%) favored testing only for mutations of known significance. 79.1% supported the use of NIPT as a screen for Down syndrome for all women with 47.9% viewing NIPT as a complete substitution for invasive testing. Most supported expansion to other aneuploidies (97.5%) and severe early onset Mendelian disorders (90.4%), but not for adult onset disorders (29.8%), or non-medical sex identification (15.7%). A majority (73.2%) believed NIPT would increase pregnancy terminations for mild disease states. Respondants favored a role for professional societies in providing regulatory oversight. CONCLUSION: Rapid incorporation of new genetic technologies may be limited by the availability of genetic counseling, concerns regarding inclusion of clinically mild disorders, results of unknown significance, and costs. This article is protected by copyright. All rights reserved.

Full text available from Prenatal Diagnosis (USD 6.00-38.00) PMID: 24222397

C) DNA Sequencing Versus Standard Prenatal Aneuploidy Screening Bianchi DW, Parker RL, Wentworth J, et al. DNA sequencing versus standard prenatal aneuploidy screening. N Engl J Med. 2014; 370(9): 799- 808.

Summary: BACKGROUND: In high-risk pregnant women, noninvasive prenatal testing with the use of massively parallel sequencing of maternal plasma cell-free DNA (cfDNA testing) accurately detects fetal autosomal aneuploidy. Its performance in low-risk women is unclear. METHODS: At 21 centers in the United States, we collected blood samples from women with singleton pregnancies who were undergoing standard aneuploidy screening (serum biochemical assays with or without nuchal translucency measurement). We performed massively parallel sequencing in a blinded fashion to determine the chromosome dosage for each sample. The primary end point was a comparison of the false positive rates of detection of fetal trisomies 21 and 18 with the use of standard screening and cfDNA testing. Birth outcomes or karyotypes were the reference standard. RESULTS: The primary series included 1914 women (mean age, 29.6 years) with an eligible sample, a singleton fetus without aneuploidy, results from cfDNA testing, and a risk classification based on standard screening. For trisomies 21 and 18, the false positive rates with cfDNA testing were significantly lower than those

with standard screening (0.3% vs. 3.6% for trisomy 21, P<0.001; and 0.2% vs. 0.6% for trisomy 18, P=0.03). The use of cfDNA testing detected all cases of aneuploidy (5 for trisomy 21, 2 for trisomy 18, and 1 for trisomy 13; negative predictive value, 100% [95% confidence interval, 99.8 to 100]). The positive predictive values for cfDNA testing versus standard screening were 45.5% versus 4.2% for trisomy 21 and 40.0% versus 8.3% for trisomy 18. CONCLUSIONS: In a general obstetrical population, prenatal testing with the use of cfDNA had significantly lower false positive rates and higher positive predictive values for detection of trisomies 21 and 18 than standard screening. (Funded by Illumina; ClinicalTrials.gov number, NCT01663350.).

Full text available from New England Journal of Medicine PMID: 24571752

D) A New Era in Noninvasive Prenatal Testing Morain S, Greene MF,Mello MM. A new era in noninvasive prenatal testing. N Engl J Med. 2013; 369(6): 499-501.

Summary: Cell-free fetal DNA testing permits earlier detection of common trisomies and generally provides earlier information about a fetus's sex. But with its clinical utility in the general population uncertain, such testing is drifting into routine practice ahead of the evidence.

Full text available from New England Journal of Medicine (subscription required) PMID: 23862975

E) Microarray-Based Prenatal Diagnosis for the Identification of Fetal Chromosome Abnormalities Shaffer LG, Rosenfeld JA. Microarray-based prenatal diagnosis for the identification of fetal chromosome abnormalities. Expert Rev Mol Diagn. 2013; 13(6): 601-611.

Summary: The goal of prenatal cytogenetic testing is to provide reassurance to the couple seeking testing for their pregnancy, identify chromosome abnormalities in the fetus, if present, and provide treatments and medical management for affected babies. Cytogenetic analysis of banded chromosomes has been the standard for identifying chromosome abnormalities in the fetus for over 40 years. With chromosome analysis, whole chromosome aneuploidies and large structural rearrangements can be identified. The sequencing of the human genome has provided the resources to develop molecular tools that allow higher resolution observations of human chromosomes. The future holds the promise of sequencing that may identify

chromosomal imbalances and deleterious single nucleotide variants. This review will focus on the use of genomic microarrays for the testing and identification of chromosome anomalies in prenatal diagnosis and will discuss the future directions of fetal testing.

Full text available from Expert Review of Molecular Diagnostics (USD 89.00 for 24 hours) PMID: 23895129

3.10.2 Short Presentations on Emerging Concepts: Prenatal Screening for Down Syndrome

A) CAP Short Presentations on Emerging Concepts (SPECS): Prenatal Screening for Down Syndrome: Past, Present and Emerging Practices [PowerPoint slides] Moyer AM, Palomaki G. CAP Short Presentations on Emerging Concepts (SPECS): Prenatal Screening for Down Syndrome: Past, Present and Emerging Practices (v 3.0e rev 7/7/15) [PowerPoint slides]. Caughron SK, Chandra PK, Misialek MJ, Nowak JA, eds. Northfield, IL: College of American Pathologists; 2015.

Access the slides

3.11 Mitochondrial Disorders

MitoExome sequencing in 102 patients with suspected mitochondrial disease From Lieber DS, et al190, Targeted exome sequencing of suspected mitochondrial disorders, Neurology, 80(19), 1766.

A) Diagnosis of Mitochondrial Disorders by Concomitant Next-generation Sequencing of the Exome and Mitochondrial Genome Dinwiddie DL, Smith LD, Miller NA, et al. Diagnosis of mitochondrial disorders by concomitant next-generation sequencing of the exome and mitochondrial genome. Genomics. 2013; 102(3): 148-156.

Summary: Mitochondrial diseases are notoriously difficult to diagnose due to extreme locus and allelic heterogeneity, with both nuclear and mitochondrial genomes potentially liable. Using exome sequencing we demonstrate the ability to rapidly and cost effectively evaluate both the nuclear and mitochondrial genomes to obtain a molecular diagnosis for four patients with three distinct mitochondrial disorders. One patient was found to have Leigh syndrome due to a mutation in MT-ATP6, two affected siblings were discovered to be compound heterozygous for mutations in the NDUFV1 gene, which causes mitochondrial complex I deficiency, and one patient was found to have coenzyme Q10 deficiency due to compound heterozygous mutations in COQ2. In all cases conventional diagnostic testing failed to identify a molecular diagnosis. We suggest that additional studies should be conducted to evaluate exome sequencing as a primary diagnostic test for mitochondrial diseases, including those due to mtDNA mutations.

Free full text available from Genomics PMID: 23631824

B) The Development of Next-Generation Sequencing Assays for the Mitochondrial Genome and 108 Nuclear Genes Associated with Mitochondrial Disorders Dames S, Chou LS, Xiao Y, et al. The development of next-generation sequencing assays for the mitochondrial genome and 108 nuclear genes associated with mitochondrial disorders. J Mol Diagn. 2013; 15(4): 526-534.

Summary: Sanger sequencing of multigenic disorders can be technically challenging, time consuming, and prohibitively expensive. High-throughput next-generation sequencing (NGS) can provide a cost-effective method for sequencing targeted genes associated with multigenic disorders. We have developed a NGS clinical targeted gene assay for the mitochondrial genome and for 108 selected nuclear genes associated with mitochondrial disorders. Mitochondrial disorders have a reported incidence of 1 in 5000 live births, encompass a broad range of phenotypes, and are attributed to mutations in the mitochondrial and nuclear genomes. Approximately 20% of mitochondrial disorders result from mutations in mtDNA, with the remaining 80% found in nuclear genes that affect mtDNA levels or mitochondrion protein assembly. In our NGS approach, the 16,569-bp mtDNA is enriched by long-range PCR and the 108 nuclear genes (which represent 1301 amplicons and 680 kb) are enriched by RainDance emulsion PCR. Sequencing is performed on Illumina HiSeq 2000 or MiSeq platforms, and bioinformatics analysis is performed using commercial and in-house developed bioinformatics pipelines. A total of 16 validation and 13 clinical samples were examined. All previously reported variants associated with mitochondrial disorders were found in validation samples, and 5 of the 13 clinical samples were found to have mutations associated with mitochondrial disorders in either the mitochondrial genome or the 108 nuclear genes. All variants were confirmed by Sanger sequencing.

Free full text available from Journal of Molecular Diagnostics PMID: 23665194

C) Targeted Exome Sequencing of Suspected Mitochondrial Disorders Lieber DS, Calvo SE, Shanahan K, et al. Targeted exome sequencing of suspected mitochondrial disorders. Neurology. 2013; 80(19): 1762-1770.

Summary: OBJECTIVE: To evaluate the utility of targeted exome sequencing for the molecular diagnosis of mitochondrial disorders, which exhibit marked phenotypic and genetic heterogeneity. METHODS: We considered a diverse set of 102 patients with suspected mitochondrial disorders based on clinical, biochemical, and/or molecular findings, and whose disease ranged from mild to severe, with varying age at onset. We sequenced the mitochondrial genome (mtDNA) and the exons of 1,598

nuclear-encoded genes implicated in mitochondrial biology, mitochondrial disease, or monogenic disorders with phenotypic overlap. We prioritized variants likely to underlie disease and established molecular diagnoses in accordance with current clinical genetic guidelines. RESULTS: Targeted exome sequencing yielded molecular diagnoses in established disease loci in 22% of cases, including 17 of 18 (94%) with prior molecular diagnoses and 5 of 84 (6%) without. The 5 new diagnoses implicated 2 genes associated with canonical mitochondrial disorders (NDUFV1, POLG2), and 3 genes known to underlie other neurologic disorders (DPYD, KARS, WFS1), underscoring the phenotypic and biochemical overlap with other inborn errors. We prioritized variants in an additional 26 patients, including recessive, X-linked, and mtDNA variants that were enriched 2-fold over background and await further support of pathogenicity. In one case, we modeled patient mutations in yeast to provide evidence that recessive mutations in ATP5A1 can underlie combined respiratory chain deficiency. CONCLUSION: The results demonstrate that targeted exome sequencing is an effective alternative to the sequential testing of mtDNA and individual nuclear genes as part of the investigation of mitochondrial disease. Our study underscores the ongoing challenge of variant interpretation in the clinical setting.

Free full text available from PubMed PMID: 23596069

3.12 Hereditary Cancer Syndromes

Quick Reference Table: Hereditary Cancers and Associated Genes

This table is for quick reference only. Clinical decision making should not be based solely on this information. Check the CAP website for the most up-to-date CAP Proficiency Testing Menu

Disease name (MIM Gene (MIM CAP PT ACMG Number*) Number*) Available Incidental Findings Recommen- dations** Adenomatous polyposis coli APC (MIM x (MIM 175100) 611731) Birt-Hogg-Dube syndrome FLCN (MIM (MIM 135150) 607273)

Breast-ovarian cancer, BRCA1 (MIM x x familial 1 (MIM 604370) 113705) Breast-ovarian cancer, BRCA2 (MIM x x familial 2 (MIM 612555) 600185) Breast cancer, familial (MIM114480)/Hereditary CDH1 (MIM diffuse gastric cancer 192090) (MIM137215) Familial medullary thyroid RET (MIM x carcinoma (MIM 155240) 164761) BMPR1A (MIM Juvenile polyposis (MIM 601299) 174900) SMAD4 (MIM 600993) Li-Fraumeni syndrome 1 TP53 (MIM x (MIM 151623) 191170) MLH1 (MIM x 120436) MSH2 (MIM x Lynch syndrome (MIM 609309) 120435) MSH6 (MIM x 600678) PMS2 (MIM x 600259) Multiple endocrine neoplasia, MEN1 (MIM x type 1 (MIM 131100) 613733) Multiple endocrine neoplasia, RET (MIM x x type 2a (MIM 171400) 164761) Multiple endocrine neoplasia, RET (MIM x x type 2b (MIM 162300) 164761) MYH-associated polyposis MUTYH (MIM x (MIM 608456) 604933) Oligodontia-colorectal cancer AXIN2 (MIM syndrome (MIM608615) 604025) Peutz-Jeghers syndrome STK11 (MIM x (MIM 175200) 602216)

SDHB (MIM 185470) SDHD (MIM 602690) VHL (MIM 608537) RET (MIM 164761) Pheochromocytoma (MIM MAX 171300) (MIM154950) TMEM127 (MIM 613403) KIF1B (MIM 605995) GDNF (MIM 600837) Pilomatrixoma (MIM 132600)/MYH-associated MUTYH (MIM polyposis x 604933) (MIM608456)/Neoplasms of Stomach (MIM613659) PTEN hamartoma tumor PTEN (MIM x syndrome (MIM 153480) 601728) MET (MIM Renal cell carcinoma, 164860) papillary 1, familial (MIM PRCC 605074) (MIM179755) Hereditary leiomyomatosis FH and renal cell cancer (MIM (MIM136850) 150800) Retinoblastoma (MIM RB1 (MIM x 180200) 614041) WT1 (MIM Wilms' tumor (MIM 194070) x 607102) Source: Ann M Moyer, MD, PhD *MIM numbers can be entered at www.omim.org to query the Online Mendelial Inheritance in Man Online Catalog of Human Genes and Genetic Disorders **Genet Med. 2013;15(7):565-74

Additional References: • 2016 Surveys. College of American Pathologists website. https://estore.cap.org/OA_HTML/ibeCCtpSctDspRte.jsp?section=10060&sitex=10020:2237 2:US. Accessed July 15, 2016.

Return to Table of Contents Section 3

• GeneReviews (www.ncbi.nlm.nih.gov/books/NBK1116/), editors: Pagon RA, Adam MP, Ardinger HH, Wallace SE, Amemiya A, Bean LJH, Bird TD, Fong C, Mefford HC, Smith RJH, Stephens K. Seattle (WA): University of Washington, Seattle; 1993-2016. • Online Mendelian Inheritance in Man (www.omim.org), McKusic-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, directed by Hamosh, A. • Green RC, Berg JS, Grody WW, et al. Genet Med. 2013 Jul; 15(7):565-74.

A) Targeted Therapy for Hereditary Cancer Syndromes: Hereditary Breast and Ovarian Cancer Syndrome, Lynch Syndrome, Familial Adenomatous Polyposis, and Li-Fraumeni Syndrome Agarwal R, Liebe S, Turski ML, et al. Targeted therapy for hereditary cancer syndromes: hereditary breast and ovarian cancer syndrome, Lynch syndrome, familial adenomatous polyposis, and Li-Fraumeni syndrome. Discov Med. 2014; 18(101): 331-339.

Summary: Cancer genetics has rapidly evolved in the last two decades. Understanding and exploring the several genetic pathways in the cancer cell is the foundation of targeted therapy. Several genomic aberrations have been identified and their role in carcinogenesis is being explored. In contrast to most cancers where these mutations are acquired, patients with hereditary cancer syndromes have inherited genomic aberrations. The understanding of the molecular pathobiology in hereditary cancer syndromes has advanced dramatically. In addition, many molecularly targeted therapies have been developed that could have potential roles in the treatment of patients with hereditary cancer syndromes. In this review, we outline the presentation, molecular biology, and possible targeted therapies for two of the most widely recognized hereditary cancer syndromes -- hereditary breast and ovarian cancer syndrome and hereditary non-polyposis colorectal cancer syndrome (Lynch syndrome). We will also discuss other syndromes such as familial adenomatous polyposis and Li-Fraumeni syndrome (TP53).

Free full text available from Discovery Medicine PMID: 25549704

B) A Practice Guideline from the American College of Medical Genetics and Genomics and the National Society of Genetic Counselors: Referral Indications for Cancer Predisposition Assessment Hampel H, Bennett RL, Buchanan A, Pearlman R, Wiesner GL. A practice guideline from the American College of Medical Genetics and Genomics and the National Society of Genetic Counselors: referral indications for cancer predisposition assessment. Genet Med. 2015;17(1):70-87.

Free full text available from Genetics in Medicine PMID: 25394175 NOTE: Also cited in Section 3.9.1

3.13 Post Mortem

A) Confirmation of Cause and Manner of Death Via a Comprehensive Cardiac Autopsy Including Whole Exome Next-Generation Sequencing Loporcaro CG, Tester DJ, Maleszewski JJ, Kruisselbrink T, Ackerman MJ. Confirmation of cause and manner of death via a comprehensive cardiac autopsy including whole exome next-generation sequencing. Arch Pathol Lab Med. 2014; 138(8): 1083-1089.

Summary: Annually, the sudden death of thousands of young people remains inadequately explained despite medicolegal investigation. Postmortem genetic testing for channelopathies/cardiomyopathies may illuminate a potential cardiac mechanism and establish a more accurate cause and manner of death and provide an actionable genetic marker to test surviving family members who may be at risk for a fatal arrhythmia. Whole exome sequencing allows for simultaneous genetic interrogation of an

individual's entire estimated library of approximately 30000 genes. Following an inconclusive autopsy, whole exome sequencing and gene-specific surveillance of all known major cardiac channelopathy/cardiomyopathy genes (90 total) were performed on autopsy blood-derived genomic DNA from a previously healthy 16-year-old adolescent female found deceased in her bedroom. Whole exome sequencing analysis revealed a R249Q-MYH7 mutation associated previously with familial hypertrophic cardiomyopathy, sudden death, and impaired beta-myosin heavy chain (MHC-beta) actin- translocating and actin-activated ATPase (adenosine triphosphatase) activity. Whole exome sequencing may be an efficient and cost-effective approach to incorporate molecular studies into the conventional postmortem examination.

Free full text available from the CAP’s Archives PMID: 24298987

B) Phenotype Driven Molecular Autopsy for Sudden Cardiac Death Cann F, Corbett M, O'Sullivan D, et al. Phenotype Driven Molecular Autopsy for Sudden Cardiac Death. Clin Genet. 2016 Mar 22. doi: 10.1111/cge.12778. [Epub ahead of print]

Summary: A phenotype driven approach to molecular autopsy based in a multidisciplinary team comprising clinical and laboratory genetics, forensic medicine and cardiology is described. Over a 13 year period, molecular autopsy was undertaken in 96 sudden cardiac death cases. 46 cases aged 1-40 years had normal hearts and suspected arrhythmic death. 7 (15%) had likely pathogenic variants in ion channelopathy genes (KCNQ1 (1), KCNH2 (4), SCN5A (1), RyR2(1)). 50 cases aged between 2 and 67 had a cardiomyopathy. 25 had Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC), 10 Dilated Cardiomyopathy (DCM) and 15 Hypertrophic Cardiomyopathy (HCM). Likely pathogenic variants were found in 3 ARVC cases (12%) in PKP2, DSC2 or DSP, 2 DCM cases (20%) in MYH7, and 4 HCM cases (27%) in MYBPC3 (3) or MYH7 (1). Uptake of cascade screening in relatives was higher when a molecular diagnosis was made at autopsy. In 3 families, variants previously published as pathogenic were detected, but clinical investigation revealed no abnormalities in carrier relatives. With a conservative approach to defining pathogenicity of sequence variants incorporating family phenotype information and population genomic data, a molecular diagnosis was made in 15% of sudden arrhythmic deaths and 18% of cardiomyopathy deaths.

Full text available from Clinical Genetics (USD 6.00-38.00) PMID: 27000522

Molecular diagnostic techniques have been used to identify the presence of infectious agents in humans for over 20 years. In 1996, the Patient Preparation and Specimen Handling Editorial Board of the College of American Pathologists published fascicle VII entitled Reference Guide for Diagnostic Pathology / Flow Cytometry, of the 134 entries, 50 defined the detection of infectious diseases using molecular testing including 18 viruses, 18 bacteria, 12 parasites and 2 fungi. The field has made great progress, including the introduction of additional assays using biplex (herpes simplex viruses I and II) or multiplex (respiratory virus panels or gastrointestinal pathogen panels) strategies. The introduction of assays with rapid turnaround times in the local laboratory provides improved patient care and outcomes, reduced length of stay, and decreased costs. Therefore, this group of assays is often considered as the place to start when initially planning a molecular diagnostics laboratory.

This section includes articles on the following topics:

• Types of Testing for Infectious Diseases (Section 4.1); • Viruses (Section 4.2); • Bacteria (Section 4.3); • Fungi (Section 4.4); • Parasites (Section 4.5); • Pan-Infection Detection (Section 4.6); and • Clinical Application of Genomics in Infectious Disease (Section 4.7).

4.1 Types of Testing for Infectious Diseases

A) Nanotools and Molecular Techniques to Rapidly Identify and Fight Bacterial Infections Dinarelli S, Girasole M, Kasas S, Longo G. Nanotools and Molecular Techniques to Rapidly Identify and Fight Bacterial Infections. J Microbiol Methods. 2016. pii: S0167-7012(16)30006-9. doi: 10.1016/j.mimet.2016.01.005. [Epub ahead of print]

Summary: Reducing the emergence and spread of antibiotic-resistant bacteria is one of the major healthcare issues of our century. In addition to the increased mortality, infections caused by multi-resistant bacteria drastically enhance the healthcare costs, mainly because of the longer

duration of illness and treatment. While in the last 20 years, bacterial identification has been revolutionized by the introduction of new molecular techniques, the current phenotypic techniques to determine the susceptibilities of common Gram-positive and Gram-negative bacteria require at least two days from collection of clinical samples. Therefore, there is an urgent need for the development of new technologies to determine rapidly drug susceptibility in bacteria and to achieve faster diagnoses. These techniques would also lead to a better understanding of the mechanisms that lead to the insurgence of the resistance, greatly helping the quest for new antibacterial systems and drugs. In this review, we describe some of the tools most currently used in clinical and microbiological research to study bacteria and to address the challenge of infections. We discuss the most interesting advancements in the molecular susceptibility testing systems, with a particular focus on the many applications of the MALDI-TOF MS system. In the field of the phenotypic characterization protocols, we detail some of the most promising semi-automated commercial systems and we focus on some emerging developments in the field of nanomechanical sensors, which constitute a step towards the development of rapid and affordable point-of- care testing devices and techniques. While there is still no innovative technique that is capable of completely substituting for the conventional protocols and clinical practices, many exciting new experimental setups and tools could constitute the basis of the standard testing package of future microbiological tests.

Full text available from Journal of Microbiological Methods (USD 39.95) PMID: 26806415

B) Next-Generation Sequencing for Infectious Disease Diagnosis and Management: A Report of the Association for Molecular Pathology Lefterova MI, Suarez CJ, Banaei N,Pinsky BA. Next-Generation Sequencing for Infectious Disease Diagnosis and Management: A Report of the Association for Molecular Pathology. J Mol Diagn. 2015; 17(6): 623-634.

Summary: Next-generation sequencing (NGS) technologies are increasingly being used for diagnosis and monitoring of infectious diseases. Herein, we review the application of NGS in clinical microbiology, focusing on genotypic resistance testing, direct detection of unknown disease-associated pathogens in clinical specimens, investigation of microbial population diversity in the human host, and strain typing. We have organized the review into three main sections: i) applications in clinical virology, ii) applications in clinical bacteriology, mycobacteriology, and mycology, and iii) validation, quality control, and maintenance of proficiency. Although NGS holds enormous promise for clinical infectious disease testing, many challenges

remain, including automation, standardizing technical protocols and bioinformatics pipelines, improving reference databases, establishing proficiency testing and quality control measures, and reducing cost and turnaround time, all of which would be necessary for widespread adoption of NGS in clinical microbiology laboratories.

Free full text available from Journal of Molecular Diagnostics PMID: 26433313

C) Comparison of 16S rRNA Gene PCR and Blood Culture for Diagnosis of Neonatal Sepsis Liu CL, Ai HW, Wang WP, et al. Comparison of 16s rRNA Gene PCR and Blood Culture for Diagnosis of Neonatal Sepsis. Arch Pediatr. 2014; 21(2): 162-169.

Summary: Septicemia is a common cause of morbidity and mortality among newborns in the developing world. However, accurate clinical diagnosis of neonatal sepsis is often difficult because symptoms and signs are often nonspecific. Blood culture has been the gold standard for confirmation of the diagnosis. However, the sensitivity is low and results are usually not promptly obtained. Therefore, the diagnosis of sepsis is often based on clinical signs in association with laboratory tests such as platelets count, immature/total neutrophils ratio (I/T), and a rise in C-reactive protein (CRP). Polymerase chain reaction (PCR) methods for the detection of neonatal sepsis represent new diagnostic tools for the early identification of pathogens. METHODS: During a 4-month prospective study, 16S rRNA PCR was compared with conventional blood culture for the diagnosis of neonatal bacterial sepsis. In addition, the relationship between known risk factors, clinical signs, laboratory parameters, and the diagnosis of sepsis was considered. RESULTS: Sepsis was suspected in 706 infants from the intensive neonatal care unit. They all were included in the study. The number of positive cultures and positive PCR results were 95 (13.5%) and 123 (17.4%), respectively. Compared with blood culture, the diagnosis of bacterial sepsis by PCR revealed a 100.0% sensitivity, 95.4% specificity, 77.2% positive predictive value, and 100.0% negative predictive value. In this study, Apgar scores at 5 min, weight, icterus, irritability, feeding difficulties, gestational age (GA), premature rupture of membrane (PRM), platelets count, I/T, and a marked rise in CRP were important in establishing the diagnosis of sepsis in the newborn. In addition, weight, GA, PRM, irritability, duration of antibiotic usage, mortality rate, and number of purulent meningitis cases were significantly different between early-onset sepsis and late-onset sepsis. CONCLUSION: 16S rRNA PCR increased the sensitivity in detecting bacterial DNA in newborns with signs of sepsis, allowed a rapid detection of

the pathogens, and led to shorter antibiotic courses. However, uncertainty about the bacterial cause of sepsis was not reduced by this method. 16S rRNA PCR needs to be further developed and improved. Blood culture is currently irreplaceable, since pure isolates are essential for antimicrobial drug susceptibility testing.

Full text available from Archives de Pédiatrie (USD 35.95) PMID: 24388336

D) Molecular Diagnostic Methods for Invasive Fungal Disease: The Horizon Draws Nearer? Halliday CL, Kidd SE, Sorrell TC, Chen SC. Molecular Diagnostic Methods for Invasive Fungal Disease: The Horizon Draws Nearer? Pathology. 2015; 47(3): 257-269.

Summary: Rapid, accurate diagnostic laboratory tests are needed to improve clinical outcomes of invasive fungal disease (IFD). Traditional direct microscopy, culture and histological techniques constitute the 'gold standard' against which newer tests are judged. Molecular diagnostic methods, whether broad-range or fungal-specific, have great potential to enhance sensitivity and speed of IFD diagnosis, but have varying specificities. The use of PCR-based assays, DNA sequencing, and other molecular methods including those incorporating proteomic approaches such as matrix-assisted laser desorption ionisation-time of flight mass spectroscopy (MALDI-TOF MS) have shown promising results. These are used mainly to complement conventional methods since they require standardisation before widespread implementation can be recommended. None are incorporated into diagnostic criteria for defining IFD. Commercial assays may assist standardisation. This review provides an update of molecular-based diagnostic approaches applicable to biological specimens and fungal cultures in microbiology laboratories. We focus on the most common pathogens, Candida and Aspergillus, and the mucormycetes. The position of molecular-based approaches in the detection of azole and echinocandin antifungal resistance is also discussed.

Full text available from Pathology (subscription required) PMID: 25719852

E) Quantitative Nucleic Acid Amplification Methods for Viral Infections Gullett JC, Nolte FS. Quantitative Nucleic Acid Amplification Methods for Viral Infections. Clin Chem. 2015; 61(1): 72-78.

Summary: BACKGROUND: Over the past 2 decades there have been substantial improvements in the methods used to quantify viral nucleic acid in body fluids and in our understanding of how to use viral load measurements in the diagnosis and management of patients with a number of viral infections. These methods are now integrated into a wide range of diagnostic and treatment guidelines and commonly deployed in a variety of clinical settings. CONTENT: Quantitative nucleic acid amplification methods that are used to measure viral load are described along with key issues and important variables that affect their performance. Particular emphasis is placed on those methods used in clinical laboratories as US Food and Drug Administration-cleared or laboratory-developed tests. We discuss the clinical applications of these methods in patients with HIV-1, hepatitis C virus, hepatitis B virus, cytomegalovirus, Epstein-Barr virus, and BK polyomavirus infections. Finally, the current challenges and future directions of viral load testing are examined. SUMMARY: Quantitative nucleic acid amplification tests provide important information that can be used to predict disease progression, distinguish symptomatic from asymptomatic infection, and assess the efficacy of antiviral therapy. Despite the advances in technology, large challenges remain for viral testing related to accuracy, precision, and standardization. Digital PCR, a direct method of quantification of nucleic acids that does not rely on rate-based measurements or calibration curves, may address many of the current challenges.

Free full text available from Clinical Chemistry

PMID: 25403817

F) Molecular Methods and Platforms for Infectious Diseases Testing: A Review of FDA-Approved and Cleared Assays Emmadi R, Boonyaratanakornkit JB, Selvarangan R, et al. Molecular methods and platforms for infectious diseases testing: a review of FDA- approved and cleared assays. J Mol Diagn. 2011; 13(6):583–604.

Summary: The superior sensitivity and specificity associated with the use of molecular assays has greatly improved the field of infectious disease diagnostics by providing clinicians with results that are both accurate and rapidly obtained. Herein, we review molecularly based infectious disease diagnostic tests that are Food and Drug Administration approved or cleared and commercially available in the United States as of December 31, 2010. We describe specific assays and their performance, as stated in the Food and Drug Administration's Summary of Safety and Effectiveness Data or the Office of In Vitro Diagnostic Device Evaluation and Safety's decision summaries, product inserts, or peer-reviewed literature. We summarize indications for testing, limitations, and challenges related to implementation in a clinical laboratory setting for a wide variety of common pathogens. The information presented in this review will be particularly useful for laboratories that plan to implement or expand their molecular offerings in the near term.

Free full text available from Journal of Molecular Diagnostics PMID: 21871973

G) Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing Espy MJ, Uhl JR, Sloan LM, et al. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev. 2006; 19(1):165-256.

Summary: Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-

time PCR technology an appealing alternative to culture-or immunoassay- based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory.

Free full text available from PubMed PMID: 16418529

4.2 Viruses

This section includes articles on the following topics:

• Cytomegalovirus (Section 4.2.1); • Enterovirus (Section 4.2.2); • Epstein Barr Virus (Section 4.2.3); • Hepatitis B Virus (Section 4.2.4); • Hepatitis C Virus (Section 4.2.5); • Human Immunodeficiency Virus (Section 4.2.6); • Respiratory Viruses (Section 4.2.7); • Herpes Simplex I and II (Section 4.2.8); • Norovirus (Section 4.2.9); • Ebola Virus (Section 4.2.10); and • Influenza (Section 4.2.11).

4.2.1 Cytomegalovirus

A) The Expanding Role of Cytomegalovirus as a Human Pathogen Navarro D. The Expanding Role of Cytomegalovirus as a Human Pathogen. J Med Virol. 2016; 88(7):1103-12.

Summary: Cytomegalovirus (CMV) continues to be a leading cause of morbidity and mortality in transplant recipients, despite major advancements in preventative strategies. Antiviral prophylaxis and pre-emptive antiviral therapeutic regimens are associated with a high incidence of late-onset end- organ disease and cause drug-related toxicity when overused. Therefore, the identification of risk factors for CMV replication is required. Genetic and immunological factors that predispose individuals to CMV-related clinical complications have been identified and may be instrumental for optimizing CMV treatment in the future. Evidence suggests a causal pathogenetic link between CMV-related complications and inflammatory diseases in non-

canonically immunosuppressed individuals such as patients with lung injury and critically ill and cancer patients. However, a randomized clinical trial is required to determine if a causal relationship exists.

Full text available from Journal of Medical Virology (USD 6.00-38.00) PMID: 26681168

B) Comparison of Two Quantitative Real-Time CMV-PCR Tests Calibrated Against the 1st WHO International Standard for Viral Load Monitoring of Renal Transplant Patients Mannonen L, Loginov R, Helantera I, et al. Comparison of Two Quantitative Real-Time CMV-PCR Tests Calibrated against the 1st WHO International Standard for Viral Load Monitoring of Renal Transplant Patients. J Med Virol. 2014; 86(4): 576-584.

Summary: Cytomegalovirus (CMV) replication in organ transplant recipients is commonly diagnosed by quantitative PCR methods. However, there has been a poor inter-laboratory correlation of viral load values due to the lack of an international reference standard. In a recent study, the COBAS(R) AmpliPrep/COBAS(R) TaqMan(R) (CAP/CTM) CMV test calibrated to the 1st WHO CMV standard, showed good reproducibility in CMV load values across multiple laboratories. Fifty-seven follow-up plasma specimens from 10 kidney transplant recipients with CMV replication were examined using the new quantitative CAP/CTM CMV test and the "in-house" quantitative CMV real- time PCR method, also calibrated against the 1st WHO CMV standard for their clinical applicability for monitoring CMV load in renal transplant patients. By CAP/CTM CMV test 49/57 specimens were CMV-DNA positive compared to 44/57 by the "in-house" PCR test. The "in-house" PCR and CAP/CTM CMV test correlated well in monitoring individual kidney transplant patients. Conversion of the CMV-DNA copies to IUs made the results of the "in-house" PCR and CAP/CTM CMV test less uniform in analysis of the patient samples. In specimens of one patient, significant underquantification of CMV load with "in-house" PCR emerged during follow-up due to a point mutation in the "in- house" PCR primer sequence. The CAP/CTM CMV test was found suitable for diagnosing and monitoring CMV replication in renal transplant patients. Multicenter studies are needed to provide more information of the commutability of the 1st WHO CMV standard and to define the clinical thresholds.

Full text available from Journal of Medical Virology (USD 6.00-38.00) PMID: 24026892

C) Clinical Utility of Cytomegalovirus Viral Load in Solid Organ Transplant Recipients Dioverti MV, Razonable RR. Clinical Utility of Cytomegalovirus Viral Load in Solid Organ Transplant Recipients. Curr Opin Infect Dis. 2015; 28(4): 317- 322.

Summary: PURPOSE OF REVIEW: Quantitative nucleic acid testing (QNAT) to measure viral load has become a mainstay in the management of cytomegalovirus (CMV) infection and disease in solid organ transplant recipients. In this article, we review the clinical applications of CMV QNAT in the management of solid organ transplant recipients. RECENT FINDINGS: Because several platforms were available for CMV QNAT, there was a wide inter-assay variability in the viral load reporting, and this limited the generation of widely applicable viral load thresholds that can be used for various clinical applications. With the recent availability of international standard and certified reference materials, there is now opportunity to standardize viral load reporting, with the goal of deriving viral load thresholds for various clinical applications, such as rapid diagnosis of CMV infection and disease, predicting the risk of disease and assessing the severity of illness, monitoring efficacy of antiviral therapies and assessing the risk of viral relapse and drug resistance. SUMMARY: Recent advances in the field such as CMV QNAT standardization, as discussed in this review, are anticipated to optimize the management of CMV infection and disease in solid organ transplant recipients.

Full text available from Current Opinion in Infectious Disease (subscription required) PMID: 26098497

D) Overview: Cytomegalovirus and the Herpesviruses in Transplantation Fishman JA. Overview: cytomegalovirus and the herpesviruses in transplantation. Am J Transplant. 2013; 13 Suppl 3(1-8); quiz 8.

Summary: Herpesviruses infect most animal species. Infections due to the eight human herpesviruses (HHV) are exacerbated by immunosuppression in organ transplantation. The special features of the herpesvirus life cycle include the ability to establish latent, nonproductive infection and the life-long capacity for reactivation to productive, lytic infection. Interactions between latent virus and the immune system determine the frequency and severity of symptomatic infections. The immunologic and cellular effects of herpesvirus infections contribute to risk for opportunistic infections and graft rejection. Among the most important advances in transplantation are laboratory assays for the diagnosis and monitoring of herpesvirus infections and antiviral

agents with improved efficacy in prophylaxis and therapy. For herpes simplex virus, varicella zoster virus and cytomegalovirus, these advances have significantly reduced the morbidity of infection. The syndromes of EBV- associated posttransplant lymphoproliferative disorders (PTLD) and Kaposi's sarcoma remain important complications of immunosuppression. The epidemiology and essential biology of human herpesvirus is reviewed.

Free full text abstract available from American Journal of Transplantation PMID: 23347210

4.2.2 Enterovirus

A) Molecular Evolution and Intra-Clade Recombination of Enterovirus D68 During the 2014 Outbreak in the United States Tan Y, Hassan F, Schuster JE, et al. Molecular Evolution and Intra-Clade Recombination of Enterovirus D68 During the 2014 Outbreak in the United States. J Virol. 2015; 90(4):1997-2007.

Summary: In August 2014 an outbreak of enterovirus D68 (EV-D68) occurred in North America, causing severe respiratory disease in children. Due to a lack of complete genome sequence data there is only a limited understanding of the molecular evolution and epidemiology of EV-D68 during this outbreak, and it is uncertain whether the differing clinical manifestations of EV-D68 infection are associated with specific viral lineages. We developed a high-throughput complete genome sequencing pipeline for EV-D68 that produced a total of 59 complete genomes from respiratory samples with a 95% success rate, including 57 genomes from Kansas City, Missouri collected during the 2014 outbreak. With these data in-hand we performed phylogenetic analyses of complete genome and VP1 capsid protein sequences. Notably, we observed considerable genetic diversity among EV- D68 isolates in Kansas City, manifest as phylogenetically distinct lineages, indicative of multiple introductions of this virus into the city. In addition, we identified an inter-subclade recombination event within EV-D68, the first recombinant in this virus reported to date. Finally, we found no significant association between EV-D68 genetic variation, either lineages or individual mutations, and a variety of demographic and clinical variables, suggesting that host factors likely play a major role in determining disease severity. Overall, our study revealed the complex pattern of viral evolution within a single geographic locality during a single outbreak, which has implications for the design of effective intervention and prevention strategies. IMPORTANCE: Until recently EV-D68 was considered to be an uncommon human pathogen, associated with mild respiratory illness. However, in 2014 EV-D68 was

responsible for more than 1000 disease cases in North America, including severe respiratory illness in children and acute flaccid myelitis, raising concerns about its potential impact on public health. Despite the emergence of EV-D68, a lack of full-length genome sequences means that little is known about the molecular evolution of this virus within a single geographic locality during a single outbreak. Herein, we doubled the number of publically available complete genome sequences of EV-D68 by performing high- throughput next-generation sequencing, characterized the evolutionary history of this outbreak in detail, identified a recombination event, and investigated whether there was any correlation between the demographic and clinical characteristics of the patients and the viral variant that infected them. Overall, these results will help inform the design of intervention strategies for EV-D68.

Full text available from Journal of Virology (USD 25.00 for 24 hour access) PMID: 26656685

4.2.3 Epstein Barr Virus

A) The Biology and Clinical Utility of EBV Monitoring in Blood Kanakry J, Ambinder R. The Biology and Clinical Utility of EBV Monitoring in Blood. Curr Top Microbiol Immunol. 2015; 391(475-499).

Summary: Epstein-Barr virus (EBV) DNA in blood can be quantified in peripheral blood mononuclear cells, in circulating cell-free (CCF) DNA specimens, or in whole blood. CCF viral DNA may be actively released or extruded from viable cells, packaged in virions or passively shed from cells during apoptosis or necrosis. In infectious mononucleosis, viral DNA is detected in each of these kinds of specimens, although it is only transiently detected in CCF specimens. In nasopharyngeal carcinoma, CCF EBV DNA is an established tumor marker. In EBV-associated Hodgkin lymphoma and in EBV-associated extranodal NK-/T-cell lymphoma, there is growing evidence for the utility of CCF DNA as a tumor marker.

Book available from Springer (USD 149.00) PMID: 26428386

B) Determining EBV Load: Current Best Practice and Future Requirements Ruf S, Wagner HJ. Determining EBV load: current best practice and future requirements. Expert Rev Clin Immunol. 2013; 9(2): 139-151.

Summary: EBV, a gammaherpesvirus and the pathogenic agent for infectious mononucleosis, is also associated with a broad spectrum of lymphoid and epithelial malignancies in immunocompetent and immunosuppressed individuals. EBV-DNA-load measurement by PCR has been shown to be a potential tool for the diagnosis of these diseases, a prognostic factor of their outcome and a successful method to monitor immunosuppressed patients. Since the end of 2011, there is an international WHO standard reference for EBV quantification available; however, many questions still remain; for instance about the optimal amplified region of the EBV genome, or the best-used specimen for EBV detection. Additionally, the optimal specimen and amplified region may vary in different malignancies. In this article, the authors review the different methods to measure EBV load, focus on the best-used specimen for the different EBV-associated malignancies and discuss future requirements and opportunities for EBV- load measurement.

Full text available from Expert Review of Clinical Immunology (USD 89.00) PMID: 23390945

C) Epstein-Barr Virus Infection and Posttransplant Lymphoproliferative Disorder Green M, Michaels MG. Epstein-Barr virus infection and posttransplant lymphoproliferative disorder. Am J Transplant. 2013; 13 Suppl 3:41-54; quiz 54.

Summary: Epstein-Barr virus (EBV) is an important pathogen in recipients of solid organ transplants (SOT). Infection with EBV manifests as a spectrum of diseases/malignancies ranging from asymptomatic viremia through infectious mononucleosis to posttransplant lymphoproliferative disorder (PTLD). EBV disease and its associated PTLD is more frequently seen when primary EBV infection occurs after transplant, a common scenario in pediatric SOT recipients. Intensity of immunosuppressive therapies also influences the risk for PTLD. The use of EBV viral load monitoring facilitates the diagnosis and management of EBV/PTLD as well as being used to inform preemptive therapy with reduction of immunosuppression, the most effective intervention for prevention of and treatment for PTLD. Other therapies, including the rituximab (anti-CD20 monoclonal antibody) and traditional chemotherapy, are also useful in the treatment of established PTLD. The future development of standards for management based on EBV viral load and routine monitoring of EBV-specific CTL responses promise further improvement in outcomes with EBV and PTLD.

Free full text abstract available from American Journal of Transplantation

PMID: 23347213

4.2.4 Hepatitis B Virus

A) Rapid and Quantitative Detection of Hepatitis B Virus Liu YP,Yao CY. Rapid and Quantitative Detection of Hepatitis B Virus. World J Gastroenterol. 2015; 21(42): 11954-11963.

Summary: Despite availability of a universal vaccine, hepatitis B virus (HBV) infection has a huge impact on public health worldwide. Accurate and timely diagnosis of HBV infection is needed. Rapid developments have been made in the diagnostic and monitoring methods for HBV infection, including serological and molecular assays. In clinical practice, qualitative hepatitis B surface antigen (HBsAg) testing has long served as a diagnostic marker for individuals infected with HBV. More recently, HBsAg level has been used to predict treatment outcome when determined early during treatment or at baseline. However, identification of HBV DNA positive cases that do not have detectable HBsAg has encouraged the application of molecular tests. Hence, combination of quantitative detection of HBV DNA and HBsAg can be used to discriminate patients during the course of HBV infection and to monitor therapy. This article reviews the most commonly used quantitative methods for HBsAg and HBV DNA.

Free full text available from PubMed PMID: 26576084

B) Recent Advances in Molecular Diagnostics of Hepatitis B Virus Datta S, Chatterjee S, Veer V. Recent Advances in Molecular Diagnostics of Hepatitis B Virus. World J Gastroenterol. 2014; 20(40): 14615-14625.

Summary: Hepatitis B virus (HBV) is one of the important global health problems today. Infection with HBV can lead to a variety of clinical manifestations including severe hepatic complications like liver cirrhosis and hepatocellular carcinoma. Presently, routine HBV screening and diagnosis is primarily based on the immuno-detection of HBV surface antigen (HBsAg). However, identification of HBV DNA positive cases, who do not have detectable HBsAg has greatly encouraged the use of nucleic acid amplification based assays, that are highly sensitive, specific and are to some extent tolerant to sequence variation. In the last few years, the field of HBV molecular diagnostics has evolved rapidly with advancements in the molecular biology tools, such as polymerase chain reaction (PCR) and real- time PCR. Recently, apart of PCR based amplification methods, a number of

isothermal amplification assays, such as loop mediated isothermal amplification, transcription mediated amplification, ligase chain reaction, and rolling circle amplification have been utilized for HBV diagnosis. These assays also offer options for real time detection and integration into biosensing devices. In this manuscript, we review the molecular technologies that are presently available for HBV diagnostics, with special emphasis on isothermal amplification based technologies. We have also included the recent trends in the development of biosensors and use of next generation sequencing technologies for HBV.

Free full text available from PubMed PMID: 25356025

C) Detection of Hepatitis B Virus Infection: A Systematic Review Ghosh M, Nandi S, Dutta S, Saha MK. Detection of Hepatitis B Virus Infection: A Systematic Review. World J Hepatol. 2015; 7(23): 2482-2491.

Summary: AIM: To review published methods for detection of hepatitis B virus (HBV) infection. METHODS: A thorough search on Medline database was conducted to find original articles describing different methods or techniques of detection of HBV, which are published in English in last 10 years. Articles outlining methods of detection of mutants or drug resistance were excluded. Full texts and abstracts (if full text not available) were reviewed thoroughly. Manual search of references of retrieved articles were also done. We extracted data on different samples and techniques of detection of HBV, their sensitivity (Sn), specificity (Sp) and applicability. RESULTS: A total of 72 studies were reviewed. HBV was detected from dried blood/plasma spots, hepatocytes, ovarian tissue, cerumen, saliva, parotid tissue, renal tissue, oocytes and embryos, cholangiocarcinoma tissue, etc. Sensitivity of dried blood spot for detecting HBV was > 90% in all the studies. In case of seronegative patients, HBV DNA or serological markers have been detected from hepatocytes or renal tissue in many instances. Enzyme linked immunosorbent assay and Chemiluminescent immunoassay (CLIA) are most commonly used serological tests for detection. CLIA systems are also used for quantitation. Molecular techniques are used qualitatively as well as for quantitative detection. Among the molecular techniques version 2.0 of the CobasAmpliprep/CobasTaqMan assay and Abbott's real time polymerase chain reaction kit were found to be most sensitive with a lower detection limit of only 6.25 IU/mL and 1.48 IU/mL respectively. CONCLUSION: Serological and molecular assays are predominant and reliable methods for HBV detection. Automated systems are highly sensitive and quantify HBV DNA and serological markers for monitoring.

Free full text available from PubMed PMID: 26483870

D) Low-Level Persistence of Drug Resistance Mutations in Hepatitis B Virus-Infected Subjects with a Past History of Lamivudine Treatment Margeridon-Thermet S, Svarovskaia ES, Babrzadeh F, et al. Low-level persistence of drug resistance mutations in hepatitis B virus-infected subjects with a past history of Lamivudine treatment. Antimicrob Agents Chemother. 2013; 57(1): 343-349.

Summary: We sought to determine the prevalence of hepatitis B virus (HBV) lamivudine (LAM)-resistant minority variants in subjects who once received LAM but had discontinued it prior to virus sampling. We performed direct PCR Sanger sequencing and ultradeep pyrosequencing (UDPS) of HBV reverse transcriptase (RT) of plasma viruses from 45 LAM-naive subjects and 46 LAM-experienced subjects who had discontinued LAM a median of 24 months earlier. UDPS was performed to a depth of approximately 3,000 reads per nucleotide. Minority variants were defined as differences from the Sanger sequence present in >/=0.5% of UDPS reads in a sample. Sanger sequencing identified >/=1 LAM resistance mutations (rtL80I/V, rtM204I, and rtA181T) in samples from 5 (11%) of 46 LAM-experienced and none of 45 LAM-naive subjects (0%; P = 0.06). UDPS detected >/=1 LAM resistance mutations (rtL80I/V, rtV173L, rtL180M, rtA181T, and rtM204I/V) in 10 (22%) of the 46 LAM-experienced subjects, including 5 in whom LAM resistance mutations were not identified by Sanger sequencing. Overall, LAM resistance mutations were more likely to be present in LAM-experienced (10/46, 22%) than LAM-naive subjects (0/45, 0%; P = 0.001). The median time since LAM discontinuation was 12.8 months in the 10 subjects with a LAM resistance mutation compared to 30.5 months in the 36 LAM-experienced subjects without a LAM resistance mutation (P < 0.001). The likelihood of detecting a LAM resistance mutation was significantly increased using UDPS compared to Sanger sequencing and was inversely associated with the time since LAM discontinuation.

Free full text available from PubMed PMID: 23114756 NOTE: Also cited in Section 4.7.2

4.2.5 Hepatitis C Virus

A) Update on Hepatitis B and C Virus Diagnosis Villar LM, Cruz HM, Barbosa JR, Bezerra CS, Portilho MM, Scalioni L de P. Update on Hepatitis B and C Virus Diagnosis. World J Virol. 2015; 4(4): 323- 342.

Summary: Viral hepatitis B and C virus (HBV and HCV) are responsible for the most of chronic liver disease worldwide and are transmitted by parenteral route, sexual and vertical transmission. One important measure to reduce the burden of these infections is the diagnosis of acute and chronic cases of HBV and HCV. In order to provide an effective diagnosis and monitoring of antiviral treatment, it is important to choose sensitive, rapid, inexpensive, and robust analytical methods. Primary diagnosis of HBV and HCV infection is made by using serological tests for detecting antigens and antibodies against these viruses. In order to confirm primary diagnosis, to quantify viral load, to determine genotypes and resistance mutants for antiviral treatment, qualitative and quantitative molecular tests are used. In this manuscript, we review the current serological and molecular methods for the diagnosis of hepatitis B and C.

Free full text available from PubMed PMID: 26568915

B) Virologic Tools for HCV Drug Resistance Testing Fourati S, Pawlotsky JM. Virologic Tools for HCV Drug Resistance Testing. Viruses. 2015; 7(12): 6346-6359.

Summary: Recent advances in molecular biology have led to the development of new antiviral drugs that target specific steps of the Hepatitis C Virus (HCV) lifecycle. These drugs, collectively termed direct-acting antivirals (DAAs), include non-structural (NS) HCV protein inhibitors, NS3/4A protease inhibitors, NS5B RNA-dependent RNA polymerase inhibitors (nucleotide analogues and non-nucleoside inhibitors), and NS5A inhibitors. Due to the high genetic variability of HCV, the outcome of DAA-based therapies may be altered by the selection of amino-acid substitutions located within the targeted proteins, which affect viral susceptibility to the administered compounds. At the drug developmental stage, preclinical and clinical characterization of HCV resistance to new drugs in development is mandatory. In the clinical setting, accurate diagnostic tools have become available to monitor drug resistance in patients who receive treatment with DAAs. In this review, we describe tools available to investigate drug resistance in preclinical studies, clinical trials and clinical practice.

Free full text available from PubMed PMID: 26690198

4.2.6 Human Immunodeficiency Virus (HIV)

A) Routine HIV Testing, Public Health, and the USPSTF—An End to the Debate Bayer R, Oppenheimer GM. Routine HIV testing, public health, and the USPSTF--an end to the debate. N Engl J Med. 2013; 368(10): 881-884.

Summary: The U.S. Preventive Services Task Force (USPSTF) is poised to release recommendations on screening for human immunodeficiency virus (HIV) infection that will endorse the routine testing of adults and adolescents, a position first adopted by the Centers for Disease Control and Prevention (CDC) in 2006. Based on an exacting systematic examination of the new evidence on clinical and public health benefits of early identification of HIV infection that has emerged since 2005, when the initial USPSTF review led to rejection of routine screening, the new recommendations will be a critical guide to clinical practice. They will also carry important policy implications, since the Affordable Care Act (ACA) mandates that all public and private health plans provide coverage for USPSTF-recommended preventive services without patient copayments.

Free full text available from New England Journal of Medicine PMID: 23425134

B) Identifying Recent HIV Infections: From Serological Assays to Genomics Moyo S, Wilkinson E, Novitsky V, et al. Identifying Recent HIV Infections: From Serological Assays to Genomics. Viruses. 2015; 7(10): 5508-5524.

Summary: In this paper, we review serological and molecular based methods to identify HIV infection recency. The accurate identification of recent HIV infection continues to be an important research area and has implications for HIV prevention and treatment interventions. Longitudinal cohorts that follow HIV negative individuals over time are the current gold standard approach, but they are logistically challenging, time consuming and an expensive enterprise. Methods that utilize cross-sectional testing and biomarker information have become an affordable alternative to the longitudinal approach. These methods use well-characterized biological makers to differentiate between recent and established HIV infections.

However, recent results have identified a number of limitations in serological based assays that are sensitive to the variability in immune responses modulated by HIV subtypes, viral load and antiretroviral therapy. Molecular methods that explore the dynamics between the timing of infection and viral evolution are now emerging as a promising approach. The combination of serological and molecular methods may provide a good solution to identify recent HIV infection in cross-sectional data. As part of this review, we present the advantages and limitations of serological and molecular based methods and their potential complementary role for the identification of HIV infection recency.

Free full text available from PubMed PMID: 26512688

4.2.7 Respiratory Viruses

A) Molecular Detection of Respiratory Viruses Buller RS. Molecular detection of respiratory viruses. Clin Lab Med. 2013; 33(3): 439-460.

Summary: Over the past several years a wide variety of molecular assays for the detection of respiratory viruses has reached the market. The tests described herein range from kits containing primers and probes detecting specific groups of viruses, to self-contained systems requiring specialized instruments that extract nucleic acids and perform the polymerase chain reaction with little operator input. Some of the tests target just the viruses involved in large yearly epidemics such as influenza, or specific groups of viruses such as the adenoviruses or parainfluenza viruses; others can detect most of the known respiratory viruses and some bacterial agents.

Full text available from Clinics in Laboratory Medicine (USD 31.50) PMID: 23931834

B) Point-of-Care Testing for Respiratory Viruses in Adults: The Current Landscape and Future Potential Brendish NJ, Schiff HF, Clark TW. Point-of-Care Testing for Respiratory Viruses in Adults: The Current Landscape and Future Potential. J Infect. 2015; 71(5): 501-510.

Summary: Respiratory viruses are responsible for a large proportion of acute respiratory illness in adults as well as children, and are associated with a huge socio-economic burden worldwide. Development of accurate point-of-

care tests (POCT) for respiratory viruses has been listed as a priority by the World Health Organisation and replacing the current paradigm of empirical antimicrobial use with directed use is a listed goal of the movement for reduction in antimicrobial resistance. POCTs for respiratory viruses have previously been limited by the poor sensitivity of antigen detection based tests and by a limited range of detectable viruses. Highly accurate molecular platforms are now able to test for a comprehensive range of viruses, can be operated by non-laboratory staff and can generate a result in approximately 1 h, making them potentially deployable as POCTs. The potential clinical benefits of POC testing for respiratory viruses in adults include a reduction in unnecessary antibiotic use, improved antiviral prescribing for influenza and rationalisation of isolation facilities. We review here the burden of disease, the currently available molecular platforms with potential for POCT use and the existing evidence for clinical and economic benefits of testing for respiratory viruses in adults.

Full text available from Journal of Infection (USD 31.50 for 24 hour access) PMID: 26215335

C) Identification of New Respiratory Viruses in the New Millennium Berry M, Gamieldien J, Fielding BC. Identification of New Respiratory Viruses in the New Millennium. Viruses. 2015; 7(3): 996-1019.

Summary: The rapid advancement of molecular tools in the past 15 years has allowed for the retrospective discovery of several new respiratory viruses as well as the characterization of novel emergent strains. The inability to characterize the etiological origins of respiratory conditions, particularly in children, led several researchers to pursue the discovery of the underlying etiology of disease. In 2001, this led to the discovery of human metapneumovirus (hMPV) and soon following that the outbreak of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) promoted an increased interest in coronavirology and the latter discovery of human coronavirus (HCoV) NL63 and HCoV-HKU1. Human bocavirus, with its four separate lineages, discovered in 2005, has been linked to acute respiratory tract infections and gastrointestinal complications. Middle East Respiratory Syndrome coronavirus (MERS-CoV) represents the most recent outbreak of a completely novel respiratory virus, which occurred in Saudi Arabia in 2012 and presents a significant threat to human health. This review will detail the most current clinical and epidemiological findings to all respiratory viruses discovered since 2001.

Free full text available from PubMed PMID: 25757061

D) Impact of a Rapid Respiratory Panel Test on Patient Outcomes Rogers BB, Shankar P, Jerris RC, et al. Impact of a Rapid Respiratory Panel Test on Patient Outcomes. Arch Pathol Lab Med. 2015; 139(5):636-41

Summary: Context .- Evolution of polymerase chain reaction testing for infectious pathogens has occurred concurrent with a focus on value-based medicine. Objective .- To determine if implementation of the FilmArray rapid respiratory panel (BioFire Diagnostics, Salt Lake City, Utah) (hereafter RRP), with a shorter time to the test result and expanded panel, results in different outcomes for children admitted to the hospital with an acute respiratory tract illness. Design .- Patient outcomes were compared before implementation of the RRP (November 1, 2011, to January 31, 2012) versus after implementation of the RRP (November 1, 2012, to January 31, 2013). The study included inpatients 3 months or older with an acute respiratory tract illness, most admitted through the emergency department. Testing before RRP implementation used batched polymerase chain reaction analysis for respiratory syncytial virus and influenza A and B, with additional testing for parainfluenza 1 through 3 in approximately 11% of patients and for human metapneumovirus in less than 1% of patients. The RRP tested for respiratory syncytial virus, influenza A and B, parainfluenza 1 through 4, human metapneumovirus, adenovirus, rhinovirus/enterovirus, and coronavirus NL62. Results .- The pre-RRP group had 365 patients, and the post-RRP group had 771 patients. After RRP implementation, the mean time to the test result was shorter (383 minutes versus 1119 minutes, P < .001), and the percentage of patients with a result in the emergency department was greater (51.6% versus 13.4%, P < .001). There was no difference in whether antibiotics were prescribed, but the duration of antibiotic use was shorter after RRP implementation (P = .003) and was dependent on receiving test results within 4 hours. If the test result was positive, the inpatient length of stay (P = .03) and the time in isolation (P = .03) were decreased after RRP implementation compared with before RRP implementation. Conclusion .- The RRP decreases the duration of antibiotic use, the length of inpatient stay, and the time in isolation.

Free full text available from the CAP’s Archives PMID: 25152311 NOTE: Also cited in Section 4.6.2

E) Comparison of the Biofire FilmArray RP, Genmark eSensor RVP, Luminex xTAG RVPv1, and Luminex xTAG RVP Fast Multiplex Assays for Detection of Respiratory Viruses Popowitch EB, O'Neill SS, Miller MB. Comparison of the Biofire FilmArray RP, Genmark eSensor RVP, Luminex xTAG RVPv1, and Luminex xTAG

RVP Fast Multiplex Assays for Detection of Respiratory Viruses. J. Clin. Microbiol. 2013; 51(5): 1528-33.

Summary: There are several U.S. FDA-cleared molecular respiratory virus panels available today, each with advantages and disadvantages. This study compares four multiplex panels, the BioFire Diagnostics FilmArray RP (respiratory panel), the GenMark Dx eSensor RVP (respiratory viral panel), the Luminex xTAG RVPv1, and the Luminex xTAG RVP fast. Three hundred specimens (200 retrospective and 100 consecutive) were tested using all four platforms to determine performance characteristics. The overall sensitivity and specificity, respectively, and 95% confidence interval (CI; in parentheses) for each panel were as follows: FilmArray RP, 84.5% (79.2, 88.6) and 100% (96.2, 100); eSensor RVP, 98.3% (95.5, 99.5) and 99.2% (95.4, 100); xTAG RVPv1, 92.7% (88.5, 95.4) and 99.8% (96.0, 100); and xTAG RVP fast, 84.4% (78.5, 88.9) and 99.9% (96.1, 100). The sensitivity of each assay fluctuated by viral target, with the greatest discrepancies noted for adenovirus and influenza virus B detection. Hands-on time and time to result were recorded and ease of use was assessed to generate a complete profile of each assay.

Free full text available from PubMed PMID: 23486707

4.2.7.1 Short Presentations on Emerging Concepts: Respiratory Viruses

A) CAP Short Presentations on Emerging Concepts (SPECS): Emerging Concepts in the Diagnosis of Respiratory Viruses [PowerPoint slides] Moyer AM. CAP Short Presentations on Emerging Concepts (SPECS): Emerging Concepts in the Diagnosis of Respiratory Viruses (v 4.0e rev 6/17/15) [PowerPoint slides]. Caughron SK, Misialek MJ, Nowak JA, eds. Northfield, IL: College of American Pathologists; 2015.

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4.2.8 Herpes Simplex I and II

A) Molecular Diagnostics and Newborns at Risk for Genital Herpes Simplex Virus Chua C, Arnolds M, Niklas V. Molecular Diagnostics and Newborns at Risk for Genital Herpes Simplex Virus. Pediatr Ann. 2015; 44(5): e97-102.

Summary: Herpes simplex virus (HSV) infection in the newborn carries a high mortality rate and can result in lifelong neurologic impairment. The severity of HSV infection in the newborn has always dictated conservative management when prodromal symptoms or active genital lesions (or those suggestive of genital herpes) are present during labor and delivery. The risk of intrapartum infection, however, is related to the presence or absence of maternal immunity (neutralizing antibody) to HSV. The most significant risk of transmission is in first-episode primary infections with active lesions at delivery. Recent recommendations from the American Academy of Pediatrics Committees on Infectious Diseases and the Fetus and Newborn use rapid serologic and virologic screening in the management of asymptomatic infants born to mothers with active genital herpes. The revised guidelines highlight infants at greatest risk for HSV disease but do not apply to asymptomatic infants born to mothers with a history of HSV but no genital lesions at delivery. The current guidelines also stipulate that maternal serologic screening and molecular assays for HSV in newborn blood and cerebrospinal fluid must be available and reported in a timely fashion.

Full text available from Pediatric Annals (USD 30.00) PMID: 25996200

B) Rapid and Direct Detection of Herpes Simplex Virus in Cerebrospinal Fluid by Use of a Commercial Real-Time PCR Assay Binnicker MJ, Espy MJ,Irish CL. Rapid and direct detection of herpes simplex virus in cerebrospinal fluid by use of a commercial real-time PCR assay. J Clin Microbiol. 2014; 52(12): 4361-4362.

Summary: Central nervous system infection due to herpes simplex virus (HSV) is a medical emergency and requires rapid diagnosis and initiation of therapy. In this study, we compared a routine real-time PCR assay for HSV types 1 (HSV-1) and 2 (HSV-2) to a recently FDA-approved direct PCR assay (Simplexa HSV-1/2 Direct; Focus Diagnostics, Cypress, CA) using cerebrospinal fluid samples (n = 100). The Simplexa HSV-1/2 assays demonstrated a combined sensitivity and specificity of 96.2% (50/52) and 97.9% (47/48), respectively. In addition, the Simplexa assay does not require nucleic acid extraction, and the results are available in 60 min.

Free full text available from Journal of Clinical Microbiology PMID: 25274992

4.2.9 Norovirus

A) Laboratory Diagnosis of Noroviruses: Present and Future Pang X, Lee BE. Laboratory Diagnosis of Noroviruses: Present and Future. Clin Lab Med. 2015; 35(2): 345-362.

Summary: Norovirus is an important cause of gastroenteritis outbreaks globally and the most prevalent cause of sporadic gastroenteritis in many regions. Rapid and accurate identification of causative viral agents is critical for outbreak investigation, disease surveillance, and management. Because norovirus is not cultivable and has a highly diversified and variable genome, it is difficult to develop diagnostic assays. Detection methods have evolved from electron microscopy to conventional end-point reverse transcription polymerase chain reaction (RT-PCR), immunoassay, real-time RT-PCR, other molecular technologies, and nanotechnology array-based assays. The status and features of various testing methods are summarized in this review.

Full text available from Clinics in Laboratory Medicine (USD 31.50) PMID: 26004647

B) Personalized Genetic Testing and Norovirus Susceptibility Prystajecky N, Brinkman FS, Auk B, Isaac-Renton JL,Tang P. Personalized Genetic Testing and Norovirus Susceptibility. Can J Infect Dis Med Microbiol. 2014; 25(4): 222-224.

Summary: BACKGROUND: The availability of direct-to-consumer personalized genetic testing has enabled the public to access and interpret their own genetic information. Various genetic traits can be determined including resistance to norovirus through a nonsense mutation (G428A) in the FUT2 gene. Although this trait is believed to confer resistance to the most dominant norovirus genotype (GII.4), the spectrum of resistance to other norovirus strains is unknown. The present report describes a cluster of symptomatic norovirus GI.6 infection in a family identified to have norovirus resistance through personalized genetic testing. CASE PRESENTATION: In January 2013, four members of a family determined by a direct-to-consumer genetic test to be homozygous for the norovirus resistance trait (A/A genotype for single nucleotide polymorphism rs601338) developed

symptoms consistent with acute viral gastroenteritis. Stool and vomitus samples were submitted for enteric viral pathogen testing. Samples were positive for norovirus GI.6 in three of the four cases. CONCLUSIONS: The present report is the first to describe norovirus GI.6 infection in patients with the G428A nonsense mutation in FUT2; this cluster of cases suggests that the G428A mutation in FUT2 may not confer resistance to norovirus GI.6. Direct-to-consumer genetic testing is empowering members of the public to identify novel associations with their genetic traits. Expert consultation is important for the interpretation of personalized genetic test results, and follow-up laboratory testing can confirm any potentially novel associations.

Free full text available from PubMed PMID: 25285128

4.2.10 Ebola Virus

A) Recent Advances in the Development of Vaccines for Ebola Virus Disease Ohimain EI. Recent Advances in the Development of Vaccines for Ebola Virus Disease. Virus Res. 2016; 211: 174-185.

Summary: Ebola virus is one of the most dangerous microorganisms in the world causing hemorrhagic fevers in humans and non-human primates. Ebola virus (EBOV) is a zoonotic infection, which emerges and re-emerges in human populations. The 2014 outbreak was caused by the Zaire strain, which has a kill rate of up to 90%, though 40% was recorded in the current outbreak. The 2014 outbreak is larger than all 20 outbreaks that have occurred since 1976, when the virus was first discovered. It is the first time that the virus was sustained in urban centers and spread beyond Africa into Europe and USA. Thus far, over 22,000 cases have been reported with about 50% mortality in one year. There are currently no approved therapeutics and preventive vaccines against Ebola virus disease (EVD). Responding to the devastating effe1cts of the 2014 outbreak and the potential risk of global spread, has spurred research for the development of therapeutics and vaccines. This review is therefore aimed at presenting the progress of vaccine development. Results showed that conventional inactivated vaccines produced from EBOV by heat, formalin or gamma irradiation appear to be ineffective. However, novel vaccines production techniques have emerged leading to the production of candidate vaccines that have been demonstrated to be effective in preclinical trials using small animal and non-human primates (NHP) models. Some of the promising vaccines have undergone phase 1 clinical trials, which demonstrated their

safety and immunogenicity. Many of the candidate vaccines are vector based such as Vesicular Stomatitis Virus (VSV), Rabies Virus (RABV), Adenovirus (Ad), Modified Vaccinia Ankara (MVA), Cytomegalovirus (CMV), human parainfluenza virus type 3 (HPIV3) and Venezuelan Equine Encephalitis Virus (VEEV). Other platforms include virus like particle (VLP), DNA and subunit vaccines.

Full text available from Virus Research (USD 41.95) PMID: 26596227

B) Molecular Detection and Point-of-Care Testing in Ebola Virus Disease and Other Threats: A New Global Public Health Framework to Stop Outbreaks Kost GJ, Ferguson W, Truong AT, et al. Molecular Detection and Point-of- Care Testing in Ebola Virus Disease and Other Threats: A New Global Public Health Framework to Stop Outbreaks. Expert Rev Mol Diagn. 2015; 15(10): 1245-1259.

Summary: Ultrahigh sensitivity and specificity assays that detect Ebola virus disease or other highly contagious and deadly diseases quickly and successfully upstream in Spatial Care Paths can stop outbreaks from escalating into devastating epidemics ravaging communities locally and countries globally. Even had the WHO and CDC responded more quickly and not misjudged the dissemination of Ebola in West Africa and other world regions, mobile rapid diagnostics were, and still are, not readily available for immediate and definitive diagnosis, a stunning strategic flaw that needs correcting worldwide. This article strategizes point-of-care testing for diagnosis, triage, monitoring, recovery and stopping outbreaks in the USA and other countries; reviews Ebola molecular diagnostics, summarizes USA FDA emergency use authorizations and documents why they should not be stop-gaps; and reduces community risk from internal and external infectious disease threats by enabling public health at points of need.

Full text available from Expert Review of Molecular Diagnostics (USD 89.00) PMID: 26367243

C) Infectious Disease. Genomes Reveal Start of Ebola Outbreak Vogel G. Infectious Disease. Genomes reveal start of Ebola outbreak. Science. 2014; 345(6200): 989-990.

Summary: When the young woman arrived at the Kenema Government Hospital in Sierra Leone in late May, she had high fever and had just miscarried. The hospital suspected she had contracted Lassa fever, because

the viral disease is endemic in the region and often causes miscarriages. But Ebola virus disease, another hemorrhagic fever illness, had been spreading in neighboring Guinea for months, so when she began bleeding profusely, staff tested her for that virus as well. The results were positive, making her the first confirmed case of Ebola in Sierra Leone.

Free full text available from Science PMID: 25170128

4.2.11 Influenza

A) Rapid Diagnosis of Influenza: State of the Art Peaper DR, Landry ML. Rapid Diagnosis of Influenza: State of the Art. Clin Lab Med. 2014; 34(2): 365-385.

Summary: Much effort has been expended developing testing modalities for influenza viruses that are capable of providing rapid results to clinicians. Antigen-detection techniques, historically the only methods able to deliver results quickly, are still widely used despite concerns about sensitivity. Recently, nucleic acid amplification tests (NAATs), which can achieve rapid turnaround times and high sensitivity, have become available. In addition, NAATs can detect other respiratory pathogens. Although there are many theoretical advantages to rapid influenza testing, the clinical impact of testing in various patient populations must be considered against the cost and the analytical performance of the tests.

Full text available from Clinics in Laboratory Medicine (USD 31.50) PMID: 24856533

B) Survey of Influenza and Other Respiratory Viruses Diagnostic Testing in US Hospitals, 2012-2013 Su S, Fry AM, Kirley PD, et al. Survey of Influenza and Other Respiratory Viruses Diagnostic Testing in US Hospitals, 2012-2013. Influenza Other Respir Viruses. 2016; 10(2): 86-90.

Summary: BACKGROUND: Little is known about laboratory capacity to routinely diagnose influenza and other respiratory viruses at clinical laboratories and hospitals. AIMS: We sought to assess diagnostic practices for influenza and other respiratory virus in a survey of hospitals and laboratories participating in the US Influenza Hospitalization Surveillance Network in 2012-2013. MATERIALS AND METHODS: All hospitals and their associated laboratories participating in the Influenza Hospitalization

Surveillance Network (FluSurv-NET) were included in this evaluation. The network covers more than 80 counties in 15 states, CA, CO, CT, GA, MD, MN, NM, NY, OR, TN, IA, MI, OH, RI, and UT, with a catchment population of ~28 million people. We administered a standardized questionnaire to key personnel, including infection control practitioners and laboratory departments, at each hospital through telephone interviews. RESULTS: Of the 240 participating laboratories, 67% relied only on commercially available rapid influenza diagnostic tests to diagnose influenza. Few reported the availability of molecular diagnostic assays for detection of influenza (26%) and other viral pathogens (

Free full text available from PubMed PMID: 26505742

4.3 Bacteria

This section includes articles on the following topics:

• Chlamydia Trachomatis/Neisseria Gonorrhoeae (Section 4.3.1); • Clostridium Difficile (Section 4.3.2); • Methicillin-Resistant Staphylococcus Areus (Section 4.3.3); • Mycobacterium (Section 4.3.4); and • Bordetella Pertussis (Section 4.3.5).

4.3.1 Chlamydia Trachomatis/Neisseria Gonorrhoeae

A) Recommendations for the Laboratory-Based Detection of Chlamydia Trachomatis and Neisseria Gonorrhoeae—2014 Centers for Disease Control and Prevention. Recommendations for the laboratory-based detection of Chlamydia trachomatis and Neisseria gonorrhoeae--2014. MMWR Recomm Rep. 2014; 63(RR-02): 1-19.

Summary: This report updates CDC's 2002 recommendations regarding screening tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae infections (CDC. Screening tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae infections-2002. MMWR 2002;51[No. RR-15]) and provides new recommendations regarding optimal specimen types, the use of tests to detect rectal and oropharyngeal C. trachomatis and N.

gonorrhoeae infections, and circumstances when supplemental testing is indicated. The recommendations in this report are intended for use by clinical laboratory directors, laboratory staff, clinicians, and disease control personnel who must choose among the multiple available tests, establish standard operating procedures for collecting and processing specimens, interpret test results for laboratory reporting, and counsel and treat patients. The performance of nucleic acid amplification tests (NAATs) with respect to overall sensitivity, specificity, and ease of specimen transport is better than that of any of the other tests available for the diagnosis of chlamydial and gonococcal infections. Laboratories should use NAATs to detect chlamydia and gonorrhea except in cases of child sexual assault involving boys and rectal and oropharyngeal infections in prepubescent girls and when evaluating a potential gonorrhea treatment failure, in which case culture and susceptibility testing might be required. NAATs that have been cleared by the Food and Drug Administration (FDA) for the detection of C. trachomatis and N. gonorrhoeae infections are recommended as screening or diagnostic tests because they have been evaluated in patients with and without symptoms. Maintaining the capability to culture for both N. gonorrhoeae and C. trachomatis in laboratories throughout the country is important because data are insufficient to recommend nonculture tests in cases of sexual assault in prepubescent boys and extragenital anatomic site exposure in prepubescent girls. N. gonorrhoeae culture is required to evaluate suspected cases of gonorrhea treatment failure and to monitor developing resistance to current treatment regimens. Chlamydia culture also should be maintained in some laboratories to monitor future changes in antibiotic susceptibility and to support surveillance and research activities such as detection of lymphogranuloma venereum or rare infections caused by variant or mutated C. trachomatis.

Free full text available from PubMed PMID: 24622331

B) Evaluation of the Hologic Gen-Probe PANTHER, APTIMA Combo 2 Assay in a Tertiary Care Teaching Hospital Cheng A, Kirby JE. Evaluation of the Hologic Gen-Probe PANTHER, APTIMA Combo 2 Assay in a Tertiary Care Teaching Hospital. Am J Clin Pathol. 2014; 141(3): 397-403.

Summary: OBJECTIVES: To evaluate the performance of the Hologic Gen- Probe (San Diego, CA) PANTHER system. METHODS: The performance of PANTHER was compared with the Hologic Gen-Probe TIGRIS and/or Roche (Indianapolis, IN) COBAS AMPLICOR systems through testing of patient specimens and the spiked-urine matrix. RESULTS: After discrepant

resolution, PANTHER demonstrated a 99.3% (95% confidence interval [CI], 96.0%-99.9%) positive and 100% (98.5%-100.0%) negative agreement for Chlamydia trachomatis (CT) and 100% (96.6%-100.0%) positive and 100% (98.6%-100.0%) negative agreement for Neisseria gonorrhoeae (NG) for all male, female, unsexed, and NG-spiked female urine specimens combined. For other specimen types collectively, the PANTHER demonstrated 100% (95% CI, 90.6%-100.0%) positive and 100% (88.3%-100.0%) negative agreement for CT and 90.9% (62.8%-98.4%) positive and 100% (93.5%- 100.0%) negative agreement for NG. Analytical sensitivity of the PANTHER in urine matrix was similar to the TIGRIS system. CONCLUSIONS: The PANTHER system provides an excellent new addition to options for detecting CT and NG, is appropriate for testing urine samples, and will facilitate high- throughput testing in the clinical laboratory.

Free full text available from American Journal of Clinical Pathology PMID: 24515768

C) The Swedish New Variant of Chlamydia Trachomatis (nvCT) Remains Undetected by Many European Laboratories as Revealed in the Recent PCR/NAT Ring Trial Organized by INSTAND e.V., Germany Reischl U, Straube E, Unemo M. The Swedish new variant of Chlamydia trachomatis (nvCT) remains undetected by many European laboratories as revealed in the recent PCR/NAT ring trial organised by INSTAND e.V., Germany. Euro Surveill. 2009; 14(32):pii:19302.

Summary: The May 2009 round of INSTAND's ring trial "Chlamydia trachomatis detection PCR/NAT" included a sample with high amount of the Swedish new variant of C. trachomatis (nvCT). A spectrum of at least 12 different commercial diagnostic nucleic acid amplification tests (NAATs) and many different in house NAATs were applied by the 128 participating laboratories which reported 152 results. Approximately 80% of the results correctly reported the presence of C. trachomatis in the nvCT specimen. The nvCT sample was mainly missed, as expected, by participants using the Roche COBAS Amplicor CT/NG (15.5% of reported results) but also by several participants using in house NAATs. The trend towards using nvCT- detecting NAATs is obvious and in addition to the new dual-target NAATs from Roche and Abbott, and BD ProbeTec ET, also a number of new CE mark-certified commercial tests from smaller diagnostic companies as well as many different in house NAATs were used. Laboratories using commercial or in house NAATs that do not detect the nvCT are encouraged to carefully monitor their C. trachomatis incidence, participate in appropriate external quality assurance and controls schemes, and consider altering their testing system. The reliable detection of low amounts of the wildtype C. trachomatis

strain in other samples of the ring trial set indicates a good diagnostic performance of all applied commercial NAATs while also detecting the nvCT strain.

Free full text available from Eurosurveillance PMID: 19679035

4.3.2 Clostridium Difficile

A) Guidelines for Diagnosis, Treatment, and Prevention of Clostridium Difficile Infections Surawicz CM, Brandt LJ, Binion DG, et al. Guidelines for Diagnosis, Treatment, and Prevention of Clostridium Difficile Infections. Am J Gastroenterol. 2013; 108(4): 478-498; quiz 499.

Summary: Clostridium difficile infection (CDI) is a leading cause of hospital- associated gastrointestinal illness and places a high burden on our healthcare system. Patients with CDI typically have extended lengths-of-stay in hospitals, and CDI is a frequent cause of large hospital outbreaks of disease. This guideline provides recommendations for the diagnosis and management of patients with CDI as well as for the prevention and control of outbreaks while supplementing previously published guidelines. New molecular diagnostic stool tests will likely replace current enzyme immunoassay tests. We suggest treatment of patients be stratified depending on whether they have mild-to-moderate, severe, or complicated disease. Therapy with metronidazole remains the choice for mild-to-moderate disease but may not be adequate for patients with severe or complicated disease. We propose a classification of disease severity to guide therapy that is useful for clinicians. We review current treatment options for patients with recurrent CDI and recommendations for the control and prevention of outbreaks of CDI.

Full text available from American Journal of Gastroenterology (subscription required) PMID: 23439232

B) C. Difficile Infection: Changing Epidemiology and Management Paradigms Vindigni SM, Surawicz CM. C. Difficile Infection: Changing Epidemiology and Management Paradigms. Clin Transl Gastroenterol. 2015; 6:e99.

Summary: The incidence of Clostridium difficile infection (CDI) has been rising in hospitals, long-term care facilities, and within the community. Cases

have been more severe with more complications, deaths, and higher healthcare-associated costs. With the emergence of a hypervirulent strain of C. difficile and the increasing prevalence of community-acquired CDI among healthy patients without traditional risk factors, the epidemiology of C. difficile has been evolving. This changing epidemiology requires a change in management. Taking into account new risk factors for CDI and growing subpopulations of affected individuals, diagnostic, treatment, and prevention approaches need to be adjusted.

Free full text available from PubMed PMID: 26158611

C) Molecular Methods for Detecting and Typing of Clostridium Difficile Collins DA, Elliott B, Riley TV. Molecular Methods for Detecting and Typing of Clostridium Difficile. Pathology. 2015; 47(3): 211-218.

Summary: Since the early 2000s, Clostridium difficile has emerged as a major international pathogen. Recently, strains of C. difficile in circulation appear to be changing, with greater diversity, leading to challenges for diagnostics and surveillance. Currently molecular diagnostic methods are favoured for their high sensitivity and rapid processing times; however, a number of issues still exist with molecular tests, in particular high cost, low clinical specificity and failure to detect some variant C. difficile strains. Molecular typing methods are used to determine the continually evolving epidemiology of C. difficile infection. Typing methods including PCR ribotyping and pulsed field gel electrophoresis are currently popular in Europe and North America, respectively, while high-throughput next- generation sequencing is likely to become more widely used in years to come. This review discusses current molecular detection and typing techniques for C. difficile.

Full text available from Pathology (subscription required) PMID: 25719853

4.3.3 Methicillin-Resistant Staphylococcus Areus (MRSA)

A) Evaluation of Multiple Real-Time PCR Tests on Nasal Samples in a Large MRSA Surveillance Program Patel PA, Robicsek A, Grayes A, et al. Evaluation of Multiple Real-Time PCR Tests on Nasal Samples in a Large MRSA Surveillance Program. Am J Clin Pathol. 2015; 143(5): 652-658.

Summary: OBJECTIVES: We evaluated the LightCycler MRSA Advanced Test (Roche Molecular Diagnostics, Pleasanton, CA), the BD MAX MRSA assay (Becton Dickinson, Franklin Lakes, NJ), and the Xpert MRSA assay (Cepheid, Sunnyvale, CA) on nasal samples using the same population. METHODS: Admission and discharge nasal swabs were collected from inpatients using a double-headed swab. One swab was plated onto CHROMagar MRSA (CMA; Becton Dickinson, Sparks, MD) and then broken off into tryptic soy broth (TSB) for enrichment. TSB was incubated for 24 hours and then plated to CMA. The molecular tests were performed on the second swab. We analyzed the cost benefit of testing to evaluate what parameters affect hospital resources. RESULTS: A total of 27,647 specimens were enrolled. The sensitivity/specificity was 98.3%/98.9% for the LightCycler MRSA Advanced Test and 95.7%/98.8% for the Xpert MRSA assay, but the difference was not significant. The positive predictive value was 86.7% for the LightCycler MRSA Advanced Test, 82.7% for the Xpert MRSA assay (P > .1), and 72.2% and for the BD MAX MRSA test (P < .001 compared with the LightCycler MRSA Advanced Test). All three assays were cost-effective, with the LightCycler MRSA Advanced Test having the highest economic return. CONCLUSIONS: Our results suggest that the performance of the three commercial assays is similar. When assessing economic cost benefit of methicillin-resistant Staphylococcus aureus screening, the two measures with the most impact are the cost of the test and the specificity of the assay results.

Free full text available from American Journal of Clinical Pathology PMID: 25873498

B) Costs and Benefits of Rapid Screening of Methicillin-Resistant Staphylococcus Aureus Carriage in Intensive Care Units: A Prospective Multicenter Study Wassenberg M, Kluytmans J, Erdkamp S, et al. Costs and benefits of rapid screening of methicillin-resistant Staphylococcus aureus carriage in intensive care units: a prospective multicenter study. Crit Care. 2012; 16(1):R22.

Summary: Pre-emptive isolation of suspected methicillin-resistant Staphylococcus aureus (MRSA) carriers is a cornerstone of successful MRSA control policies. Implementation of such strategies is hampered when using conventional cultures with diagnostic delays of three to five days, as many non-carriers remain unnecessarily isolated. Rapid diagnostic testing (RDT) reduces the amount of unnecessary isolation days, but costs and benefits have not been accurately determined in intensive care units (ICUs). METHODS: Embedded in a multi-center hospital-wide study in 12 Dutch hospitals we quantified cost per isolation day avoided using RDT for MRSA,

added to conventional cultures, in ICUs. BD GeneOhm MRSA PCR (IDI) and Xpert MRSA (GeneXpert) were subsequently used during 17 and 14 months, and their test characteristics were calculated with conventional culture results as reference. We calculated the number of pre-emptive isolation days avoided and incremental costs of adding RDT. RESULTS: A total of 163 patients at risk for MRSA carriage were screened and MRSA prevalence was 3.1% (n=5). Duration of isolation was 27.6 and 21.4 hours with IDI and GeneXpert, respectively, and would have been 96.0 hours when based on conventional cultures. The negative predictive value was 100% for both tests. Numbers of isolation days were reduced by 44.3% with PCR-based screening at the additional costs of euro327.84 (IDI) and euro252.14 (GeneXpert) per patient screened. Costs per isolation day avoided were euro136.04 (IDI) and euro121.76 (GeneXpert). CONCLUSIONS: In a low endemic setting for MRSA, RDT safely reduced the number of unnecessary isolation days on ICUs by 44%, at the costs of euro121.76 to euro136.04 per isolation day avoided.

Free full text available from PubMed PMID: 22314204

C) Novel Antibody-Antibiotic Conjugate Eliminates Intracellular S. Aureus Lehar SM, Pillow T, Xu M, et al. Novel Antibody-Antibiotic Conjugate Eliminates Intracellular S. Aureus. Nature. 2015; 527(7578): 323-328.

Summary: Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.

Full text available from Nature (USD 32.00) PMID: 26536114

D) Polymerase Chain Reaction-Based Active Surveillance of MRSA in Emergency Department Patients Seki M, Takahashi H, Yamamoto N, et al. Polymerase Chain Reaction-Based Active Surveillance of MRSA in Emergency Department Patients. Infect Drug Resist. 2015; 8:113-118.

Summary: Conventional culture methods to detect methicillin-resistant Staphylococcus aureus (MRSA) take a few days, and their sensitivity and usefulness also need to be improved. In this study, active screening was performed using the polymerase chain reaction (PCR) for colonization with MRSA on admission and follow-up surveillance after admission to an emergency department between June 2012 and August 2012, and the backgrounds of PCR and/or culture-method-positive patients were compared. Among 95 patients, 15 (15.8%) patients were positive for MRSA on PCR and/or culture; 6.3% (6/95) of patients were positive on admission, and 9.5% (9/95) became positive during the stay after admission. The major primary diagnoses in MRSA-positive patients were trauma and cerebrovascular diseases. Nine (60%) of 15 patients were MRSA-positive on both PCR and culture, compared with three (20%) of 15 who were PCR- positive but culture-negative. The other three (20%) of 15 patients were PCR-negative but culture-positive. Furthermore, there was a tendency for younger age and shorter stay to be associated with PCR-positive but culture- negative results. These findings suggest that active surveillance with PCR may be highly sensitive and useful for the early diagnosis of MRSA colonization to prevent nosocomial transmission from the emergency department to the regular inpatient wards of the hospital.

Free full text available from PubMed PMID: 25999747

E) PBP2a Mutations Causing High-Level Ceftaroline Resistance in Clinical Methicillin-Resistant Staphylococcus Aureus Isolates Long SW, Olsen RJ, Mehta SC, et al. PBP2a mutations causing high-level Ceftaroline resistance in clinical methicillin-resistant Staphylococcus aureus isolates. Antimicrob Agents Chemother. 2014; 58(11): 6668-6674.

patient and five MRSA strains from the respiratory tract of this patient. Whole-genome sequencing identified two amino acid-altering mutations uniquely present in the ceftaroline-binding pocket of the transpeptidase region of penicillin-binding protein 2a (PBP2a) in ceftaroline-resistant isolates. Biochemical analyses and the study of isogenic mutant strains confirmed that these changes caused ceftaroline resistance. Thus, we identified the molecular mechanism of ceftaroline resistance in the first MRSA strain with high-level ceftaroline resistance isolated in the United States.

Free full text available from Antimicrobial Agents and Chemotherapy PMID: 25155594 NOTE: Also cited in Section 4.7.2

F) Alternative Mutational Pathways to Intermediate Resistance to Vancomycin in Methicillin-Resistant Staphylococcus Aureus Vidaillac C, Gardete S, Tewhey R, et al. Alternative mutational pathways to intermediate resistance to vancomycin in methicillin-resistant Staphylococcus aureus. J Infect Dis. 2013; 208(1): 67-74.

Summary: BACKGROUND: We used 2 in vitro experimental systems to compare phenotypic and genotypic changes that accompany selection of mutants of methicillin-resistant Staphylococcus aureus (MRSA) strain JH1 with low-level vancomycin resistance similar to the type found in vancomycin- intermediate S. aureus (VISA). METHODS: The previously described MRSA strain JH1 and its vancomycin-intermediate mutant derivative JH2, both of which were recovered from a patient undergoing vancomycin chemotherapy, were used in this study. Mutants of JH1 were selected in vitro by means of a pharmacokinetic/pharmacodynamic (PK/PD) model of simulated endocardial vegetations (SEVs) and by exposure to vancomycin in laboratory growth medium. Phenotypic abnormalities of JH1 mutants generated by each in vitro experimental system were compared to those of JH2, and whole genomes of 2 in vitro JH1 mutants were sequenced to identify mutations that may be associated with an increased vancomycin minimum inhibitory concentration. RESULTS: JH1R1 was selected from the PK/PD model, and JH1R2 was selected in laboratory growth medium. Both mutants displayed reduced vancomycin and daptomycin susceptibility and phenotypic alterations (eg, thicker cell walls and abnormal autolysis) that are typical of in vivo VISA mutants. Genome sequencing of JH1R1 identified point mutations in 4 genes, all of which were different from the mutations described in JH2, including 1 mutation in yycG, a component of the WalKR sensory regulatory system. Sequencing of the JH1R2 genome identified mutations in 7 genes,

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including 2 in rpoB. CONCLUSION: Our findings indicate that JH1 is able to develop VISA-type resistance through several alternative genetic pathways.

Free full text available from Journal of Infectious Disease PMID: 23539745 NOTE: Also cited in Section 4.7.2

4.3.4 Mycobacterium

A) Clinical Usefulness of PCR for Differential Diagnosis of Tuberculosis and Nontuberculous Mycobacterial Infection in Paraffin-Embedded Lung Tissues Kim YN, Kim KM, Choi HN, et al. Clinical Usefulness of PCR for Differential Diagnosis of Tuberculosis and Nontuberculous Mycobacterial Infection in Paraffin-Embedded Lung Tissues. J Mol Diagn. 2015; 17(5): 597-604.

Summary: The need for isolation of nontuberculous mycobacteria (NTM) from clinical specimens has increased in recent years. Our aim was to determine the clinical usefulness of PCR for differential diagnosis of tuberculosis and nontuberculous mycobacterial infection in lung tissue that show chronic granulomatous inflammation. A total of 199 formalin-fixed, paraffin-embedded specimens, including 137 Mycobacterium tuberculosis (MTB), 17 NTM cases, and 45 other than mycobacterial cases were collected. We performed acid-fast staining, MTB and NTM nested PCRs, and MTB and NTM real-time PCRs. No histologic difference between MTB and NTM infections was observed. Sensitivity and specificity for detecting MTB were 70.1% and 95.1% by nested PCR, respectively, and 70.8% and 100.0% by real-time PCR, respectively. Sensitivity and specificity for detecting NTM were 52.9% and 96.15% by nested PCR, respectively, and 35.3% and 100.0% by real-time PCR, respectively. Mycobacteria were identified by acid- fast staining in 50 of 154 cases (32.5%). All 50 acid-fast staining-positive cases showed positive nested and real-time PCR results (n = 47 MTB PCR positive; n = 3 NTM PCR positive), and results agreed with final diagnosis. PCR will be useful for the rapid diagnosis of mycobacterial infection and differentiation of MTB from NTM in formalin-fixed, paraffin-embedded specimens, especially in acid-fast staining-positive specimens.

Full text available from Journal of Molecular Diagnostics (USD 31.50 for 24 hour access) PMID: 26163897

B) Molecular Diagnosis of TB in the HIV Positive Population Vittor AY, Garland JM, Gilman RH. Molecular Diagnosis of TB in the HIV Positive Population. Ann Glob Health. 2014; 80(6): 476-485.

Summary: BACKGROUND: Tuberculosis (TB) remains a major contributor to morbidity and mortality in HIV-positive individuals, causing 1.1 million incident cases and 0.32 million deaths in 2012. Diagnosis of TB is particularly challenging in HIV-coinfected individuals, due to a high frequency of smear- negative disease, atypical presentations, and extrapulmonary TB. OBJECTIVE: The aim of this article was to review the current literature on molecular diagnostics for TB with an emphasis on the performance of these diagnostic tests in the HIV-positive population. METHODS: We searched the PubMed database using at least one of the terms TB, HIV, diagnostics, Xpert MTB/RIF, nucleic acid amplification tests, drug susceptibility testing, RNA transcription, and drew on World Health Organization publications. FINDINGS: With increased focus on reducing TB prevalence worldwide, a new set of tools for diagnosing the disease have emerged. Molecular tools such as Xpert MTB/RIF and line-probe assays are now in use or are being rolled out in many regions. The diagnostic performance of these and other molecular assays are discussed here as they pertain to the HIV-positive population. CONCLUSIONS: Molecular diagnostics offer a useful addition and at times, alternative, to traditional culture methods for the diagnosis of TB. However, most of these tests suffer from decreased accuracy in the HIV- positive population.

Free full text available from Annals of Global Health PMID: 25960097

C) Acid-Fast Smear and Histopathology Results Provide Guidance for the Appropriate Use of Broad-Range Polymerase Chain Reaction and Sequencing for Mycobacteria Miller K, Harrington SM, Procop GW. Acid-Fast Smear and Histopathology Results Provide Guidance for the Appropriate Use of Broad-Range Polymerase Chain Reaction and Sequencing for Mycobacteria. Arch Pathol Lab Med. 2015; 139(8): 1020-1023.

Summary: CONTEXT: New molecular diagnostic tests are attractive because of the potential they hold for improving diagnostics in microbiology. The value of these tests, which is often assumed, should be investigated to determine the best use of these potentially powerful tools. OBJECTIVE: To investigate the usefulness of broad-range polymerase chain reaction (PCR), followed by sequencing, in mycobacterial infections. DESIGN: We reviewed the test performance of acid-fast bacilli (AFB) PCR and traditional diagnostic

methods (histopathology, AFB smear, and culture). We assessed the diagnostic effect and cost of the unrestricted ordering of broad-range PCR for the detection and identification of mycobacteria in clinical specimens. RESULTS: The AFB PCR was less sensitive than culture and histopathology and was less specific than culture, AFB smear, and histopathology. During 18 months, $93 063 was spent on 183 patient specimens for broad-range PCR and DNA sequencing for mycobacteria to confirm one culture-proven Mycobacterium tuberculosis infection that was also known to be positive by AFB smear and histopathology. In this cohort, there was a false-negative AFB PCR for M tuberculosis and a false-positive AFB PCR for Mycobacterium lentiflavum . CONCLUSION: Testing of AFB smear-negative specimens from patients without an inflammatory response supportive of a mycobacterial infection is costly and has not been proven to improve patient care. Traditional diagnostics (histopathology, AFB smear, and culture) should remain the primary methods for the detection of mycobacteria in clinical specimens.

Free full text available from the CAP’s Archives PMID: 25574585

D) Revised Device Labeling for the Cepheid Xpert MTB/RIF Assay for Detecting Mycobacterium Tuberculosis Division of Microbiology Devices, Office of In Vitro Diagnostics and Radiological Health, Center for Devices and Radiological Health, Food and Drug Administration. Revised Device Labeling for the Cepheid Xpert MTB/RIF Assay for Detecting Mycobacterium tuberculosis. MMWR Morb Mortal Wkly Rep. 2015; 64(07): 193.

Summary: The Food and Drug Administration (FDA) has cleared the Xpert MTB/RIF Assay (Cepheid; Sunnyvale, California) with an expanded intended use that includes testing of either one or two sputum specimens as an alternative to examination of serial acid-fast stained sputum smears to aid in the decision of whether continued airborne infection isolation (AII) is warranted for patients with suspected pulmonary tuberculosis. This change reflects the outcome of a recent multicenter international study demonstrating that negative Xpert MTB/RIF Assay results from either one or two sputum specimens are highly predictive of the results of two or three negative acid- fast sputum smears.

Free full text available from Morbidity and Mortality Weekly Report PMID: 25719683

4.3.5 Bordetella Pertussis

A) Optimizing Polymerase Chain Reaction Testing for the Diagnosis of Pertussis: Current Perspectives Arbefeville S, Ferrieri P. Optimizing Polymerase Chain Reaction Testing for the Diagnosis of Pertussis: Current Perspectives. Pathology and Laboratory Medicine International. 2015; 2015(7): 67-73.

Summary: Nucleic acid testing has revolutionized the diagnosis of pertussis in the clinical microbiology laboratory and has become the main avenue of testing for pertussis infection. Real-time polymerase chain reaction (RT-PCR) is an important tool for timely diagnosis of pertussis and is more sensitive than culture. The most commonly amplified targets are the insertion- sequence (IS) genes, which are found in multiple copies in the genome of Bordetella species. Some strains of Bordetella pertussis have more than 200 copies of IS481 in their genome. This high number of repeats allows RT-PCR assays to be very sensitive and makes nucleic acid testing two to three times more sensitive than culture. Despite these advantages, RT-PCR can give inaccurate results due to contamination or lack of specificity. Contamination can easily happen during specimen collection, DNA extraction, or nucleic acid amplification steps. To avoid contamination, laboratories need to have quality controls and good workflows in place. The poor specificity of the nucleic acid assays amplifying the IS genes is because they are found in various Bordetella species and, thus, not unique to a specific species. Bordetella holmesii, a more recently described Bordetella species found to be responsible for respiratory symptoms similar to pertussis in adolescents and adults, can be misidentified as B. pertussis in RT-PCR assays that amplify only the IS481 target. Use of multiple targets may improve specificity of RT-PCR assays for pertussis. In the past few years, the US Food and Drug Administration has cleared three commercial assays for the detection of B. pertussis in respiratory specimens. Several commercial assays and analyte-specific reagents, which are not US Food and Drug Administration cleared, are available for the detection of one or more Bordetella species by nucleic acid testing. Because of the diversity of nucleic acid amplification assays used, pertussis testing is not standardized across clinical laboratories.

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4.4 Fungi

This section includes articles on the following topics:

• Aspergillus Species (Section 4.4.1); • Candida Species (Section 4.4.2); and • Pneumocystis Species (Section 4.4.3).

A) Improved Characterization of Medically Relevant Fungi in the Human Respiratory Tract Using Next-Generation Sequencing Bittinger K, Charlson ES, Loy E, et al. Improved characterization of medically relevant fungi in the human respiratory tract using next-generation sequencing. Genome Biol. 2014; 15(10): 487.

Summary: Background: Fungi are important pathogens but challenging to enumerate using next-generation sequencing because of low absolute abundance in many samples and high levels of fungal DNA from contaminating sources.Results: Here, we analyze fungal lineages present in the human airway using an improved method for contamination filtering. We use DNA quantification data, which is routinely acquired during DNA library preparation, to annotate output sequence data and improve the identification and filtering of contaminants. We compare fungal communities and bacterial communities from healthy subjects, HIV+ subjects, and lung transplant recipients, providing a gradient of increasing lung impairment for comparison. We use deep sequencing to characterize ribosomal rRNA gene segments from fungi and bacteria in DNA extracted from bronchiolar lavage samples and oropharyngeal wash. Comparison to clinical culture data documents improved detection after applying the filtering procedure.Conclusions: We find increased representation of medically relevant organisms, including Candida, Cryptococcus, and Aspergillus, in subjects with increasingly severe pulmonary and immunologic deficits. We analyze covariation of fungal and bacterial taxa, and find that oropharyngeal communities rich in Candida are also rich in mitis group Streptococci, a community pattern associated with pathogenic polymicrobial biofilms. Thus, using this approach, it is possible to characterize fungal communities in the human respiratory tract more accurately and explore their interactions with bacterial communities in health and disease.

Free full text available from PubMed PMID: 25344286

4.4.1 Aspergillus Species

A) Retrospective Comparison of Nucleic Acid Sequence-Based Amplification, Real-Time PCR, and Galactomannan Test for Diagnosis of Invasive Aspergillosis Wang L, He Y, Xia Y, Su X, Wang H, Liang S. Retrospective Comparison of Nucleic Acid Sequence-Based Amplification, Real-Time PCR, and Galactomannan Test for Diagnosis of Invasive Aspergillosis. J Mol Diagn. 2014; 16(5): 584-590.

Summary: Invasive aspergillosis is a life-threatening infection in immunocompromised patients, and treating these infections at an early stage is often crucial for a favorable outcome. Early diagnosis, however, remains challenging. We performed a retrospective comparison of three methods: real-time quantitative PCR (qPCR), nucleic acid sequence-based amplification (NASBA), and galactomannan enzyme-linked immunosorbent assay (GM-ELISA); these detect circulating Aspergillus DNA, RNA, and galactomannan, respectively. Blood samples from 80 patients at high risk for invasive aspergillosis were tested by each assay. The sensitivity of NASBA, qPCR, and GM-ELISA was 76.47% (95% CI, 58.4-88.6%), 67.65% (95% CI, 49.4-82.0%), and 52.94% (95% CI, 35.4-69.8%), respectively, and the specificity was 80.43% (95% CI, 65.6-90.1%), 89.13% (95% CI, 75.6-95.9%), and 80.43% (95% CI, 65.6-90.1%), respectively. We also evaluated the efficiency of the three tests in various combinations. Perfect specificity (100%; 95% CI, 90.4-100%) and perfect positive predictive value (100%; 95% CI, 77.1-100%) were achieved by combining NASBA and qPCR testing in series. Testing with both NASBA and qPCR in parallel was the most sensitive and had the highest Youden index. Our data support the great potential of NASBA and qPCR, singly or in combination, for diagnosis of invasive aspergillosis in high-risk populations.

Free full text available from Journal of Molecular Diagnostics PMID: 25129364

B) Validation of a New Aspergillus Real-Time PCR Assay for Direct Detection of Aspergillus and Azole Resistance of Aspergillus Fumigatus on Bronchoalveolar Lavage Fluid Chong GL, van de Sande WW, Dingemans GJ, et al. Validation of a New Aspergillus Real-Time PCR Assay for Direct Detection of Aspergillus and Azole Resistance of Aspergillus Fumigatus on Bronchoalveolar Lavage Fluid. J Clin Microbiol. 2015; 53(3): 868-874.

Summary: Azole resistance in Aspergillus fumigatus is increasingly reported. Here, we describe the validation of the AsperGenius, a new multiplex real- time PCR assay consisting of two multiplex real-time PCRs, one that identifies the clinically relevant Aspergillus species, and one that detects the TR34, L98H, T289A, and Y121F mutations in CYP51A and differentiates susceptible from resistant A. fumigatus strains. The diagnostic performance of the AsperGenius assay was tested on 37 bronchoalveolar lavage (BAL) fluid samples from hematology patients and 40 BAL fluid samples from intensive care unit (ICU) patients using a BAL fluid galactomannan level of >/=1.0 or positive culture as the gold standard for detecting the presence of Aspergillus. In the hematology and ICU groups combined, there were 22 BAL fluid samples from patients with invasive aspergillosis (IA) (2 proven, 9 probable, and 11 nonclassifiable). Nineteen of the 22 BAL fluid samples were positive, according to the gold standard. The optimal cycle threshold value for the presence of Aspergillus was <36. Sixteen of the 19 BAL fluid samples had a positive PCR (2 Aspergillus species and 14 A. fumigatus samples). This resulted in a sensitivity, specificity, and positive and negative predictive values of 88.9%, 89.3%, 72.7%, and 96.2%, respectively, for the hematology group and 80.0%, 93.3%, 80.0%, and 93.3%, respectively, in the ICU group. The CYP51A real-time PCR confirmed 12 wild-type and 2 resistant strains (1 TR34-L98H and 1 TR46-Y121F-T289A mutant). Voriconazole therapy failed for both patients. The AsperGenius multiplex real-time PCR assay allows for sensitive and fast detection of Aspergillus species directly from BAL fluid samples. More importantly, this assay detects and differentiates wild-type from resistant strains, even if BAL fluid cultures remain negative.

Free full text available from PubMed PMID: 25568431

C) Multicenter Comparison of Serum and Whole-Blood Specimens for Detection of Aspergillus DNA in High-Risk Hematological Patients Springer J, Morton CO, Perry M, et al. Multicenter comparison of serum and whole-blood specimens for detection of Aspergillus DNA in high-risk hematological patients. J Clin Microbiol. 2013; 51(5): 1445-1450.

Summary: Samples from patients at high risk for invasive aspergillosis (IA) were prospectively collected and analyzed for the presence of molecular markers of fungal infection. Serum specimens were screened for galactomannan and Aspergillus DNA, and whole-blood specimens were screened only for Aspergillus DNA. Fungal infections were categorized according to the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group, National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG)

criteria. Forty-seven cases (proven and probable IA) and 31 controls (no evidence of IA) were selected retrospectively for this case-control study, comprising 803 samples, in order to determine the performance of whole- blood PCR, serum PCR, and serum galactomannan testing. Although no single assay was able to detect every case of IA, a combination of different assays provided the best performance. There was no significant difference between the use of whole-blood and serum specimens for PCR-based diagnosis of IA, but there was a trend for whole blood to be more sensitive (85% versus 79%) and to yield an earlier positive result (36 days versus 15 days) than for serum. However, DNA extraction from serum specimens is easier and faster than that from whole-blood specimens, and it allows the same specimen to be used for both galactomannan and PCR assays. In conclusion, the appropriate sample type for DNA extraction should be determined by the local requirements and the technical platforms available at each individual center. A combination of biomarker tests offered the best diagnostic utility for detecting IA.

Free full text available from PubMed PMID: 23426930

4.4.2 Candida Species

A) Application of PCR and High-Resolution Melting for Rapid Identification of Yeasts Routinely Isolated in a Clinical Microbiology Laboratory Ninghui G, Bing W, Wei R, et al. Application of PCR and High-Resolution Melting for Rapid Identification of Yeasts Routinely Isolated in a Clinical Microbiology Laboratory. Ann Clin Lab Sci. 2015; 45(6): 680-685.

Summary: This study aimed to develop a method for rapid and accurate identification of yeasts obtained in the clinic, especially from immunocompromised patients, in order to provide a timely and appropriate antifungal therapy. A total of 112 Candida isolates were analyzed in this study; 28 of them were used to validate the PCR-HRM method in species identification in a blinded manner. Strains were identified by conventional techniques that use VITEK 2 YST cards and Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). These methods were compared to the newly developed technique based on real-time polymerase-chain reaction high-resolution melting (PCR-HRM). Discordant results were resolved with internal transcribed spacer (ITS) gene sequencing, the "golden standard" used to evaluate the reliability of all methods in identifying yeasts at the species level. PCR-HRM sensitivity was assessed with the isolated strains. VITEK 2, MALDI-TOF-MS, and PCR-HRM

accurately identified 89.2% (74/83), 97.6% (81/83), 100% (83/83) of the isolates, respectively. PCR-HRM detection limit was 1fg/mul of yeasts. In validation assays, a 100% accuracy rate was achieved by the use of PCR- HRM. Therefore, the PCR-HRM method is a rapid, sensitive, and specific diagnostic approach, which provides a cost-effective and more suitable alternative for yeast identification in a clinical laboratory. Future research is needed for automation of data acquisition.

Full text available from Annals of Clinical & Laboratory Science (USD 7.00) PMID: 26663799

B) T2 Magnetic Resonance Assay for the Rapid Diagnosis of Candidemia in Whole Blood: A Clinical Trial Mylonakis E, Clancy CJ, Ostrosky-Zeichner L, et al. T2 magnetic resonance assay for the rapid diagnosis of candidemia in whole blood: a clinical trial. Clin Infect Dis. 2015; 60(6): 892-899.

Summary: BACKGROUND: Microbiologic cultures, the current gold standard diagnostic method for invasive Candida infections, have low specificity and take up to 2-5 days to grow. We present the results of the first extensive multicenter clinical trial of a new nanodiagnostic approach, T2 magnetic resonance (T2MR), for diagnosis of candidemia. METHODS: Blood specimens were collected from 1801 hospitalized patients who had a blood culture ordered for routine standard of care; 250 of them were manually supplemented with concentrations from <1 to 100 colony-forming units (CFUs)/mL for 5 different Candida species. RESULTS: T2MR demonstrated an overall specificity per assay of 99.4% (95% confidence interval [CI], 99.1%-99.6%) with a mean time to negative result of 4.2 +/- 0.9 hours. Subanalysis yielded a specificity of 98.9% (95% CI, 98.3%-99.4%) for Candida albicans/Candida tropicalis, 99.3% (95% CI, 98.7%-99.6%) for Candida parapsilosis, and 99.9% (95% CI, 99.7%-100.0%) for Candida krusei/Candida glabrata. The overall sensitivity was found to be 91.1% (95% CI, 86.9%-94.2%) with a mean time of 4.4 +/- 1.0 hours for detection and species identification. The subgroup analysis showed a sensitivity of 92.3% (95% CI, 85.4%-96.6%) for C. albicans/C. tropicalis, 94.2% (95% CI, 84.1%- 98.8%) for C. parapsilosis, and 88.1% (95% CI, 80.2%-93.7%) for C. krusei/C. glabrata. The limit of detection was 1 CFU/mL for C. tropicalis and C. krusei, 2 CFU/mL for C. albicans and C. glabrata, and 3 CFU/mL for C. parapsilosis. The negative predictive value was estimated to range from 99.5% to 99.0% in a study population with 5% and 10% prevalence of candidemia, respectively. CONCLUSIONS: T2MR is the first fully automated technology that directly analyzes whole blood specimens to identify species without the need for prior isolation of Candida species, and represents a

breakthrough shift into a new era of molecular diagnostics. CLINICAL TRIALS REGISTRATION: NCT01752166.

Full text available from Clinical Infectious Disease (USD 39.00) PMID: 25586686

C) Candida Identification: A Journey from Conventional to Molecular Methods in Medical Mycology Alam MZ, Alam Q, Jiman-Fatani A, et al. Candida identification: a journey from conventional to molecular methods in medical mycology. World J Microbiol Biotechnol. 2014; 30(5): 1437-1451.

Summary: The incidence of Candida infections have increased substantially in recent years due to aggressive use of immunosuppressants among patients. Use of broad-spectrum antibiotics and intravascular catheters in the intensive care unit have also attributed with high risks of candidiasis among immunocompromised patients. Among Candida species, C. albicans accounts for the majority of superficial and systemic infections, usually associated with high morbidity and mortality often caused due to increase in antimicrobial resistance and restricted number of antifungal drugs. Therefore, early detection of candidemia and correct identification of Candida species are indispensable pre-requisites for appropriate therapeutic intervention. Since blood culture based methods lack sensitivity, and species-specific identification by conventional method is time-consuming and often leads to misdiagnosis within closely related species, hence, molecular methods may provide alternative for accurate and rapid identification of Candida species. Although, several molecular approaches have been developed for accurate identification of Candida species but the internal transcribed spacer 1 and 2 (ITS1 and ITS2) regions of the rRNA gene are being used extensively in a variety of formats. Of note, ITS sequencing and PCR-RFLP analysis of ITS region seems to be promising as a rapid, easy, and cost-effective method for identification of Candida species. Here, we review a number of existing techniques ranging from conventional to molecular approaches currently in use for the identification of Candida species. Further, advantages and limitations of these methods are also discussed with respect to their discriminatory power, reproducibility, and ease of performance.

Full text available from World Journal of Microbiology and Biotechnology (USD 39.95) PMID: 24379160

D) Invasive Candidiasis Kullberg BJ,Arendrup MC. Invasive Candidiasis. N Engl J Med. 2015; 373(15): 1445-1456.

Summary: Despite advances in antifungal therapy, the mortality associated with invasive candidiasis remains as high as 40%. This review summarizes recent trends and current strategies, including early treatment and the emergence of resistance against triazoles and echinocandins.

Full text available from New England Journal of Medicine (subscription required) PMID: 26444731

4.4.3 Pneumocystis Species

A) Diagnosis of Pneumocystis Jirovecii Pneumonia in Immunocompromised Patients by Real-Time PCR: A 4-Year Prospective Study Robert-Gangneux F, Belaz S, Revest M, et al. Diagnosis of Pneumocystis Jirovecii Pneumonia in Immunocompromised Patients by Real-Time PCR: A 4-Year Prospective Study. J Clin Microbiol. 2014; 52(9): 3370-3376.

Summary: Pneumocystis jirovecii pneumonia (PCP) is a life-threatening infection in immunocompromised patients. Quantitative real-time PCR (qPCR) is more sensitive than microscopic examination for the detection of P. jirovecii but also detects colonized patients. Hence, its positive predictive value (PPV) needs evaluation. In this 4-year prospective observational study, all immunocompromised patients with acute respiratory symptoms who were investigated for PCP were included, totaling 659 patients (814 bronchoalveolar lavage fluid samples). Patients with negative microscopy but positive qPCR were classified through medical chart review as having retained PCP, possible PCP, or colonization, and their clinical outcomes were compared to those of patients with microscopically proven PCP. Overall, 119 patients were included for analysis, of whom 35, 41, and 43 were classified as having retained PCP, possible PCP, and colonization, respectively. The 35 patients with retained PCP had clinical findings similar to those with microscopically proven PCP but lower fungal loads (P < 0.001) and were mainly non-HIV-infected patients (P < 0.05). Although the mean amplification threshold was higher in colonized patients, it was not possible to determine a discriminant qPCR cutoff. The PPV of qPCR in patients with negative microscopy were 29.4% and 63.8% when considering retained PCP and retained plus possible PCP, respectively. Patients with possible PCP

had a higher mortality rate than patients with retained PCP or colonization (63% versus 3% and 16%, respectively); patients who died had not received co-trimoxazole. In conclusion, qPCR is a useful tool to diagnose PCP in non- HIV patients, and treatment might be better targeted through a multicomponent algorithm including both clinical/radiological parameters and qPCR results.

Free full text available from PubMed PMID: 25009050

B) Development and Validation of a Pneumocystis Jirovecii Real-Time Polymerase Chain Reaction Assay for Diagnosis of Pneumocystis Pneumonia Church DL, Ambasta A, Wilmer A, et al. Development and Validation of a Pneumocystis Jirovecii Real-Time Polymerase Chain Reaction Assay for Diagnosis of Pneumocystis Pneumonia. Can J Infect Dis Med Microbiol. 2015; 26(5): 263-267.

Summary: BACKGROUND: Pneumocystis jirovecii (PJ), a pathogenic fungus, causes severe interstitial Pneumocystis pneumonia (PCP) among immunocompromised patients. A laboratory-developed real-time polyermase chain reaction (PCR) assay was validated for PJ detection to improve diagnosis of PCP. METHODS: Forty stored bronchoalveolar lavage (BAL) samples (20 known PJ positive [PJ+] and 20 known PJ negative [PJ-]) were initially tested using the molecular assay. Ninety-two sequentially collected BAL samples were then analyzed using an immunofluorescence assay (IFA) and secondarily tested using the PJ real-time PCR assay. Discrepant results were resolved by retesting BAL samples using another real-time PCR assay with a different target. PJ real-time PCR assay performance was compared with the existing gold standard (ie, IFA) and a modified gold standard, in which a true positive was defined as a sample that tested positive in two of three methods in a patient suspected to have PCP. RESULTS: Ninety of 132 (68%) BAL fluid samples were collected from immunocompromised patients. Thirteen of 92 (14%) BALs collected were PJ+ when tested using IFA. A total of 40 BAL samples were PJ+ in the present study including: all IFA positive samples (n=13); all referred PJ+ BAL samples (n=20); and seven additional BAL samples that were IFA negative, but positive using the modified gold standard. Compared with IFA, the PJ real-time PCR had sensitivity, specificity, and positive and negative predictive values of 100%, 91%, 65% and 100%, respectively. Compared with the modified gold standard, PJ real- time PCR had a sensitivity, specificity, and positive and negative predictive values of 100%. CONCLUSION: PJ real-time PCR improved detection of PJ in immunocompromised patients.

Free full text available from PubMed PMID: 26600815

C) Loop-Mediated Isothermal Amplification with the Procedure for Ultra Rapid Extraction Kit for the Diagnosis of Pneumocystis Pneumonia Kawano S, Maeda T, Suzuki T, et al. Loop-Mediated Isothermal Amplification with the Procedure for Ultra Rapid Extraction Kit for the Diagnosis of Pneumocystis Pneumonia. J Infect Chemother. 2015; 21(3): 224-226.

Summary: Loop-mediated isothermal amplification (LAMP) is an innovative molecular technique requiring only a heating device and isothermal conditions to amplify a specific target gene. The results of current microscopic diagnostic tools for pneumocystis pneumonia are not sufficiently consistent for detecting infection with a low-density of Pneumocystis jirovecii. Although polymerase chain reaction (PCR) is highly sensitive, it is not suitable for resource-limited facilities. LAMP is a potential diagnostic replacement for PCR in such settings but a critical disadvantage of DNA extraction was still remained. Therefore, we employed the Procedure for Ultra Rapid Extraction (PURE) kit, which uses a porous material, to isolate the DNA from clinical samples in a simple way in combination with previously reported LAMP procedure for diagnosing PCP. The detection limit of the PURE-LAMP method applied to artificial bronchoalveolar lavage fluid samples was 100 copies/tube, even with the use of massive blood- contaminated solutions. In addition, we concluded the diagnostic procedure within 1 h without the need for additional equipment. PURE-LAMP coupled with suitable primers for specific pathogens has good potential for diagnosing various infectious diseases.

Full text available from Journal of Infection and Chemotherapy (USD 31.50 for 24 hour access) PMID: 25455747

D) An Improved Single-Round PCR Leads to Rapid and Highly Sensitive Detection of Pneumocystis Spp Chabé M, Khalife S, Gantois N, Even G, Audebert C. An improved single- round PCR leads to rapid and highly sensitive detection of Pneumocystis spp. Med Mycol. 2014; 52(8): 841-846.

Summary: In order to standardize a polymerase chain reaction (PCR)-based method of Pneumocystis detection, we describe the development of an improved PCR method that targets the Pneumocystis mtLSUrRNA gene. Design of a new primer pair and PCR program with suitable parameters and

optimization resulted in a simpler and faster single-round amplification assay. The sensitivity of the novel Pneumocystis genus-specific PCR proved comparable to the reference nested PCR. The improvement that this new PCR assay offers in the detection and epidemiological studies of Pneumocystis spp. infection in research laboratories is discussed.

Full text available from Medical Mycology (USD 39.00) PMID: 24965947

4.5 Parasites

This section includes articles on the following topics:

• Malaria (Section 4.5.1); • Leishmania Species (Section 4.5.2); and • Schistosomiasis (Section 4.5.3).

A) Identification of Parasitic Communities Within European Ticks Using Next-Generation Sequencing Bonnet S, Michelet L, Moutailler S, et al. Identification of parasitic communities within European ticks using next-generation sequencing. PLoS Negl Trop Dis. 2014; 8(3): e2753.

Summary: BACKGROUND: Risk assessment of tick-borne and zoonotic disease emergence necessitates sound knowledge of the particular microorganisms circulating within the communities of these major vectors. Assessment of pathogens carried by wild ticks must be performed without a priori, to allow for the detection of new or unexpected agents. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the potential of Next-Generation Sequencing techniques (NGS) to produce an inventory of parasites carried by questing ticks. Sequences corresponding to parasites from two distinct genera were recovered in Ixodes ricinus ticks collected in Eastern France: Babesia spp. and Theileria spp. Four Babesia species were identified, three of which were zoonotic: B. divergens, Babesia sp. EU1 and B. microti; and one which infects cattle, B. major. This is the first time that these last two species have been identified in France. This approach also identified new sequences corresponding to as-yet unknown organisms similar to tropical Theileria species. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate the capability of NGS to produce an inventory of live

tick-borne parasites, which could potentially be transmitted by the ticks, and uncovers unexpected parasites in Western Europe.

Free full text available from PubMed PMID: 24675738

4.5.1 Malaria

A) Simple Real-Time PCR and Amplicon Sequencing Method for Identification of Plasmodium Species in Human Whole Blood Lefterova MI, Budvytiene I, Sandlund J, Farnert A, Banaei N. Simple Real- Time PCR and Amplicon Sequencing Method for Identification of Plasmodium Species in Human Whole Blood. J Clin Microbiol. 2015; 53(7): 2251-2257.

Summary: Malaria is the leading identifiable cause of fever in returning travelers. Accurate Plasmodium species identification has therapy implications for P. vivax and P. ovale, which have dormant liver stages requiring primaquine. Compared to microscopy, nucleic acid tests have improved specificity for species identification and higher sensitivity for mixed infections. Here, we describe a SYBR green-based real-time PCR assay for Plasmodium species identification from whole blood, which uses a panel of reactions to detect species-specific non-18S rRNA gene targets. A pan- Plasmodium 18S rRNA target is also amplified to allow species identification or confirmation by sequencing if necessary. An evaluation of assay accuracy, performed on 76 clinical samples (56 positives using thin smear microscopy as the reference method and 20 negatives), demonstrated clinical sensitivities of 95.2% for P. falciparum (20/21 positives detected) and 100% for the Plasmodium genus (52/52), P. vivax (20/20), P. ovale (9/9), and P. malariae (6/6). The sensitivity of the P. knowlesi-specific PCR was evaluated using spiked whole blood samples (100% [10/10 detected]). The specificities of the real-time PCR primers were 94.2% for P. vivax (49/52) and 100% for P. falciparum (51/51), P. ovale (62/62), P. malariae (69/69), and P. knowlesi (52/52). Thirty-three specimens were used to test species identification by sequencing the pan-Plasmodium 18S rRNA PCR product, with correct identification in all cases. The real-time PCR assay also identified two samples with mixed P. falciparum and P. ovale infection, which was confirmed by sequencing. The assay described here can be integrated into a malaria testing algorithm in low-prevalence areas, allowing definitive Plasmodium species identification shortly after malaria diagnosis by microscopy.

Free full text available from PubMed PMID: 25972416

B) Molecular-Based Isothermal Tests for Field Diagnosis of Malaria and Their Potential Contribution to Malaria Elimination Oriero EC, Jacobs J, Van Geertruyden JP, Nwakanma D, D'Alessandro U. Molecular-Based Isothermal Tests for Field Diagnosis of Malaria and Their Potential Contribution to Malaria Elimination. J Antimicrob Chemother. 2015; 70(1): 2-13.

Summary: In countries where malaria transmission has decreased substantially, thanks to the scale-up of control interventions, malaria elimination may be feasible. Nevertheless, this goal requires new strategies such as the active detection and treatment of infected individuals. As the detection threshold for the currently used diagnostic methods is 100 parasites/muL, most low-density, asymptomatic infections able to maintain transmission cannot be detected. Identifying them by molecular methods such as PCR is a possible option but the field deployment of these tests is problematic. Isothermal amplification of nucleic acids (at a constant temperature) offers the opportunity of addressing some of the challenges related to the field deployment of molecular diagnostic methods. One of the novel isothermal amplification methods for which a substantial amount of work has been done is the loop-mediated isothermal amplification (LAMP) assay. The present review describes LAMP and several other isothermal nucleic acid amplification methods, such as thermophilic helicase-dependent amplification, strand displacement amplification, recombinase polymerase amplification and nucleic acid sequence-based amplification, and explores their potential use as high-throughput, field-based molecular tests for malaria diagnosis.

Free full text available from Journal of Antimicrobial Chemotherapy PMID: 25223973

C) A Lab-on-Chip for Malaria Diagnosis and Surveillance Taylor BJ, Howell A, Martin KA, et al. A lab-on-chip for malaria diagnosis and surveillance. Malar J. 2014; 13:179.

Summary: BACKGROUND: Access to timely and accurate diagnostic tests has a significant impact in the management of diseases of global concern such as malaria. While molecular diagnostics satisfy this need effectively in developed countries, barriers in technology, reagent storage, cost and expertise have hampered the introduction of these methods in developing countries. In this study a simple, lab-on-chip PCR diagnostic was created for

Return to Table of Contents Section 4

malaria that overcomes these challenges. METHODS: The platform consists of a disposable plastic chip and a low-cost, portable, real-time PCR machine. The chip contains a desiccated hydrogel with reagents needed for Plasmodium specific PCR. Chips can be stored at room temperature and used on demand by rehydrating the gel with unprocessed blood, avoiding the need for sample preparation. These chips were run on a custom-built instrument containing a Peltier element for thermal cycling and a laser/camera setup for amplicon detection. RESULTS: This diagnostic was capable of detecting all Plasmodium species with a limit of detection for Plasmodium falciparum of 2 parasites/muL of blood. This exceeds the sensitivity of microscopy, the current standard for diagnosis in the field, by ten to fifty-fold. In a blind panel of 188 patient samples from a hyper-endemic region of malaria transmission in Uganda, the diagnostic had high sensitivity (97.4%) and specificity (93.8%) versus conventional real-time PCR. The test also distinguished the two most prevalent malaria species in mixed infections, P. falciparum and Plasmodium vivax. A second blind panel of 38 patient samples was tested on a streamlined instrument with LED-based excitation, achieving a sensitivity of 96.7% and a specificity of 100%. CONCLUSIONS: These results describe the development of a lab-on-chip PCR diagnostic from initial concept to ready-for-manufacture design. This platform will be useful in front-line malaria diagnosis, elimination programmes, and clinical trials. Furthermore, test chips can be adapted to detect other pathogens for a differential diagnosis in the field. The flexibility, reliability, and robustness of this technology hold much promise for its use as a novel molecular diagnostic platform in developing countries.

Free full text available from PubMed PMID: 24885206

D) Genome Sequencing Sheds Light on Emerging Drug Resistance in Malaria Parasites Neafsey DE. Genome sequencing sheds light on emerging drug resistance in malaria parasites. Nat Genet. 2013; 45(6): 589-590.

Summary: Plasmodium falciparum in Southeast Asia are gradually becoming resistant to artemesinin, a standard first-line treatment for malaria. Whole-genome sequencing offers a chance to better understand and perhaps undermine the parasite's evolutionary response to this drug.

Full text article available from Nature Genetics (USD 18.00) PMID: 23715326 NOTE: Also cited in Section 4.7.2

E) Impact of Routine Real-Time PCR Testing of Imported Malaria Over 4 Years of Implementation in a Clinical Laboratory Shokoples S, Mukhi SN, Scott AN,Yanow SK. Impact of routine real-time PCR testing of imported malaria over 4 years of implementation in a clinical laboratory. J Clin Microbiol. 2013; 51(6): 1850-1854.

Summary: In clinical laboratories, diagnosis of imported malaria is commonly performed by microscopy. However, the volume of specimens is generally low and maintaining proficiency in reading blood smears, particularly at the species level, is challenging in this setting. To address this problem, the Provincial Laboratory for Public Health (ProvLab) in Alberta, Canada, implemented real-time PCR for routine confirmation of all smear- positive samples in the province. Here we report our experience over a 4- year period (2008 to 2012) with this new diagnostic algorithm. While detection of Plasmodium falciparum by microscopy alone was accurate, real- time PCR served as an important adjunct to microscopy for the identification of non-falciparum species. In 18% of cases, the result was reported as non- falciparum or the species could not be identified by microscopy alone, and in all cases, the species was resolved by real-time PCR. In another 4% of cases, the species was misidentified by microscopy. To enhance surveillance for malaria, we integrated our demographic, clinical, and laboratory data into a new system developed by the Canadian Network for Public Health Intelligence, called the Malaria System for Online Surveillance (SOS). Using this application, we characterized our patient populations and travel history to identify risk factors associated with malaria infection abroad.

Free full text available from PubMed PMID: 23554200

4.5.2 Leishmania Species

A) A Rapid Molecular Diagnosis of Cutaneous Leishmaniasis by Colorimetric Malachite Green-Loop-Mediated Isothermal Amplification (LAMP) Combined with an FTA Card as a Direct Sampling Tool Nzelu CO, Caceres AG, Guerrero-Quincho S, et al. A Rapid Molecular Diagnosis of Cutaneous Leishmaniasis by Colorimetric Malachite Green- Loop-Mediated Isothermal Amplification (LAMP) Combined with an FTA Card as a Direct Sampling Tool. Acta Trop. 2016; 153:116-119.

Summary: Leishmaniasis remains one of the world's most neglected diseases, and early detection of the infectious agent, especially in developing countries, will require a simple and rapid test. In this study, we established a

quick, one-step, single-tube, highly sensitive loop-mediated isothermal amplification (LAMP) assay for rapid detection of Leishmania DNA from tissue materials spotted on an FTA card. An FTA-LAMP with pre-added malachite green was performed at 64 degrees C for 60min using a heating block and/or water bath and DNA amplification was detected immediately after incubation. The LAMP assay had high detection sensitivity down to a level of 0.01 parasites per mul. The field- and clinic-applicability of the colorimetric FTA-LAMP assay was demonstrated with 122 clinical samples collected from patients suspected of having cutaneous leishmaniasis in Peru, from which 71 positives were detected. The LAMP assay in combination with an FTA card described here is rapid and sensitive, as well as simple to perform, and has great potential usefulness for diagnosis and surveillance of leishmaniasis in endemic areas.

Full text available from Acta Tropica (USD 39.95) PMID: 26516109

B) Comparative Genomic Analysis of Leishmania (Viannia) Peruviana and Leishmania (Viannia) Braziliensis Valdivia HO, Reis-Cunha JL, Rodrigues-Luiz GF, et al. Comparative Genomic Analysis of Leishmania (Viannia) Peruviana and Leishmania (Viannia) Braziliensis. BMC Genomics. 2015; 16:715.

Summary: BACKGROUND: The Leishmania (Viannia) braziliensis complex is responsible for most cases of New World tegumentary leishmaniasis. This complex includes two closely related species but with different geographic distribution and disease phenotypes, L. (V.) peruviana and L. (V.) braziliensis. However, the genetic basis of these differences is not well understood and the status of L. (V.) peruviana as distinct species has been questioned by some. Here we sequenced the genomes of two L. (V.) peruviana isolates (LEM1537 and PAB-4377) using Illumina high throughput sequencing and performed comparative analyses against the L. (V.) braziliensis M2904 reference genome. Comparisons were focused on the detection of Single Nucleotide Polymorphisms (SNPs), insertions and deletions (INDELs), aneuploidy and gene copy number variations. RESULTS: We found 94,070 variants shared by both L. (V.) peruviana isolates (144,079 in PAB-4377 and 136,946 in LEM1537) against the L. (V.) braziliensis M2904 reference genome while only 26,853 variants separated both L. (V.) peruviana genomes. Analysis in coding sequences detected 26,750 SNPs and 1,513 indels shared by both L. (V.) peruviana isolates against L. (V.) braziliensis M2904 and revealed two L. (V.) braziliensis pseudogenes that are likely to have coding potential in L. (V.) peruviana. Chromosomal read density and allele frequency profiling showed a

heterogeneous pattern of aneuploidy with an overall disomic tendency in both L. (V.) peruviana isolates, in contrast with a trisomic pattern in the L. (V.) braziliensis M2904 reference. Read depth analysis allowed us to detect more than 368 gene expansions and 14 expanded gene arrays in L. (V.) peruviana, and the likely absence of expanded amastin gene arrays. CONCLUSIONS: The greater numbers of interspecific SNP/indel differences between L. (V.) peruviana and L. (V.) braziliensis and the presence of different gene and chromosome copy number variations support the classification of both organisms as closely related but distinct species. The extensive nucleotide polymorphisms and differences in gene and chromosome copy numbers in L. (V.) peruviana suggests the possibility that these may contribute to some of the unique features of its biology, including a lower pathology and lack of mucosal development.

Free full text available from PubMed PMID: 26384787

C) Fast, Sensitive and Specific Diagnosis of Infections with Leishmania Spp. In Formalin-Fixed, Paraffin-Embedded Skin Biopsies by Cytochrome B Polymerase Chain Reaction Gebhardt M, Ertas B, Falk TM, Blodorn-Schlicht N, Metze D, Boer-Auer A. Fast, Sensitive and Specific Diagnosis of Infections with Leishmania Spp. In Formalin-Fixed, Paraffin-Embedded Skin Biopsies by Cytochrome B Polymerase Chain Reaction. Br J Dermatol. 2015; 173(5): 1239-1249.

Summary: BACKGROUND: Northern spread of sandflies and Leishmania spp. has been observed in Europe. Diagnosis can be difficult owing to the various clinical manifestations. Species identification is important for patient management and therapy. Molecular diagnostics is increasingly used for pan-Leishmania detection but species identification remains challenging in formalin-fixed material. OBJECTIVES: To apply cytochrome b (cytb) polymerase chain reaction (PCR) and sequencing for identification of Leishmania species on formalin-fixed, paraffin-embedded (FFPE) skin biopsies; and to identify species-specific histological patterns. METHODS: Sixty-nine biopsies (48 patients) diagnosed with leishmaniasis based on the presence of amastigotes in the tissue (n = 41) or granulomatous infiltrates with positive pan-Leishmania real-time PCR (n = 28) were analysed with cytb PCR, sequencing and phylogenetic analysis. Histological sections were analysed; epidemiological data were collected. RESULTS: Cytb PCR identified Leishmania in all specimens: L. infantum (79%), L. major (8%), L. panamensis (4%), L. tropica (4%), L. killicki (2%) and L. aethiopica (2%). Of the detected species 95% were endemic to the country in which the infection was acquired. Amastigotes were found in 59%. Infiltrates were mainly

tuberculoid granulomatous (65%), interstitial (15%) and sarcoidal (10%). Pseudolymphomatous features and pseudocarcinomatous hyperplasia were more common in L. major infections than in L. infantum (P < 0.01). CONCLUSIONS: Cytb PCR and sequencing is a fast, reliable and sensitive assay for identification of Leishmania spp. in FFPE biopsies. Leishmania infantum is the main cause of cutaneous leishmaniasis in Germany. Tuberculoid granulomas, other types of granulomas and pseudolymphomatous infiltrates may be encountered; the latter being indicative of infection with L. major.

Full text available from British Journal of Dermatology (USD 6.00-38.00) PMID: 26286104

D) Comparison of Conventional, Molecular, and Immunohistochemical Methods in Diagnosis of Typical and Atypical Cutaneous Leishmaniasis Shirian S, Oryan A, Hatam GR, Panahi S, Daneshbod Y. Comparison of conventional, molecular, and immunohistochemical methods in diagnosis of typical and atypical cutaneous leishmaniasis. Arch Pathol Lab Med. 2014; 138(2): 235-240.

Summary: CONTEXT: Localized cutaneous leishmaniasis (CL) typically presents as papules, crusted nodules, plaques, or noduloulcerative lesions. Atypical CL does not show these features or mimic malignant lesion. In atypical forms, CL may be overlooked because of its similarity to other dermal diseases. OBJECTIVE: To compare conventional, molecular, and immunohistochemical methods in the diagnosis of typical and atypical CL. DESIGN: The kinetoplast DNA, nested, polymerase chain reaction assay and immunohistochemical methods were compared and validated against conventional methods, including cytology and pathology, using 100 specimens of typical and atypical lesions of suspected CL. RESULTS: Compared with other methods, polymerase chain reaction of the kinetoplast DNA showed the highest sensitivity (typical positive, 100%, 67 of 67; atypical positive, 94%, 31 of 33) and specificity (100%), followed by immunohistochemistry (typical positive, 97%, 65 of 67, with 100% specificity; atypical positives, 94%, 31 of 33, with 100% specificity), and cytology (typical positive, 79%, 53 of 67, with 100% specificity; atypical positive, 58%, 19 of 33, with 100% specificity), followed by pathology (typical positive, 70%, 47 of 67, with 100% specificity; atypical positive, 42%, 14 of 33, with 100% specificity). In addition, polymerase chain reaction enabled identification of 98% (98 of 100) of the positive samples that included strains of Leishmania major (99% [99 of 100] cases) and Leishmania tropica (1% [1 of 100] cases). CONCLUSIONS: Because cytology is cheap and easy to perform with high sensitivity, it is the preferred, primary approach for typical CL, but cytology

and pathology do not have sufficient sensitivity for diagnosis of atypical CL cases. Nested polymerase chain reaction and immunohistochemistry are sensitive tests for diagnosis of both typical and atypical CL and are recommended as complementary tests in suspected CL with negative conventional microscopy results.

Free full text available from the CAP’s Archives PMID: 24476521

4.5.3 Schistosomiasis

A) Isothermal Recombinase Polymerase Amplification (RPA) of Schistosoma Haematobium DNA and Oligochromatographic Lateral Flow Detection Rosser A, Rollinson D, Forrest M, Webster BL. Isothermal Recombinase Polymerase Amplification (RPA) of Schistosoma Haematobium DNA and Oligochromatographic Lateral Flow Detection. Parasit Vectors. 2015; 8:446.

Summary: BACKGROUND: Accurate diagnosis of urogenital schistosomiasis is vital for surveillance/control programs. Amplification of schistosome DNA in urine by PCR is sensitive and specific but requires infrastructure, financial resources and skilled personnel, often not available in endemic areas. Recombinase Polymerase Amplification (RPA) is an isothermal DNA amplification/detection technology that is simple, rapid, portable and needs few resources. FINDINGS: Here a Schistosoma haematobium RPA assay was developed and adapted so that DNA amplicons could be detected using oligochromatographic Lateral Flow (LF) strips. The assay successfully amplified S. haematobium DNA at 30-45 degrees C in 10 mins and was sensitive to a lower limit of 100 fg of DNA. The assay was also successful with the addition of crude urine, up to 5% of the total reaction volume. Cross amplification occurred with other schistosome species but not with other common urine microorganisms. CONCLUSION: The LF-RPA assay developed here can amplify and detect low levels of S. haematobium DNA. Reactions are rapid, require low temperatures and positive reactions are interpreted using lateral flow strips, reducing the need for infrastructure and resources. This together with an ability to withstand inhibitors within urine makes RPA a promising technology for further development as a molecular diagnostic tool for urogenital schistosomiasis.

Free full text available from PubMed PMID: 26338510

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B) Detection of Schistosoma Mansoni and Schistosoma Haematobium by Real-Time PCR with High Resolution Melting Analysis Sady H, Al-Mekhlafi HM, Ngui R, et al. Detection of Schistosoma Mansoni and Schistosoma Haematobium by Real-Time PCR with High Resolution Melting Analysis. Int J Mol Sci. 2015; 16(7): 16085-16103.

Summary: The present study describes a real-time PCR approach with high resolution melting-curve (HRM) assay developed for the detection and differentiation of Schistosoma mansoni and S. haematobium in fecal and urine samples collected from rural Yemen. The samples were screened by microscopy and PCR for the Schistosoma species infection. A pair of degenerate primers were designed targeting partial regions in the cytochrome oxidase subunit I (cox1) gene of S. mansoni and S. haematobium using real-time PCR-HRM assay. The overall prevalence of schistosomiasis was 31.8%; 23.8% of the participants were infected with S. haematobium and 9.3% were infected with S. mansoni. With regards to the intensity of infections, 22.1% and 77.9% of S. haematobium infections were of heavy and light intensities, respectively. Likewise, 8.1%, 40.5% and 51.4% of S. mansoni infections were of heavy, moderate and light intensities, respectively. The melting points were distinctive for S. mansoni and S. haematobium, categorized by peaks of 76.49 +/- 0.25 degrees C and 75.43 +/- 0.26 degrees C, respectively. HRM analysis showed high detection capability through the amplification of Schistosoma DNA with as low as 0.0001 ng/microL. Significant negative correlations were reported between the real-time PCR-HRM cycle threshold (Ct) values and microscopic egg counts for both S. mansoni in stool and S. haematobium in urine (p < 0.01). In conclusion, this closed-tube HRM protocol provides a potentially powerful screening molecular tool for the detection of S. mansoni and S. haematobium. It is a simple, rapid, accurate, and cost-effective method. Hence, this method is a good alternative approach to probe-based PCR assays.

Free full text available from PubMed PMID: 26193254

C) Prospective European-Wide Multicentre Study on a Blood Based Real- Time PCR for the Diagnosis of Acute Schistosomiasis Wichmann D, Poppert S, Von Thien H, et al. Prospective European-Wide Multicentre Study on a Blood Based Real-Time PCR for the Diagnosis of Acute Schistosomiasis. BMC Infect Dis. 2013; 13:55.

Summary: BACKGROUND: Acute schistosomiasis constitutes a rare but serious condition in individuals experiencing their first prepatent Schistosoma

infection. To circumvent costly and time-consuming diagnostics, an early and rapid diagnosis is required. So far, classic diagnostic tools such as parasite microscopy or serology lack considerable sensitivity at this early stage of Schistosoma infection. To validate the use of a blood based real-time polymerase chain reaction (PCR) test for the detection of Schistosoma DNA in patients with acute schistosomiasis who acquired their infection in various endemic regions we conducted a European-wide prospective study in 11 centres specialized in travel medicine and tropical medicine. METHODS: Patients with a history of recent travelling to schistosomiasis endemic regions and freshwater contacts, an episode of fever (body temperature >/=38.5 degrees C) and an absolute or relative eosinophil count of >/=700/mul or 10%, were eligible for participation. PCR testing with DNA extracted from serum was compared with results from serology and microscopy. RESULTS: Of the 38 patients with acute schistosomiasis included into the study, PCR detected Schistosoma DNA in 35 patients at initial presentation (sensitivity 92%). In contrast, sensitivity of serology (enzyme immunoassay and/or immunofluorescence assay) or parasite microscopy was only 70% and 24%, respectively. CONCLUSION: For the early diagnosis of acute schistosomiasis, real-time PCR for the detection of schistosoma DNA in serum is more sensitive than classic diagnostic tools such as serology or microscopy, irrespective of the region of infection. Generalization of the results to all Schistosoma species may be difficult as in the study presented here only eggs of S. mansoni were detected by microscopy. A minimum amount of two millilitre of serum is required for sufficient diagnostic accuracy.

Free full text available from PubMed PMID: 23363565

D) Chlamydia Trachomatis and Genital Mycoplasmas: Pathogens with an Impact on Human Reproductive Health Ljubin-Sternak S, Mestrovic T. Chlamydia Trachomatis and Genital Mycoplasmas: Pathogens with an Impact on Human Reproductive Health. J Pathog. 2014; 183167.

Summary: The most prevalent, curable sexually important diseases are those caused by Chlamydia trachomatis (C. trachomatis) and genital mycoplasmas. An important characteristic of these infections is their ability to cause long-term sequels in upper genital tract, thus potentially affecting the reproductive health in both sexes. Pelvic inflammatory disease (PID), tubal factor infertility (TFI), and ectopic pregnancy (EP) are well documented complications of C. trachomatis infection in women. The role of genital mycoplasmas in development of PID, TFI, and EP requires further evaluation, but growing evidence supports a significant role for these in the

pathogenesis of chorioamnionitis, premature membrane rupture, and preterm labor in pregnant woman. Both C. trachomatis and genital mycoplasmas can affect the quality of sperm and possibly influence the fertility of men. For the purpose of this paper, basic, epidemiologic, clinical, therapeutic, and public health issue of these infections were reviewed and discussed, focusing on their impact on human reproductive health.

Free full text available from PubMed PMID: 25614838

4.6 Pan-Infection Detection

Quick Reference Table: High Multiplexed IVD Assays to Detect Important Human Pathogens

Product Name Manufacturer Regulatory Gastrointestinal Pathogen Panels BD MAX Enteric Bacterial Panel BD IVD FilmArray GI Panel bioMerieux IVD ProGastro SSCS Hologic IVD NxTAG Gastrointestinal Pathogen Panel Luminex IVD Verigene Enteric Pathogens Test Nanosphere IVD Respiratory Pathogen Panels FilmArray Respiratory Panel bioMerieux IVD eSensor Respiratory Viral Panel GenMark IVD NxTAG Respiratory Pathogen Panel Luminex IVD NxTAG Respiratory Pathogen Panel Luminex IVD FAST v2 Verigene Respiratory Pathogens Flex Nanosphere IVD Bacteremia Pathogen Panels FilmArray BCID Panel bioMerieux IVD Verigene Gram-Positive Blood Culture Nanosphere IVD Test Verigene Gram-Negative Blood Culture Nanosphere IVD Test Meningitis and Ecephalitis Pathogen Panels FilmArray ME Panel bioMerieux IVD Biothreat Pathogens Panels Some pathogens available Various EUA Source: Randall J. Olsen, MD

The information presented in this table is for quick reference only. Clinical decision making should not be based solely on this information. The data is current as of Spring 2016.

This section includes articles on the following topics:

• Stool (Section 4.6.1); • Respiratory (Section 4.6.2); • Emerging Infectious Diseases (Section 4.6.3); and • Positive Blood Cultures (Section 4.6.4).

4.6.1 Stool

A) Molecular Detection of Gastrointestinal Viral Infections in Hospitalized Patients Rovida F, Campanini G, Piralla A, Adzasehoun KM, Sarasini A, Baldanti F. Molecular Detection of Gastrointestinal Viral Infections in Hospitalized Patients. Diagn Microbiol Infect Dis. 2013; 77(3): 231-235.

Summary: Gastrointestinal viral syndromes are a common cause of morbidity and mortality in humans worldwide. Etiological agents include a large number of viruses encompassing several orders, families, and genera. During the period April 2011 to April 2012, 689 stool samples from as many patients hospitalized at the Fondazione IRCCS Policlinico San Matteo of Pavia exhibiting gastrointestinal syndromes were examined for the presence of rotavirus, norovirus, astrovirus, adenovirus, rhinovirus, enterovirus, parechovirus, bocavirus, coronavirus, sapovirus, cosavirus, and aichi virus using polymerase chain reaction assays. Gastrointestinal viral agents were detected in 246 (36%) patients of the 689 analyzed. Adenovirus and norovirus were the most common viruses in this cohort, while aichi virus was the only gastrointestinal agent not detected. Surprisingly, rhinovirus was one of the most frequently detected viruses. However, a potential association with gastroenteritis remains to be confirmed.

Full text available from Diagnostic Microbiology & Infectious Disease (USD 31.50) PMID: 24035383

B) Comparative Evaluation of Two Commercial Multiplex Panels for Detection of Gastrointestinal Pathogens by Use of Clinical Stool Specimens Khare R, Espy MJ, Cebelinski E, et al. Comparative evaluation of two commercial multiplex panels for detection of gastrointestinal pathogens by use of clinical stool specimens. J Clin Microbiol. 2014; 52(10): 3667-3673.

Summary: The detection of pathogens associated with gastrointestinal disease may be important in certain patient populations, such as immunocompromised hosts, the critically ill, or individuals with prolonged disease that is refractory to treatment. In this study, we evaluated two commercially available multiplex panels (the FilmArray gastrointestinal [GI] panel [BioFire Diagnostics, Salt Lake City, UT] and the Luminex xTag gastrointestinal pathogen panel [GPP] [Luminex Corporation, Toronto, Canada]) using Cary-Blair stool samples (n = 500) submitted to our laboratory for routine GI testing (e.g., culture, antigen testing, microscopy, and individual real-time PCR). At the time of this study, the prototype (non- FDA-cleared) FilmArray GI panel targeted 23 pathogens (14 bacterial, 5 viral, and 4 parasitic), and testing of 200 mul of Cary-Blair stool was recommended. In contrast, the Luminex GPP assay was FDA cleared for the detection of 11 pathogens (7 bacterial, 2 viral, and 2 parasitic), but had the capacity to identify 4 additional pathogens using a research-use-only protocol. Importantly, the Luminex assay was FDA cleared for 100 mul raw stool; however, 100 mul Cary-Blair stool was tested by the Luminex assay in this study. Among 230 prospectively collected samples, routine testing was positive for one or more GI pathogens in 19 (8.3%) samples, compared to 76 (33.0%) by the FilmArray and 69 (30.3%) by the Luminex assay. Clostridium difficile (12.6 to 13.9% prevalence) and norovirus genogroup I (GI)/GII (5.7 to 13.9% prevalence) were two of the pathogens most commonly detected by both assays among prospective samples. Sapovirus was also commonly detected (5.7% positive rate) by the FilmArray assay. Among 270 additional previously characterized samples, both multiplex panels demonstrated high sensitivity (>90%) for the majority of targets, with the exception of several pathogens, notably Aeromonas sp. (23.8%) by FilmArray and Yersinia enterocolitica (48.1%) by the Luminex assay. Interestingly, the FilmArray and Luminex panels identified mixed infections in 21.1% and 13.0% of positive prospective samples, respectively, compared to only 8.3% by routine methods.

Free full text available from PubMed PMID: 25100818

C) Rapid and Simultaneous Detection of Three Major Diarrhea-Causing Viruses by Multiplex Real-Time Nucleic Acid Sequence-Based Amplification Mo QH, Wang HB, Dai HR, et al. Rapid and Simultaneous Detection of Three Major Diarrhea-Causing Viruses by Multiplex Real-Time Nucleic Acid Sequence-Based Amplification. Arch Virol. 2015; 160(3): 719-725.

Summary: Rotaviruses, noroviruses and astroviruses are the major viral pathogens leading to diarrhea worldwide. Epidemiological investigations of outbreaks associated with these viruses have been impeded by the lack of methods for quick diagnosis and detection. In the current study, a multiplex real-time nucleic acid sequence-based amplification (RT-NASBA) system was developed for the simultaneous detection of rotavirus A/norovirus genogroup II/astrovirus. The specificity and sensitivity of the assay were compared with multiplex RT-PCR. The results showed that the multiplex RT- NASBA assay was established successfully, and robust signals could be observed in 10 minutes with high specificity. The limit of detection of the multiplex RT-NASBA assay was 7, 100, and 200 copies per reaction for rotavirus A, norovirus genogroup II, and astrovirus, respectively. The assay was thus 10 to 100 times more sensitive than multiplex RT-PCR. Clinical evaluation indicated that the assay was 100% concordant with multiplex RT- PCR and was reliable for the detection of both single infections and multiple infections in stool samples. To the best of our knowledge, this is the first multiplex RT-NASBA assay established for the detection of three major diarrhea-causing viruses. This assay provides a valuable platform for the rapid, specific, sensitive and simultaneous diagnosis of these pathogens, especially in resource-limited countries where expensive thermocycling equipment is not available.

Full text available from Archives of Virology (USD 39.95) PMID: 25559674

D) Multicenter Evaluation of the BioFire FilmArray Gastrointestinal Panel for Etiologic Diagnosis of Infectious Gastroenteritis Buss SN, Leber A, Chapin K, et al. Multicenter Evaluation of the BioFire FilmArray Gastrointestinal Panel for Etiologic Diagnosis of Infectious Gastroenteritis. J Clin Microbiol. 2015; 53(3): 915-925.

Summary: The appropriate treatment and control of infectious gastroenteritis depend on the ability to rapidly detect the wide range of etiologic agents associated with the disease. Clinical laboratories currently utilize an array of different methodologies to test for bacterial, parasitic, and viral causes of gastroenteritis, a strategy that suffers from poor sensitivity, potentially long

turnaround times, and complicated ordering practices and workflows. Additionally, there are limited or no testing methods routinely available for most diarrheagenic Escherichia coli strains, astroviruses, and sapoviruses. This study assessed the performance of the FilmArray Gastrointestinal (GI) Panel for the simultaneous detection of 22 different enteric pathogens directly from stool specimens: Campylobacter spp., Clostridium difficile (toxin A/B), Plesiomonas shigelloides, Salmonella spp., Vibrio spp., Vibrio cholerae, Yersinia enterocolitica, enteroaggregative E. coli, enteropathogenic E. coli, enterotoxigenic E. coli, Shiga-like toxin-producing E. coli (stx1 and stx2) (including specific detection of E. coli O157), Shigella spp./enteroinvasive E. coli, Cryptosporidium spp., Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, adenovirus F 40/41, astrovirus, norovirus GI/GII, rotavirus A, and sapovirus. Prospectively collected stool specimens (n = 1,556) were evaluated using the BioFire FilmArray GI Panel and tested with conventional stool culture and molecular methods for comparison. The FilmArray GI Panel sensitivity was 100% for 12/22 targets and >/=94.5% for an additional 7/22 targets. For the remaining three targets, sensitivity could not be calculated due to the low prevalences in this study. The FilmArray GI Panel specificity was >/=97.1% for all panel targets. The FilmArray GI Panel provides a comprehensive, rapid, and streamlined alternative to conventional methods for the etiologic diagnosis of infectious gastroenteritis in the laboratory setting. The potential advantages include improved performance parameters, a more extensive menu of pathogens, and a turnaround time of as short as 1h.

Free full text available from PubMed PMID: 25588652

E) Development and Assessment of Molecular Diagnostic Tests for 15 Enteropathogens Causing Childhood Diarrhoea: A Multicentre Study Liu J, Kabir F, Manneh J, et al. Development and assessment of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: a multicentre study. Lancet Infect Dis. 2014; 14(8): 716-724.

Summary: BACKGROUND: Childhood diarrhoea can be caused by many pathogens that are difficult to assay in the laboratory. Molecular diagnostic techniques provide a uniform method to detect and quantify candidate enteropathogens. We aimed to develop and assess molecular tests for identification of enteropathogens and their association with disease. METHODS: We developed and assessed molecular diagnostic tests for 15 enteropathogens across three platforms-PCR-Luminex, multiplex real-time PCR, and TaqMan array card-at five laboratories worldwide. We judged the analytical and clinical performance of these molecular techniques against

comparator methods (bacterial culture, ELISA, and PCR) using 867 diarrhoeal and 619 non-diarrhoeal stool specimens. We also measured molecular quantities of pathogens to predict the association with diarrhoea, by univariate logistic regression analysis. FINDINGS: The molecular tests showed very good analytical and clinical performance at all five laboratories. Comparator methods had limited sensitivity compared with the molecular techniques (20-85% depending on the target) but good specificity (median 97.3%, IQR 96.5-98.9; mean 95.2%, SD 9.1). Positive samples by comparator methods usually had higher molecular quantities of pathogens than did negative samples, across almost all platforms and for most pathogens (p<0.05). The odds ratio for diarrhoea at a given quantity (measured by quantification cycle, Cq) showed that for most pathogens associated with diarrhoea-including Campylobacter jejuni and Campylobacter coli, Cryptosporidium spp, enteropathogenic Escherichia coli, heat-stable enterotoxigenic E coli, rotavirus, Shigella spp and enteroinvasive E coli, and Vibrio cholerae-the strength of association with diarrhoea increased at higher pathogen loads. For example, Shigella spp at a Cq range of 15-20 had an odds ratio of 8.0 (p<0.0001), but at a Cq range of 25-30 the odds ratio fell to 1.7 (p=0.043). INTERPRETATION: Molecular diagnostic tests can be implemented successfully and with fidelity across laboratories around the world. In the case of diarrhoea, these techniques can detect pathogens with high sensitivity and ascribe diarrhoeal associations based on quantification, including in mixed infections, providing rich and unprecedented measurements of infectious causes. FUNDING: Bill & Melinda Gates Foundation Next Generation Molecular Diagnostics Project.

Full text available from The Lancet (registration required) PMID: 25022434

F) Site-Specific Clinical Evaluation of the Luminex xTAG Gastrointestinal Pathogen Panel for Detection of Infectious Gastroenteritis in Fecal Specimens Patel A, Navidad J, Bhattacharyya S. Site-specific clinical evaluation of the Luminex xTAG gastrointestinal pathogen panel for detection of infectious gastroenteritis in fecal specimens. J Clin Microbiol. 2014; 52(8): 3068-3071.

Summary: We evaluate the clinical performance of the Luminex xTAG gastrointestinal (GI) pathogen in vitro diagnostic (IVD) assay in a comparison between clinical and public health laboratories. The site reproducibility study showed 98.7% sensitivity with high positive and negative agreement values (96.2% and 99.8%, respectively), while assay performance against confirmatory methods resulted in 96.4% sensitivity with similar positive and negative agreement values (90.1% and 99.5%, respectively). High-

throughput detection of multiple GI pathogens improved turnaround time, consolidated laboratory workflow, and simplified stool culture practices, thus reducing the overall cost and number of specimens processed.

Free full text available from PubMed PMID: 24899032

4.6.2 Respiratory

A) Rapid Point of Care Diagnostic Tests for Viral and Bacterial Respiratory Tract Infections-Needs, Advances, and Future Prospects Zumla A, Al-Tawfiq JA, Enne VI, et al. Rapid point of care diagnostic tests for viral and bacterial respiratory tract infections-needs, advances, and future prospects. Lancet Infect Dis. 2014; 14(11):1123-35

Summary: Respiratory tract infections rank second as causes of adult and paediatric morbidity and mortality worldwide. Respiratory tract infections are caused by many different bacteria (including mycobacteria) and viruses, and rapid detection of pathogens in individual cases is crucial in achieving the best clinical management, public health surveillance, and control outcomes. Further challenges in improving management outcomes for respiratory tract infections exist: rapid identification of drug resistant pathogens; more widespread surveillance of infections, locally and internationally; and global responses to infections with pandemic potential. Developments in genome amplification have led to the discovery of several new respiratory pathogens, and sensitive PCR methods for the diagnostic work-up of these are available. Advances in technology have allowed for development of single and multiplexed PCR techniques that provide rapid detection of respiratory viruses in clinical specimens. Microarray-based multiplexing and nucleic- acid-based deep-sequencing methods allow simultaneous detection of pathogen nucleic acid and multiple antibiotic resistance, providing further hope in revolutionising rapid point of care respiratory tract infection diagnostics.

Full text available from The Lancet (registration required) PMID: 25189349

B) Exploring the Potential of Next-Generation Sequencing in Detection of Respiratory Viruses Prachayangprecha S, Schapendonk CM, Koopmans MP, et al. Exploring the Potential of Next-Generation Sequencing in Detection of Respiratory Viruses. J Clin Microbiol. 2014; 52(10): 3722-3730.

Summary: Efficient detection of human respiratory viral pathogens is crucial in the management of patients with acute respiratory tract infection. Sequence-independent amplification of nucleic acids combined with next- generation sequencing technology and bioinformatics analyses is a promising strategy for identifying pathogens in clinical and public health settings. It allows the characterization of hundreds of different known pathogens simultaneously and of novel pathogens that elude conventional testing. However, major hurdles for its routine use exist, including cost, turnaround time, and especially sensitivity of the assay, as the detection limit is dependent on viral load, host genetic material, and sequencing depth. To obtain insights into these aspects, we analyzed nasopharyngeal aspirates from a cohort of 81 Thai children with respiratory disease for the presence of respiratory viruses using a sequence-independent next-generation sequencing approach and routinely used diagnostic real-time reverse transcriptase PCR (real-time RT-PCR) assays. With respect to the detection of rhinovirus and human metapneumovirus, the next-generation sequencing approach was at least as sensitive as diagnostic real-time RT-PCR in this small cohort, whereas for bocavirus and enterovirus, next-generation sequencing was less sensitive than real-time RT-PCR. The advantage of the sequencing approach over real-time RT-PCR was the immediate availability of virus-typing information. Considering the development of platforms capable of generating more output data at declining costs, next-generation sequencing remains of interest for future virus diagnosis in clinical and public health settings and certainly as an additional tool when screening results from real-time RT-PCR are negative.

Free full text available from PubMed PMID: 25100822

C) Clinical Utility of PCR for Common Viruses in Acute Respiratory Illness Rhedin S, Lindstrand A, Rotzen-Ostlund M, et al. Clinical Utility of PCR for Common Viruses in Acute Respiratory Illness. Pediatrics. 2014; 133(3): e538-545.

Summary: BACKGROUND: Acute respiratory illness (ARI) accounts for a large proportion of all visits to pediatric health facilities. Quantitative real-time polymerase chain reaction (qPCR) analyses allow sensitive detection of viral nucleic acids, but it is not clear to what extent specific viruses contribute to disease because many viruses have been detected in asymptomatic children. Better understanding of how to interpret viral findings is important to reduce unnecessary use of antibiotics. OBJECTIVE: To compare viral qPCR findings from children with ARI versus asymptomatic control subjects.

METHODS: Nasopharyngeal aspirates were collected from children aged

Free full text available from Pediatrics PMID: 24567027

D) Impact of a Rapid Respiratory Panel Test on Patient Outcomes Rogers BB, Shankar P, Jerris RC, Kotzbauer D, Anderson EJ, Watson JR. Impact of a Rapid Respiratory Panel Test on Patient Outcomes. Arch Pathol Lab Med. 2015; 139(5):636-641

Results .- The pre-RRP group had 365 patients, and the post-RRP group had 771 patients. After RRP implementation, the mean time to the test result was shorter (383 minutes versus 1119 minutes, P < .001), and the percentage of patients with a result in the emergency department was greater (51.6% versus 13.4%, P < .001). There was no difference in whether antibiotics were prescribed, but the duration of antibiotic use was shorter after RRP implementation (P = .003) and was dependent on receiving test results within 4 hours. If the test result was positive, the inpatient length of stay (P = .03) and the time in isolation (P = .03) were decreased after RRP implementation compared with before RRP implementation. Conclusion .- The RRP decreases the duration of antibiotic use, the length of inpatient stay, and the time in isolation.

Free full text available from the CAP’s Archives PMID: 25152311 NOTE: Also cited in Section 4.2.7

4.6.3 Emerging Infectious Diseases

A) First International External Quality Assessment of Molecular Diagnostics for MERS-CoV Pas SD, Patel P, Reusken C, et al. First International External Quality Assessment of Molecular Diagnostics for MERS-CoV. J Clin Virol. 2015; 69:81-85.

Summary: BACKGROUND: Since the discovery of Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, diagnostic protocols were quickly published and deployed globally. OBJECTIVES: We set out to assess the quality of MERS-CoV molecular diagnostics worldwide. STUDY DESIGN: Both sensitivity and specificity were assessed using 12 samples containing different viral loads of MERS-CoV or common coronaviruses (OC43, 229E, NL63, HKU1). RESULTS: The panel was sent to more than 106 participants, of which 99 laboratories from 6 continents returned 189 panel results.Scores ranged from 100% (84 laboratories) to 33% (1 laboratory). 15% of respondents reported quantitative results, 61% semi-quantitative (Ct-values or time to positivity) and 24% reported qualitative results. The major specific technique used was real-time RT-PCR using the WHO recommended targets upE, ORF1a and ORF1b. The evaluation confirmed that RT-PCRs targeting the ORF1b are less sensitive, and therefore not advised for primary diagnostics. CONCLUSIONS: The first external quality assessment MERS- CoV panel gives a good insight in molecular diagnostic techniques and their performances for sensitive and specific detection of MERS-CoV RNA

globally. Overall, all laboratories were capable of detecting MERS-CoV with some differences in sensitivity. The observation that 8% of laboratories reported false MERS-CoV positive single assay results shows room for improvement, and the importance of using confirmatory targets.

Free full text available from Journal of Clinical Virology PMID: 26209385

B) Evaluation of the FilmArray(®) System for Detection of Bacillus Anthracis, Francisella Tularensis and Yersinia Pestis Seiner DR, Colburn HA, Baird C, et al. Evaluation of the FilmArray(®) system for detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis. J Appl Microbiol. 2013; 114(4): 992-1000.

Summary: AIMS: To evaluate the sensitivity and specificity of the BioFire Diagnostics FilmArray((R)) system in combination with their Biothreat Panel for the detection of Bacillus anthracis (Ba), Francisella tularensis (Ft) and Yersinia pestis (Yp) DNA, and demonstrate the detection of Ba spores. METHODS AND RESULTS: DNA samples from Ba, Ft and Yp strains and near-neighbours, and live Ba spores were analysed using the FilmArray((R)) Biothreat Panel, a multiplexed PCR-based assay for 17 pathogens and toxins. Sensitivity studies with DNA indicate that the limit of detection is 250 genome equivalents (GEs) per sample or lower. Furthermore, the identification of Ft, Yp or Bacillus species was made in 63 of 72 samples tested at 25 GE or less. With samples containing 25 CFU of Ba Sterne spores, at least one of the two possible Ba markers was identified in all samples tested. We observed no cross-reactivity with near-neighbour DNAs. CONCLUSIONS: Our results indicate that the FilmArray((R)) Biothreat Panel is a sensitive and selective assay for detecting the genetic signatures of Ba, Ft and Yp. SIGNIFICANCE AND IMPACT OF THE STUDY: The FilmArray((R)) platform is a complete sample-to-answer system, combining sample preparation, PCR and data analysis. This system is particularly suited for biothreat testing where samples need to be analysed for multiple biothreats by operators with limited training.

Free full text available from PubMed PMID: 23279070

C) Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents Euler M, Wang Y, Heidenreich D, et al. Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents. J Clin Microbiol. 2013; 51(4): 1110-1117.

Summary: Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42 degrees C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms.

Free full text available from PubMed PMID: 23345286

4.6.4 Positive Blood Cultures

A) Accuracy of LightCycler(®) SeptiFast for the Detection and Identification of Pathogens in the Blood of Patients with Suspected Sepsis: A Systematic Review and Meta-Analysis Dark P, Blackwood B, Gates S, et al. Accuracy of LightCycler(®) SeptiFast for the detection and identification of pathogens in the blood of patients with suspected sepsis: a systematic review and meta-analysis. Intensive Care Med. 2015; 41(1): 21-33.

Summary: PURPOSE: There is an urgent need to develop diagnostic tests to improve the detection of pathogens causing life-threatening infection (sepsis). SeptiFast is a CE-marked multi-pathogen real-time PCR system capable of detecting DNA sequences of bacteria and fungi present in blood samples within a few hours. We report here a systematic review and meta- analysis of diagnostic accuracy studies of SeptiFast in the setting of suspected sepsis. METHODS: A comprehensive search strategy was developed to identify studies that compared SeptiFast with blood culture in suspected sepsis. Methodological quality was assessed using QUADAS. Heterogeneity of studies was investigated using a coupled forest plot of sensitivity and specificity and a scatter plot in receiver operator characteristic space. Bivariate model method was used to estimate summary sensitivity

and specificity. RESULTS: From 41 phase III diagnostic accuracy studies, summary sensitivity and specificity for SeptiFast compared with blood culture were 0.68 (95 % CI 0.63-0.73) and 0.86 (95 % CI 0.84-0.89) respectively. Study quality was judged to be variable with important deficiencies overall in design and reporting that could impact on derived diagnostic accuracy metrics. CONCLUSIONS: SeptiFast appears to have higher specificity than sensitivity, but deficiencies in study quality are likely to render this body of work unreliable. Based on the evidence presented here, it remains difficult to make firm recommendations about the likely clinical utility of SeptiFast in the setting of suspected sepsis.

Full text available from Intensive Care Medicine (USD 39.95) PMID: 25416643

B) Clinical Evaluation of the FilmArray Blood Culture Identification Panel in Identification of Bacteria and Yeasts from Positive Blood Culture Bottles Altun O, Almuhayawi M, Ullberg M, Ozenci V. Clinical evaluation of the FilmArray blood culture identification panel in identification of bacteria and yeasts from positive blood culture bottles. J Clin Microbiol. 2013; 51(12): 4130-4136.

Summary: The FilmArray platform (FA; BioFire, Salt Lake City, UT) is a closed diagnostic system allowing high-order multiplex PCR analysis with automated readout of results directly from positive blood cultures in 1 h. In the present study, we evaluated the clinical performance of the FilmArray blood culture identification (BCID) panel, which includes 19 bacteria, five yeasts, and three antibiotic resistance genes. In total, 206 blood culture bottles were included in the study. The FilmArray could identify microorganisms in 153/167 (91.6%) samples with monomicrobial growth. Thirteen of the 167 (7.8%) microorganisms were not covered by the FilmArray BCID panel. In 6/167 (3.6%) samples, the FilmArray detected an additional microorganism compared to blood culture. When polymicrobial growth was analyzed, the FilmArray could detect all target microorganisms in 17/24 (71%) samples. Twelve blood culture bottles that yielded a positive signal but showed no growth were also negative by FilmArray. In 3/206 (1.5%) bottles, the FilmArray results were invalid. The results of the FilmArray were reproducible, as demonstrated by the testing and retesting of five bottles in the same day and a longitudinal follow-up of five other blood cultures up to 4 weeks. The present study shows that the FilmArray is a rapid identification method with high performance in direct identification of bacteria and yeasts from positive blood culture bottles.

Free full text available from PubMed PMID: 24088863

C) Evaluation of the Verigene Gram-Positive Blood Culture Nucleic Acid Test for Rapid Detection of Bacteria and Resistance Determinants Wojewoda CM, Sercia L, Navas M, et al. Evaluation of the Verigene Gram- positive blood culture nucleic acid test for rapid detection of bacteria and resistance determinants. J Clin Microbiol. 2013; 51(7): 2072-2076.

Summary: Rapid identification of pathogens from blood cultures can decrease lengths of stay and improve patient outcomes. We evaluated the accuracy of the Verigene Gram-positive blood culture (BC-GP) nucleic acid test for investigational use only (Nanosphere, Inc., Northbrook, IL) for the identification of Gram-positive bacteria from blood cultures. The detection of resistance genes (mecA in Staphylococcus aureus and Staphylococcus epidermidis and vanA or vanB in Enterococcus faecium and Enterococcus faecalis) by the BC-GP assay also was assessed. A total of 186 positive blood cultures (in BacT/Alert FA bottles) with Gram-positive cocci observed with Gram staining were analyzed using the BC-GP assay. The BC-GP results were compared with the identification and susceptibility profiles obtained with routine methods in the clinical laboratory. Discordant results were arbitrated with additional biochemical, cefoxitin disk, and repeat BC-GP testing. The initial BC-GP organism identification was concordant with routine method results for 94.6% of the blood cultures. Only 40% of the Streptococcus pneumoniae identifications were correct. The detection of the mecA gene for 69 blood cultures with only S. aureus or S. epidermidis was concordant with susceptibility testing results. For 3 of 6 cultures with multiple Staphylococcus spp., mecA detection was reported but was correlated with oxacillin resistance in a species other than S. aureus or S. epidermidis. The detection of vanA agreed with susceptibility testing results for 45 of 46 cultures with E. faecalis or E. faecium. Comparison of the mean times to results for each organism group showed that BC-GP results were available 31 to 42 h earlier than phenotypic identifications and 41 to 50 h earlier than susceptibility results.

Free full text available from PubMed PMID: 23596240

4.7 Clinical Application of Genomics in Infectious Disease

This section includes articles on the following applications:

• Identification and Outbreak Investigation (Section 4.7.1); • Susceptibility and Resistance (Section 4.7.2); and • Public Health and Epidemiology (Section 4.7.3).

A) A Genomic Day in the Life of a Clinical Microbiology Laboratory Long SW, Williams D, Valson C, et al. A genomic day in the life of a clinical microbiology laboratory. J Clin Microbiol. 2013; 51(4): 1272-1277.

Summary: Next-generation sequencing technology is available to many clinical laboratories; however, it is not yet widely used in routine microbiology practice. To demonstrate the feasibility of using whole-genome sequencing in a routine clinical microbiology workflow, we sequenced the genome of every organism isolated in our laboratory for 1 day.

Free full text available from PubMed PMID: 23345298

4.7.1 Identification and Outbreak Investigation

A) Clinical Laboratory Response to a Mock Outbreak of Invasive Bacterial Infections: A Preparedness Study Olsen RJ, Fittipaldi N, Kachroo P, et al. Clinical Laboratory Response to a Mock Outbreak of Invasive Bacterial Infections: A Preparedness Study. J Clin Microbiol. 2014; 52(12): 4210-4216.

Summary: Large hospital-based clinical laboratories must be prepared to rapidly investigate potential infectious disease outbreaks. To challenge the ability of our molecular diagnostics laboratory to use whole-genome sequencing in a potential outbreak scenario and identify impediments to these efforts, we studied 84 invasive serotype emm59 group A streptococcus (GAS) strains collected in the United States. We performed a rapid-response exercise to the mock outbreak scenario using whole-genome sequencing, genome-wide transcript analysis, and mouse virulence studies. The protocol changes installed in response to the lessons learned were tested in a second iteration. The initial investigation was completed in 9 days. Whole-genome sequencing showed that the invasive infections were caused by multiple subclones of epidemic emm59 GAS strains likely spread to the United States from Canada. The phylogenetic tree showed a strong temporal-spatial structure with diversity in mobile genetic element content, features that are useful for identifying closely related strains and possible transmission events. The genome data informed the epidemiology, identifying multiple patients who likely acquired the organisms through direct person-to-person transmission. Transcriptome analysis unexpectedly revealed significantly altered expression of genes encoding a two-component regulator and the hyaluronic acid capsule virulence factor. Mouse infection studies confirmed a high-virulence capacity of these emm59 organisms. Whole-genome sequencing, coupled with transcriptome analysis and animal virulence studies, can be rapidly performed in a clinical environment to effectively contribute to patient care decisions and public health maneuvers.

Free full text available from PubMed PMID: 25253790

B) Detecting the Emergence of Novel, Zoonotic Viruses Pathogenic to Humans Rosenberg R. Detecting the Emergence of Novel, Zoonotic Viruses Pathogenic to Humans. Cell Mol Life Sci. 2015; 72(6): 1115-1125.

Summary: RNA viruses, with their high potential for mutation and epidemic spread, are the most common class of pathogens found as new causes of human illness. Despite great advances made in diagnostic technology since the 1950s, the annual rate at which novel virulent viruses have been found has remained at 2-3. Most emerging viruses are zoonoses; they have jumped from mammal or bird hosts to humans. An analysis of virus discovery indicates that the small number of novel viruses discovered annually is an artifact of inadequate surveillance in tropical and subtropical countries, where even established endemic pathogens are often misdiagnosed. Many of the

emerging viruses of the future are already infecting humans but remain to be uncovered by a strategy of disease surveillance in selected populations.

Free full text available from PubMed PMID: 25416679

C) Tracking a Hospital Outbreak of Carbapenem-Resistant Klebsiella Pneumoniae with Whole-Genome Sequencing Snitkin ES, Zelazny AM, Thomas PJ, et al. Tracking a Hospital Outbreak of Carbapenem-Resistant Klebsiella Pneumoniae with Whole-Genome Sequencing. Sci Transl Med. 2012; 4(148): 148ra116.

Summary: The Gram-negative bacteria Klebsiella pneumoniae is a major cause of nosocomial infections, primarily among immunocompromised patients. The emergence of strains resistant to carbapenems has left few treatment options, making infection containment critical. In 2011, the U.S. National Institutes of Health Clinical Center experienced an outbreak of carbapenem-resistant K. pneumoniae that affected 18 patients, 11 of whom died. Whole-genome sequencing was performed on K. pneumoniae isolates to gain insight into why the outbreak progressed despite early implementation of infection control procedures. Integrated genomic and epidemiological analysis traced the outbreak to three independent transmissions from a single patient who was discharged 3 weeks before the next case became clinically apparent. Additional genomic comparisons provided evidence for unexpected transmission routes, with subsequent mining of epidemiological data pointing to possible explanations for these transmissions. Our analysis demonstrates that integration of genomic and epidemiological data can yield actionable insights and facilitate the control of nosocomial transmission.

Free full text available from PubMed PMID: 22914622

D) The Use of Next Generation Sequencing in the Diagnosis and Typing of Respiratory Infections Thorburn F, Bennett S, Modha S, Murdoch D, Gunson R, Murcia PR. The Use of Next Generation Sequencing in the Diagnosis and Typing of Respiratory Infections. J Clin Virol. 2015; 69:96-100.

Summary: BACKGROUND: Molecular assays are the gold standard methods used to diagnose viral respiratory pathogens. Pitfalls associated with this technique include limits to the number of targeted pathogens, the requirement for continuous monitoring to ensure sensitivity/specificity is

maintained and the need to evolve to include emerging pathogens. Introducing target independent next generation sequencing (NGS) could resolve these issues and revolutionise respiratory viral diagnostics. OBJECTIVES: To compare the sensitivity and specificity of target independent NGS against the current standard diagnostic test. STUDY DESIGN: Diagnostic RT-PCR of clinical samples was carried out in parallel with target independent NGS. NGS sequences were analyzed to determine the proportion with viral origin and consensus sequences were used to establish viral genotypes and serotypes where applicable. RESULTS: 89 nasopharyngeal swabs were tested. A viral pathogen was detected in 43% of samples by NGS and 54% by RT-PCR. All NGS viral detections were confirmed by RT-PCR. CONCLUSIONS: Target independent NGS can detect viral pathogens in clinical samples. Where viruses were detected by RT-PCR alone the Ct value was higher than those detected by both assays, suggesting an NGS detection cut-off - Ct=32. The sensitivity and specificity of NGS compared with RT-PCR was 78% and 80% respectively. This is lower than current diagnostic assays but NGS provided full genome sequences in some cases, allowing determination of viral subtype and serotype. Sequencing technology is improving rapidly and it is likely that within a short period of time sequencing depth will increase in-turn improving test sensitivity.

Free full text available from PubMed PMID: 26209388

E) Whole-Genome Sequencing for Analysis of an Outbreak of Meticillin- Resistant Staphylococcus Aureus: A Descriptive Study Harris SR, Cartwright EJ, Torok ME, et al. Whole-Genome Sequencing for Analysis of an Outbreak of Meticillin-Resistant Staphylococcus Aureus: A Descriptive Study. Lancet Infect Dis. 2013; 13(2): 130-136.

Summary: BACKGROUND: The emergence of meticillin-resistant Staphylococcus aureus (MRSA) that can persist in the community and replace existing hospital-adapted lineages of MRSA means that it is necessary to understand transmission dynamics in terms of hospitals and the community as one entity. We assessed the use of whole-genome sequencing to enhance detection of MRSA transmission between these settings. METHODS: We studied a putative MRSA outbreak on a special care baby unit (SCBU) at a National Health Service Foundation Trust in Cambridge, UK. We used whole-genome sequencing to validate and expand findings from an infection-control team who assessed the outbreak through conventional analysis of epidemiological data and antibiogram profiles. We sequenced isolates from all colonised patients in the SCBU, and sequenced

MRSA isolates from patients in the hospital or community with the same antibiotic susceptibility profile as the outbreak strain. FINDINGS: The hospital infection-control team identified 12 infants colonised with MRSA in a 6 month period in 2011, who were suspected of being linked, but a persistent outbreak could not be confirmed with conventional methods. With whole- genome sequencing, we identified 26 related cases of MRSA carriage, and showed transmission occurred within the SCBU, between mothers on a postnatal ward, and in the community. The outbreak MRSA type was a new sequence type (ST) 2371, which is closely related to ST22, but contains genes encoding Panton-Valentine leucocidin. Whole-genome sequencing data were used to propose and confirm that MRSA carriage by a staff member had allowed the outbreak to persist during periods without known infection on the SCBU and after a deep clean. INTERPRETATION: Whole- genome sequencing holds great promise for rapid, accurate, and comprehensive identification of bacterial transmission pathways in hospital and community settings, with concomitant reductions in infections, morbidity, and costs. FUNDING: UK Clinical Research Collaboration Translational Infection Research Initiative, Wellcome Trust, Health Protection Agency, and the National Institute for Health Research Cambridge Biomedical Research Centre.

Free full text available from PubMed PMID: 23158674

F) Whole-Genome Sequencing of Bacterial Sexually Transmitted Infections: Implications for Clinicians Seth-Smith HM, Thomson NR. Whole-genome sequencing of bacterial sexually transmitted infections: implications for clinicians. Curr Opin Infect Dis. 2013; 26(1): 90-98.

Summary: PURPOSE OF REVIEW: Increasingly, genomics is being used to answer detailed clinical questions. Although genome analysis of bacterial sexually transmitted infections (STIs) lags far behind that of many other bacterial pathogens, genomics can reveal previously inaccessible aspects of pathogen biology. RECENT FINDINGS: Comparative genomic studies on the most common bacterial STI, chlamydia, have revolutionized our understanding of this intracellular bacterium, demonstrating that it undergoes extensive recombination and that the traditional typing schemes can be misleading. Genome projects can also help us to understand the recently observed phenomenon of 'diagnostic escape' seen in both Chlamydia trachomatis and Neisseria gonorrhoeae. SUMMARY: The routine use of genomics in clinical settings is becoming a reality. For STIs, a primary requirement is an understanding of the diversity of circulating strains and

how they change over time. This can help to inform future studies and allow us to address real clinical issues such as outbreak identification, global spread of successful clones and antimicrobial resistance monitoring.

Free full text available from Current Opinion in Infectious Diseases PMID: 23221768

G) Rapid Whole-Genome Sequencing for Investigation of a Neonatal MRSA Outbreak Köser CU, Holden MT, Ellington MJ, et al. Rapid whole-genome sequencing for investigation of a neonatal MRSA outbreak. N Engl J Med. 2012; 366(24):2267–75.

Summary: BACKGROUND: Isolates of methicillin-resistant Staphylococcus aureus (MRSA)belonging to a single lineage are often indistinguishable by means of current typing techniques. Whole-genome sequencing can provide clinically relevant data within a time frame that can influence patient care. The need for automated data interpretation and the provision of clinically meaningful reports represent hurdles to clinical implementation.

Full text available from New England Journal of Medicine (USD 15.00) PMID: 22693998

H) Rapidly Progressive, Fatal, Inhalation Anthrax-like Infection in a Human: Case Report, Pathogen Genome Sequencing, Pathology, and Coordinated Response Wright AM, Beres SB, Consamus EN, et al. Rapidly progressive, fatal, inhalation anthrax-like infection in a human: case report, pathogen genome sequencing, pathology, and coordinated response. Arch Pathol Lab Med. 2011; 135(11):1447–1459.

Summary: Context.—Ten years ago a bioterrorism event involving Bacillus anthracis spores captured the nation's interest, stimulated extensive new research on this pathogen, and heightened concern about illegitimate release of infectious agents. Sporadic reports have described rare, fulminant, and sometimes fatal cases of pneumonia in humans and nonhuman primates caused by strains of Bacillus cereus, a species closely related to Bacillus anthracis. Objectives.—To describe and investigate a case of rapidly progressive, fatal, anthrax-like pneumonia and the overwhelming infection caused by a Bacillus species of uncertain provenance in a patient residing in rural Texas. Design.—We characterized the genome of the causative strain within days of its recovery from antemortem cultures using next-generation sequencing and performed immunohistochemistry on tissues obtained at

autopsy with antibodies directed against virulence proteins of B anthracis and B cereus. Results.—We discovered that the infection was caused by a previously unknown strain of B cereus that was closely related to, but genetically distinct from, B anthracis. The strain contains a plasmid similar to pXO1, a genetic element encoding anthrax toxin and other known virulence factors. Immunohistochemistry demonstrated that several homologs of B anthracis virulence proteins were made in infected tissues, likely contributing to the patient's death. Conclusions.—Rapid genome sequence analysis permitted us to genetically define this strain, rule out the likelihood of bioterrorism, and contribute effectively to the institutional response to this event. Our experience strongly reinforced the critical value of deploying a well-integrated, anatomic, clinical, and genomic strategy to respond rapidly to a potential emerging, infectious threat to public health.

Free full text available from the CAP’s Archives PMID: 21882964

4.7.2 Susceptibility and Resistance

A) Progress on the Development of Rapid Methods for Antimicrobial Susceptibility Testing Pulido MR, Garcia-Quintanilla M, Martin-Pena R, Cisneros JM, McConnell MJ. Progress on the Development of Rapid Methods for Antimicrobial Susceptibility Testing. J Antimicrob Chemother. 2013; 68(12): 2710-2717.

Summary: Antimicrobial susceptibility testing is essential for guiding the treatment of many types of bacterial infections, especially in the current context of rising rates of antibiotic resistance. The most commonly employed methods rely on the detection of phenotypic resistance by measuring bacterial growth in the presence of the antibiotic being tested. Although these methods are highly sensitive for the detection of resistance, they require that the bacterial pathogen is isolated from the clinical sample before testing and must employ incubation times that are sufficient for differentiating resistant from susceptible isolates. Knowledge regarding the molecular determinants of antibiotic resistance has facilitated the development of novel approaches for the rapid detection of resistance in bacterial pathogens. PCR-based techniques, mass spectrometry, microarrays, microfluidics, cell lysis-based approaches and whole-genome sequencing have all demonstrated the ability to detect resistance in various bacterial species. However, it remains to be determined whether these methods can achieve sufficient sensitivity and specificity compared with standard phenotypic resistance testing to justify their use in routine clinical practice. In the present review, we discuss recent

progress in the development of methods for rapid antimicrobial susceptibility testing and highlight the limitations of each approach that still remain be addressed.

Full text available from Journal of Antimicrobial Chemotherapy PMID: 23818283

B) How to Detect Carbapenemase Producers? A Literature Review of Phenotypic and Molecular Methods Hammoudi D, Moubareck CA, Sarkis DK. How to Detect Carbapenemase Producers? A Literature Review of Phenotypic and Molecular Methods. J Microbiol Methods. 2014; 107:106-118.

Summary: This review describes the current state-of-art of carbapenemase detection methods. Identification of carbapenemases is first based on conventional phenotypic tests including antimicrobial susceptibility testing, modified-Hodge test and carbapenemase-inhibitor culture tests. Second, molecular characterization of carbapenemase genes by PCR sequencing is essential. Third, innovative biochemical and spectrometric detection may be applied.

Full text available from Journal of Microbiological Methods (USD 39.95) PMID: 25304059

C) Whole-Genome Sequencing to Control Antimicrobial Resistance Koser CU, Ellington MJ, Peacock SJ. Whole-genome sequencing to control antimicrobial resistance. Trends Genet. 2014; 30(9): 401-407.

Summary: Following recent improvements in sequencing technologies, whole-genome sequencing (WGS) is positioned to become an essential tool in the control of antibiotic resistance, a major threat in modern healthcare. WGS has already found numerous applications in this area, ranging from the development of novel antibiotics and diagnostic tests through to antibiotic stewardship of currently available drugs via surveillance and the elucidation of the factors that allow the emergence and persistence of resistance. Numerous proof-of-principle studies have also highlighted the value of WGS as a tool for day-to-day infection control and, for some pathogens, as a primary diagnostic tool to detect antibiotic resistance. However, appropriate data analysis platforms will need to be developed before routine WGS can be introduced on a large scale.

Free full text available from PubMed PMID: 25096945

D) PBP2a Mutations Causing High-Level Ceftaroline Resistance in Clinical Methicillin-Resistant Staphylococcus Aureus Isolates Long SW, Olsen RJ, Mehta SC, et al. PBP2a mutations causing high-level Ceftaroline resistance in clinical methicillin-resistant Staphylococcus aureus isolates. Antimicrob Agents Chemother. 2014; 58(11): 6668-6674.

Summary: Ceftaroline is the first member of a novel class of cephalosporins approved for use in the United States. Although prior studies have identified eight ceftaroline-resistant methicillin-resistant Staphylococcus aureus (MRSA) isolates in Europe and Asia with MICs ranging from 4 to 8 mg/liter, high-level resistance to ceftaroline (>32 mg/liter) has not been described in MRSA strains isolated in the United States. We isolated a ceftaroline- resistant (MIC > 32 mg/liter) MRSA strain from the blood of a cystic fibrosis patient and five MRSA strains from the respiratory tract of this patient. Whole-genome sequencing identified two amino acid-altering mutations uniquely present in the ceftaroline-binding pocket of the transpeptidase region of penicillin-binding protein 2a (PBP2a) in ceftaroline-resistant isolates. Biochemical analyses and the study of isogenic mutant strains confirmed that these changes caused ceftaroline resistance. Thus, we identified the molecular mechanism of ceftaroline resistance in the first MRSA strain with high-level ceftaroline resistance isolated in the United States.

Free full text available from Antimicrobial Agents and Chemotherapy PMID: 25155594 NOTE: Also cited in Section 4.3.3

E) Comparative Genomic Analysis of Mycobacterium Tuberculosis Drug Resistant Strains from Russia Ilina EN, Shitikov EA, Ikryannikova LN, et al. Comparative genomic analysis of Mycobacterium tuberculosis drug resistant strains from Russia. PLoS One. 2013; 8(2): e56577.

Summary: Tuberculosis caused by multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (MTB) strains is a growing problem in many countries. The availability of the complete nucleotide sequences of several MTB genomes allows to use the comparative genomics as a tool to study the relationships of strains and differences in their evolutionary history including acquisition of drug- resistance. In our work, we sequenced three genomes of Russian MTB strains of different phenotypes--drug susceptible, MDR and XDR. Of them, MDR and XDR strains were collected in Tomsk (Siberia, Russia) during the local TB outbreak in 1998-1999 and belonged to rare KQ and KY families in

accordance with IS6110 typing, which are considered endemic for Russia. Based on phylogenetic analysis, our isolates belonged to different genetic families, Beijing, Ural and LAM, which made the direct comparison of their genomes impossible. For this reason we performed their comparison in the broader context of all M. tuberculosis genomes available in GenBank. The list of unique individual non-synonymous SNPs for each sequenced isolate was formed by comparison with all SNPs detected within the same phylogenetic group. For further functional analysis, all proteins with unique SNPs were ascribed to 20 different functional classes based on Clusters of Orthologous Groups (COG). We have confirmed drug resistant status of our isolates that harbored almost all known drug-resistance associated mutations. Unique SNPs of an XDR isolate CTRI-4(XDR), belonging to a Beijing family were compared in more detail with SNPs of additional 14 Russian XDR strains of the same family. Only type specific mutations in genes of repair, replication and recombination system (COG category L) were found common within this group. Probably the other unique SNPs discovered in CTRI-4(XDR) may have an important role in adaptation of this microorganism to its surrounding and in escape from antituberculosis drugs treatment.

Free full text available from PubMed PMID: 23437175

F) Mycobacterium Tuberculosis Mutation Rate Estimates from Different Lineages Predict Substantial Differences in the Emergence of Drug- Resistant Tuberculosis Ford CB, Shah RR, Maeda MK, et al. Mycobacterium tuberculosis mutation rate estimates from different lineages predict substantial differences in the emergence of drug-resistant tuberculosis. Nat Genet. 2013; 45(7): 784-790.

Summary: A key question in tuberculosis control is why some strains of M. tuberculosis are preferentially associated with resistance to multiple drugs. We demonstrate that M. tuberculosis strains from lineage 2 (East Asian lineage and Beijing sublineage) acquire drug resistances in vitro more rapidly than M. tuberculosis strains from lineage 4 (Euro-American lineage) and that this higher rate can be attributed to a higher mutation rate. Moreover, the in vitro mutation rate correlates well with the bacterial mutation rate in humans as determined by whole-genome sequencing of clinical isolates. Finally, using a stochastic mathematical model, we demonstrate that the observed differences in mutation rate predict a substantially higher probability that patients infected with a drug-susceptible lineage 2 strain will harbor multidrug-resistant bacteria at the time of diagnosis. These data suggest that

interventions to prevent the emergence of drug-resistant tuberculosis should target bacterial as well as treatment-related risk factors.

Free full text available from PubMed PMID: 23749189

G) Alternative Mutational Pathways to Intermediate Resistance to Vancomycin in Methicillin-Resistant Staphylococcus Aureus Vidaillac C, Gardete S, Tewhey R, et al. Alternative mutational pathways to intermediate resistance to vancomycin in methicillin-resistant Staphylococcus aureus. J Infect Dis. 2013; 208(1): 67-74.

Free full text available from Journal of Infectious Disease PMID: 23539745 NOTE: Also cited in Section 4.3.3

H) Deep Sequencing Reveals Mutagenic Effects of Ribavirin During Monotherapy of Hepatitis C Virus Genotype 1-Infected Patients Dietz J, Schelhorn SE, Fitting D, et al. Deep sequencing reveals mutagenic effects of ribavirin during monotherapy of hepatitis C virus genotype 1- infected patients. J Virol. 2013; 87(11): 6172-6181.

Summary: The preeminent mode of action of the broad-spectrum antiviral nucleoside ribavirin in the therapy of chronic hepatitis C is currently unresolved. Particularly under contest are possible mutagenic effects of ribavirin that may lead to viral extinction by lethal mutagenesis of the hepatitis C virus (HCV) genome. We applied ultradeep sequencing to determine ribavirin-induced sequence changes in the HCV coding region (nucleotides [nt] 330 to 9351) of patients treated with 6-week ribavirin monotherapy (n = 6) in comparison to placebo (n = 6). Baseline HCV RNA levels maximally declined on average by -0.8 or -0.1 log10 IU/ml in ribavirin- versus placebo-treated patients. No general increase in rates of nucleotide substitutions in ribavirin-treated patients was observed. However, more HCV genome positions with high G-to-A and C-to-U transition rates were detected between baseline and treatment week 6 in ribavirin-treated patients in comparison to placebo-treated patients (rate of 0.0041 transitions per base pair versus rate of 0.0022 transitions per base pair; P = 0.049). Similarly, the sensitive detection of low-frequency minority variants by statistical filtering indicated significantly more positions with G-to-A and C-to-U transitions in ribavirin-treated patients than in placebo-treated patients (rate of 0.0331 transitions versus rate of 0.0186 transitions per G/C-containing position at baseline; P = 0.018). In contrast, non-ribavirin-associated A-to-G and U-to-C transitions were not enriched in the ribavirin group (P = 0.152). We conclude that ribavirin exerts a mutagenic effect on the virus in patients with chronic hepatitis C by facilitating G-to-A and C-to-U nucleotide transitions.

Free full text available from PubMed PMID: 23536652

I) Genome Sequencing Sheds Light on Emerging Drug Resistance in Malaria Parasites Neafsey DE. Genome sequencing sheds light on emerging drug resistance in malaria parasites. Nat Genet. 2013; 45(6): 589-590.

Full text article available from Nature Genetics (USD 18.00) PMID: 23715326 NOTE: Also cited in Section 4.5.1

J) Genetic Analysis of Primaquine Tolerance in a Patient with Relapsing Vivax Malaria Bright AT, Alenazi T, Shokoples S, et al. Genetic analysis of primaquine tolerance in a patient with relapsing vivax malaria. Emerg Infect Dis. 2013; 19(5): 802-805.

Summary: Patients with Plasmodium vivax malaria are treated with primaquine to prevent relapse infections. We report primaquine failure in a patient with 3 relapses without any possibility of re-infection. Using whole genome sequencing of the relapsing parasite isolates, we identified single nucleotide variants as candidate molecular markers of resistance.

Free full text available from PubMed PMID: 23648098

K) Low-Level Persistence of Drug Resistance Mutations in Hepatitis B Virus-Infected Subjects with a Past History of Lamivudine Treatment Margeridon-Thermet S, Svarovskaia ES, Babrzadeh F, et al. Low-level persistence of drug resistance mutations in hepatitis B virus-infected subjects with a past history of Lamivudine treatment. Antimicrob Agents Chemother. 2013; 57(1): 343-349.

discontinuation was 12.8 months in the 10 subjects with a LAM resistance mutation compared to 30.5 months in the 36 LAM-experienced subjects without a LAM resistance mutation (P < 0.001). The likelihood of detecting a LAM resistance mutation was significantly increased using UDPS compared to Sanger sequencing and was inversely associated with the time since LAM discontinuation.

Free full text available from PubMed PMID: 23114756 NOTE: Also cited in Section 4.2.4

4.7.3 Public Health and Epidemiology

A) Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis Rhee SY, Blanco JL, Jordan MR, et al. Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis. PLoS Med. 2015; 12(4): e1001810.

Summary: BACKGROUND: Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. METHODS AND FINDINGS: We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naive individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05-1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non- nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds

ratio [OR] = 1.16; 95% CI: 1.06-1.25), North America (OR = 1.19; 95% CI: 1.12-1.26), Europe (OR = 1.07; 95% CI: 1.01-1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12-1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92-1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions-a proxy for recent infection-yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs-K101E, K103N, Y181C, and G190A-accounted for >80% of NNRTI- associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI- associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high- level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. CONCLUSIONS: Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.

Free full text available from PubMed PMID: 25849352

B) The Current Epidemiology and Clinical Decisions Surrounding Acute Respiratory Infections Zaas AK, Garner BH, Tsalik EL, Burke T, Woods CW, Ginsburg GS. The Current Epidemiology and Clinical Decisions Surrounding Acute Respiratory Infections. Trends Mol Med. 2014; 20(10): 579-588.

Summary: Acute respiratory infection (ARI) is a common diagnosis in outpatient and emergent care settings. Currently available diagnostics are limited, creating uncertainty in the use of antibacterial, antiviral, or supportive care. Up to 72% of ambulatory care patients with ARI are treated with an

antibacterial, despite only a small fraction actually needing one. Antibiotic overuse is not restricted to ambulatory care: ARI accounts for approximately 5 million emergency department (ED) visits annually in the USA, where 52- 61% of such patients receive antibiotics. Thus, an accurate test for the presence or absence of viral or bacterial infection is needed. In this review, we focus on recent research showing that the host-response (genomic, proteomic, or miRNA) can accomplish this task.

Full text available from Trends in Molecular Medicine (USD 31.50 for 24 hour access) PMID: 25201713

C) Advanced Molecular Surveillance of Hepatitis C Virus Rossi LM, Escobar-Gutierrez A, Rahal P. Advanced Molecular Surveillance of Hepatitis C Virus. Viruses. 2015; 7(3): 1153-1188.

Summary: Hepatitis C virus (HCV) infection is an important public health problem worldwide. HCV exploits complex molecular mechanisms, which result in a high degree of intrahost genetic heterogeneity. This high degree of variability represents a challenge for the accurate establishment of genetic relatedness between cases and complicates the identification of sources of infection. Tracking HCV infections is crucial for the elucidation of routes of transmission in a variety of settings. Therefore, implementation of HCV advanced molecular surveillance (AMS) is essential for disease control. Accounting for virulence is also important for HCV AMS and both viral and host factors contribute to the disease outcome. Therefore, HCV AMS requires the incorporation of host factors as an integral component of the algorithms used to monitor disease occurrence. Importantly, implementation of comprehensive global databases and data mining are also needed for the proper study of the mechanisms responsible for HCV transmission. Here, we review molecular aspects associated with HCV transmission, as well as the most recent technological advances used for virus and host characterization. Additionally, the cornerstone discoveries that have defined the pathway for viral characterization are presented and the importance of implementing advanced HCV molecular surveillance is highlighted.

Free full text available from PubMed PMID: 25781918

D) Absence of Patient-to-Patient Intrahospital Transmission of Staphylococcus Aureus as Determined by Whole-Genome Sequencing Long SW, Beres SB, Olsen RJ, Musser JM. Absence of patient-to-patient intrahospital transmission of Staphylococcus aureus as determined by whole- genome sequencing. MBio. 2014(5): e01692-01614.

Summary: Nosocomial transmission of pathogens is a major health care challenge. The increasing spread of antibiotic-resistant strains represents an ongoing threat to public health. Previous Staphylococcus aureus transmission studies have focused on transmission of S. aureus between asymptomatic carriers or used low-resolution typing methods such as multilocus sequence typing (MLST) or spa typing. To identify patient-to- patient intrahospital transmission using high-resolution genetic analysis, we sequenced the genomes of a consecutive set of 398 S. aureus isolates from sterile-site infections. The S. aureus strains were collected from four hospitals in the Houston Methodist Hospital System over a 6-month period. Importantly, we discovered no evidence of transmission of S. aureus between patients with sterile-site infections. The lack of intrahospital transmission may reflect a fundamental difference between day-to-day transmission events in the hospital setting and the more frequently studied outbreak scenarios. Importance: Previous studies have suggested that nosocomial transmission of S. aureus is common. Our data revealed an unexpected lack of evidence for intrahospital transmission of S. aureus between patients with invasive infections. This finding has important implications for hospital infection control and public health efforts. In addition, our data demonstrate that highly related pools of S. aureus strains exist in the community which may complicate outbreak investigations.

Free full text available from PubMed PMID: 25293757

E) Rapid Bacterial Whole-genome Sequencing to Enhance Diagnostic and Public Health Microbiology Reuter S, Ellington MJ, Cartwright EJ, et al. Rapid bacterial whole-genome sequencing to enhance diagnostic and public health microbiology. JAMA Intern Med. 2013; 173(15): 1397-1404.

Summary: IMPORTANCE: The latest generation of benchtop DNA sequencing platforms can provide an accurate whole-genome sequence (WGS) for a broad range of bacteria in less than a day. These could be used to more effectively contain the spread of multidrug-resistant pathogens. OBJECTIVE: To compare WGS with standard clinical microbiology practice for the investigation of nosocomial outbreaks caused by multidrug-resistant

bacteria, the identification of genetic determinants of antimicrobial resistance, and typing of other clinically important pathogens. DESIGN, SETTING, AND PARTICIPANTS: A laboratory-based study of hospital inpatients with a range of bacterial infections at Cambridge University Hospitals NHS Foundation Trust, a secondary and tertiary referral center in England, comparing WGS with standard diagnostic microbiology using stored bacterial isolates and clinical information. MAIN OUTCOMES AND MEASURES: Specimens were taken and processed as part of routine clinical care, and cultured isolates stored and referred for additional reference laboratory testing as necessary. Isolates underwent DNA extraction and library preparation prior to sequencing on the Illumina MiSeq platform. Bioinformatic analyses were performed by persons blinded to the clinical, epidemiologic, and antimicrobial susceptibility data. RESULTS: We investigated 2 putative nosocomial outbreaks, one caused by vancomycin-resistant Enterococcus faecium and the other by carbapenem-resistant Enterobacter cloacae; WGS accurately discriminated between outbreak and nonoutbreak isolates and was superior to conventional typing methods. We compared WGS with standard methods for the identification of the mechanism of carbapenem resistance in a range of gram-negative bacteria (Acinetobacter baumannii, E cloacae, Escherichia coli, and Klebsiella pneumoniae). This demonstrated concordance between phenotypic and genotypic results, and the ability to determine whether resistance was attributable to the presence of carbapenemases or other resistance mechanisms. Whole-genome sequencing was used to recapitulate reference laboratory typing of clinical isolates of Neisseria meningitidis and to provide extended phylogenetic analyses of these. CONCLUSIONS AND RELEVANCE: The speed, accuracy, and depth of information provided by WGS platforms to confirm or refute outbreaks in hospitals and the community, and to accurately define transmission of multidrug-resistant and other organisms, represents an important advance.

Free full text available from PubMed PMID: 23857503

F) Outbreak Investigation Using High-Throughput Genome Sequencing Within a Diagnostic Microbiology Laboratory Sherry NL, Porter JL, Seemann T, Watkins A, Stinear TP, Howden BP. Outbreak investigation using high-throughput genome sequencing within a diagnostic microbiology laboratory. J Clin Microbiol. 2013; 51(5): 1396-1401.

Summary: Next-generation sequencing (NGS) of bacterial genomes has recently become more accessible and is now available to the routine diagnostic microbiology laboratory. However, questions remain regarding its feasibility, particularly with respect to data analysis in nonspecialist centers.

To test the applicability of NGS to outbreak investigations, Ion Torrent sequencing was used to investigate a putative multidrug-resistant Escherichia coli outbreak in the neonatal unit of the Mercy Hospital for Women, Melbourne, Australia. Four suspected outbreak strains and a comparator strain were sequenced. Genome-wide single nucleotide polymorphism (SNP) analysis demonstrated that the four neonatal intensive care unit (NICU) strains were identical and easily differentiated from the comparator strain. Genome sequence data also determined that the NICU strains belonged to multilocus sequence type 131 and carried the bla(CTX- M-15) extended-spectrum beta-lactamase. Comparison of the outbreak strains to all publicly available complete E. coli genome sequences showed that they clustered with neonatal meningitis and uropathogenic isolates. The turnaround time from a positive culture to the completion of sequencing (prior to data analysis) was 5 days, and the cost was approximately $300 per strain (for the reagents only). The main obstacles to a mainstream adoption of NGS technologies in diagnostic microbiology laboratories are currently cost (although this is decreasing), a paucity of user-friendly and clinically focused bioinformatics platforms, and a lack of genomics expertise outside the research environment. Despite these hurdles, NGS technologies provide unparalleled high-resolution genotyping in a short time frame and are likely to be widely implemented in the field of diagnostic microbiology in the next few years, particularly for epidemiological investigations (replacing current typing methods) and the characterization of resistance determinants. Clinical microbiologists need to familiarize themselves with these technologies and their applications.

Free full text available from PubMed PMID: 23408689

US FDA-approved drugs that contain pharmacogenetics and pharmacogenomics information in their package insert labels. Weng L, Zhang L, Peng Y, Huang RS. Pharmacogenetics and pharmacogenomics: a bridge to individualized cancer therapy. Pharmacogenomics. Feb 2013;14(3):315-324. This section includes the following topics:

• Overview of Pharmacogenomics and Its Clinical Utility and Implementation (Section 5.1); • Cardiovascular Pharmacogenomics (Section 5.2); • Pharmacogenomics in Cancer (Section 5.3); • Pharmacogenomics in Anesthesia (Section 5.4); • Pharmacogenomics of Infectious Diseases and Interactions with the Immune System (Section 5.5); • Psychiatric Pharmacogenomics (Section 5.6); and • Other Pharmacogenomics (Section 5.7).

Pharmacogenomics Quick Reference Table: Commonly Tested Gene-Drug Pairs

Gene-drug pairs presented in this table represent those for which tests are available and clinical guidelines exist. The information presented in this table is for quick reference only and is limited to genes with relevant germline variants. Clinical decision making should not be based solely on this information and additional tests relevant to other drugs may also be available. For more information, please see the references and resources listed after the table. Gene Drug Genotypes References HLA-B Abacavir *57:01 - increased risk of 6, 18 hypersensitivity Allopurinol *58:01 - increased risk of severe 6, 28 cutaneous adverse reactions Carbamazepine *15:02 - increased risk of Stevens- 6, 16 Johnson Syndrome/Toxic Epidermal Necrolysis

Return to Table of Contents 335 Section 5

HLA-B Phenytoin *15:02 - increased risk of Stevens- 5, 6 Johnson Syndrome/Toxic Epidermal Necrolysis CYP2C19 Tricyclic increased activity (*17) ‐ increased 13 Antidepressants metabolism and possibly decreased (i.e. amitriptyline) efficacy, consider different drug; decreased activity (*2‐8) ‐reduced metabolism and increased risk of side effects Clopidogrel increased activity (*17) ‐ increased 19, 26, 30 platelet inhibition; decreased activity (*2‐8) ‐reduced platelet inhibition/risk for cardiovascular events Selective increased activity (*17) ‐ increased 12 Serotonin metabolism and possibly decreased Reuptake efficacy, consider different drug; Inhibitors (i.e. decreased activity (*2‐8) ‐reduced citalopram, metabolism and increased risk of side escitalopram) effects Voriconazole decreased activity variants ‐ increased 15, 31, 32 risk of adverse effects CYP2D6 Tricyclic increased activity (*1xN, *2xN, multiple 13 Antidepressants functional copies) ‐ increased (i.e. amitriptyline) metabolism and decreased efficacy; decreased activity (*4, *5, *6) ‐ reduced metabolism and increased side effects Codine increased activity (*1xN, *2xN, multiple 8 functional copies) ‐ increased formation of morphine and higher risk of toxicity; decreased activity (*4, *5, *6) ‐ decreased morphine formation and insufficient pain relief Selective 12 Serotonin decreased/inactive (*3‐*17, *19‐21, Reuptake *29, *38, *40, *42) ‐ reduced Inhibitors (i.e. metabolism and increased side effects citalopram, escitalopram)

CYP2D6 Oxycodone poor metabolizers (2 copies of *3‐8, 8, 29 *11‐16, *19‐21, *38, *40, *42) ‐ less pain relief, neutropenia, leukopenia, moderate diarrhea Tramadol decreased/inactive (*3‐*17, *19‐21, 8 *29, *38, *40, *42) ‐ increased drug concentrations and possibly increased risk of adverse events Tamoxifen decreased/inactive (*3‐*17, *19‐21, 9, 22, 31 *29, *38, *40, *42) ‐ increased risk of breast cancer relapse, consider aromatase inhibitor for post‐ menopausal women UGT1A1 Atazanavir reduced activity (*28 [TA7], *37 [TA8], 11 *6, *80) ‐ increased likelihood of bilirubin‐related discontinuation of atazanavir Irinotecan reduced activity (*28 [TA7]) ‐ increased 3, 10, 20 risk for neutropenia TPMT Thiopurines (i.e. reduced activity (*2, *3A, *3B, *3C, *4) 22, 24 azathioprine, ‐ increased risk for myelosuppression, mercaptopurine) fatal toxicity possible

DPYD Fluoropyrimidines non‐functional variants (*2A, *13, 5, 22 (i.e. 5- rs67376798 A) ‐ increased risk for fluorouracil, severe/fatal drug toxicity capecitabine, tegafur) CFTR Ivacaftor G551D, G1244E, G1349D, G178R, 2, 7 G551S, S1251N, S1255P, S549N, S549R ‐ effective; F508del homozygotes ‐ not effective

CYP2C9 Phenytoin reduced activity (*2, *3) ‐ increased risk 4 of toxicity Warfarin reduced activity (*2, *3) ‐ increased risk 14, 26 of bleeding, lower starting dose recommended, VKORC1 genotype also of importance

G6PD Rasburicase deficiency (male with class I, II, or III 17, 25 allele; female with two class I‐III alleles) ‐ increased risk of acute hemolytic anemia SLCO1B1 Simvastatin reduced function/expression (*5, *15, 23, 26 *17) ‐ increased risk of myopathy CYP3A5 Tacrolimus extensive and intermediate 1, 27 metabolizers (*1/*1, *1/*3, *1/*6, *1/*7) ‐ decreased chance of achieving target concentrations, increased starting dose recommended VKORC1 Warfarin rs9923231 (‐1639G>A) ‐ A allele 14, 26 associated with reduced VKORC1 (target of warfarin), reduced dose of warfarin necessary IFNL3 PEG Interferon- rs12979860 CC ‐ increased likelihood of 21 Alpha response in patients with HCV genotype 1 Source: Ann M. Moyer, MD, PhD

1. Birdwell KA, Decker B, Barbarino JM, et al. Clinical Pharmacogenetics Implementation Consortium (CPIC) Guidelines for Cyp3a5 Genotype and Tacrolimus Dosing. Clin Pharmacol Ther. 2015; 98(1): 19-24. 2. Brodlie M, Haq IJ, Roberts K, Elborn JS. Targeted Therapies to Improve CFTR Function in Cystic Fibrosis. Genome Med. 2015; 7:101. 3. Butzke B, Oduncu FS, Severin F, et al. The Cost-Effectiveness of UT1A1 Genotyping before Colorectal Cancer Treatment with Irinotecan from the Perspective of the German Statutory Health Insurance. Acta Oncol. 2016; 55(3): 318-328. 4. Caudle KE, Rettie AE, Whirl-Carrillo M, et al. Clinical Pharmacogenetics Implementation Consortium Guidelines for CYP2C9 and HLA-B Genotypes and Phenytoin Dosing. Clin Pharmacol Ther. 2014; 96(5): 542-548. 5. Caudle KE, Thorn CF, Klein TE, et al. Clinical Pharmacogenetics Implementation Consortium Guidelines for Dihydropyrimidine Dehydrogenase Genotype and Fluoropyrimidine Dosing. Clin Pharmacol Ther. 2013; 94(6): 640-645. 6. Cheng CY, Su SC, Chen CH, Chen WL, Deng ST, Chung WH. HLA Associations and Clinical Implications in T-Cell Mediated Drug Hypersensitivity Reactions: An Updated Review. J Immunol Res. 2014; 2014:565320. 7. Clancy JP, Johnson SG, Yee SW, et al. Clinical Pharmacogenetics Implementation Consortium (CPIC) Guidelines for Ivacaftor Therapy in the Context of CFTR Genotype. Clin Pharmacol Ther. 2014; 95(6): 592-597. 8. Crews KR, Gaedigk A, Dunnenberger HM, et al. Clinical Pharmacogenetics Implementation Consortium Guidelines for Cytochrome P450 2D6 Genotype and Codeine Therapy: 2014 Update. Clin Pharmacol Ther. 2014; 95(4): 376-382. 9. Del Re M, Citi V, Crucitta S, et al. Pharmacogenetics of CYP2D6 and Tamoxifen Therapy: Light at the End of the Tunnel? Pharmacol Res. 2016; 107:398-406 10. Etienne-Grimaldi MC, Boyer JC, Thomas F, et al. UGT1A1 Genotype and Irinotecan Therapy: General Review and Implementation in Routine Practice. Fundam Clin Pharmacol. 2015; 29(3): 219-237.

11. Gammal RS, Court MH, Haidar CE, et al. Clinical Pharmacogenetics Implementation Consortium (CPIC) Guideline for UGT1A1 and Atazanavir Prescribing. Clin Pharmacol Ther. 2016; 99(4): 363-369. 12. Hicks JK, Bishop JR, Sangkuhl K, et al. Clinical Pharmacogenetics Implementation Consortium (CPIC) Guideline for CYP2D6 and CYP2C19 Genotypes and Dosing of Selective Serotonin Reuptake Inhibitors. Clin Pharmacol Ther. 2015; 98(2): 127-134. 13. Hicks JK, Swen JJ, Thorn CF, et al. Clinical Pharmacogenetics Implementation Consortium Guideline for CYP2D6 and CYP2C19 Genotypes and Dosing of Tricyclic Antidepressants. Clin Pharmacol Ther. 2013; 93(5): 402-408. 14. Johnson JA, Gong L, Whirl-Carrillo M, et al. Clinical Pharmacogenetics Implementation Consortium Guidelines for CYP2C9 and VKORC1 Genotypes and Warfarin Dosing. Clin Pharmacol Ther. 2011; 90(4): 625-629. 15. Lamoureux F, Duflot T, Woillard JB, et al. Impact of CYP2C19 Genetic Polymorphisms on Voriconazole Dosing and Exposure in Adult Patients with Invasive Fungal Infections. Int J Antimicrob Agents. 2016; 47(2): 124-131. 16. Leckband SG, Kelsoe JR, Dunnenberger HM, et al. Clinical Pharmacogenetics Implementation Consortium Guidelines for HLA-B Genotype and Carbamazepine Dosing. Clin Pharmacol Ther. 2013; 94(3): 324-328. 17. Luzzatto L and Seneca E. G6PD Deficiency: A Classic Example of Pharmacogenetics with on- Going Clinical Implications. Br J Haematol. 2014; 164(4): 469-480. 18. Martin MA, Hoffman JM, Freimuth RR, et al. Clinical Pharmacogenetics Implementation Consortium Guidelines for HLA-B Genotype and Abacavir Dosing: 2014 Update. Clin Pharmacol Ther. 2014; 95(5): 499-500. 19. Mega JL, Close SL, Wiviott SD, et al. Cytochrome P-450 Polymorphisms and Response to Clopidogrel. N Engl J Med. 2009; 360(4): 354-362. 20. Miyata Y, Touyama T, Kusumi T, et al. UDP-Glucuronosyltransferase 1A1*6 and *28 Polymorphisms as Indicators of Initial Dose Level of Irinotecan to Reduce Risk of Neutropenia in Patients Receiving FOLFIRI for Colorectal Cancer. Int J Clin Oncol. 2016;21(4):696-703. 21. Muir AJ, Gong L, Johnson SG, et al. Clinical Pharmacogenetics Implementation Consortium (CPIC) Guidelines for IFNL3 (IL28B) Genotype and PEG Interferon-Alpha-Based Regimens. Clin Pharmacol Ther. 2014; 95(2): 141-146. 22. Pesenti C, Gusella M, Sirchia SM and Miozzo M. Germline Oncopharmacogenetics, a Promising Field in Cancer Therapy. Cell Oncol (Dordr). 2015; 38(1): 65-89. 23. Ramsey LB, Johnson SG, Caudle KE, et al. The Clinical Pharmacogenetics Implementation Consortium Guideline for SLCO1B1 and Simvastatin-Induced Myopathy: 2014 Update. Clin Pharmacol Ther. 2014; 96(4): 423-428. 24. Relling MV, Gardner EE, Sandborn WJ, et al. Clinical Pharmacogenetics Implementation Consortium Guidelines for Thiopurine Methyltransferase Genotype and Thiopurine Dosing: 2013 Update. Clin Pharmacol Ther. 2013; 93(4): 324-325. 25. Relling MV, McDonagh EM, Chang T, et al. Clinical Pharmacogenetics Implementation Consortium (CPIC) Guidelines for Rasburicase Therapy in the Context of G6PD Deficiency Genotype. Clin Pharmacol Ther. 2014; 96(2): 169-174. 26. Roden DM. Cardiovascular Pharmacogenomics: Current Status and Future Directions. J Hum Genet. 2016; 61(1): 79-85. 27. Rojas L, Neumann I, Herrero MJ, et al. Effect of CYP3A5*3 on Kidney Transplant Recipients Treated with Tacrolimus: A Systematic Review and Meta-Analysis of Observational Studies. Pharmacogenomics J. 2015; 15(1): 38-48. 28. Saito Y, Stamp LK, Caudle KE, et al. Clinical Pharmacogenetics Implementation Consortium (CPIC) Guidelines for Human Leukocyte Antigen B (HLA-B) Genotype and Allopurinol Dosing: 2015 Update. Clin Pharmacol Ther. 2016; 99(1): 36-37. 29. Samer CF, Daali Y, Wagner M, et al. Genetic Polymorphisms and Drug Interactions Modulating CYP2D6 and CYP3A Activities Have a Major Effect on Oxycodone Analgesic Efficacy and Safety. Br J Pharmacol. 2010; 160(4): 919-930. 30. Scott SA, Sangkuhl K, Stein CM, et al. Clinical Pharmacogenetics Implementation Consortium Guidelines for CYP2C19 Genotype and Clopidogrel Therapy: 2013 Update. Clin Pharmacol Ther. 2013; 94(3): 317-323.

31. Swen JJ, Nijenhuis M, de Boer A, et al. Pharmacogenetics: From Bench to Byte--an Update of Guidelines. Clin Pharmacol Ther. 2011; 89(5): 662-673. 32. Teusink A, Vinks A, Zhang K, et al. Genotype-Directed Dosing Leads to Optimized Voriconazole Levels in Pediatric Patients Receiving Hematopoietic Stem Cell Transplantation. Biol Blood Marrow Transplant. 2016; 22(3): 482-486.

Additional Resources  Clinical Pharmacogenetics Implementation Consortium (CPIC); www.cpicpgx.org  Dutch Pharmacogenetics Working Group (DPWG); www.pharmgkb.org/page/dpwg  PharmGKB; www.pharmgkb.org  U.S. Food and Drug Administration (FDA); http://www.fda.gov/Drugs/ScienceResearch/ResearchAreas/Pharmacogenetics/ucm08337 8.htm

5.1 Overview of Pharmacogenomics and Its Clinical Utility and Implementation

A) Preemptive Pharmacogenomic Testing for Precision Medicine: A Comprehensive Analysis of Five Actionable Pharmacogenomic Genes Using Next-Generation DNA Sequencing and a Customized CYP2D6 Genotyping Cascade Ji Y, Skierka JM, Blommel JH, et al. Preemptive Pharmacogenomic Testing for Precision Medicine: A Comprehensive Analysis of Five Actionable Pharmacogenomic Genes Using Next-Generation DNA Sequencing and a Customized CYP2D6 Genotyping Cascade. J Mol Diagn. 2016; 18(3):438- 45.

Summary: Significant barriers, such as lack of professional guidelines, specialized training for interpretation of pharmacogenomics (PGx) data, and insufficient evidence to support clinical utility, prevent preemptive PGx testing from being widely clinically implemented. The current study, as a pilot project for the Right Drug, Right Dose, Right Time-Using Genomic Data to Individualize Treatment Protocol, was designed to evaluate the impact of preemptive PGx and to optimize the workflow in the clinic setting. We used an 84-gene next-generation sequencing panel that included SLCO1B1, CYP2C19, CYP2C9, and VKORC1 together with a custom-designed CYP2D6 testing cascade to genotype the 1013 subjects of the Right Drug, Right Dose, Right Time-Using Genomic Data to Individualize Treatment Protocol in laboratories approved by the Clinical Laboratory Improvement Act. Actionable PGx variants were placed in patient's electronic medical records where integrated clinical decision support rules alert providers when a relevant medication is ordered. The fraction of this cohort carrying actionable PGx variant(s) in individual genes ranged from 30% (SLCO1B1)

to 79% (CYP2D6). When considering all five genes together, 99% of the subjects carried an actionable PGx variant(s) in at least one gene. Our study provides evidence in favor of preemptive PGx testing by identifying the risk of a variant being present in the population we studied.

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B) Disease-Drug Database for Pharmacogenomic-Based Prescribing Hussain S, Kenigsberg BB, Danahey K, et al. Disease-Drug Database for Pharmacogenomic-Based Prescribing. Clin Pharmacol Ther. 2016. doi: 10.1002/cpt.364. [Epub ahead of print]

Summary: Providers have expressed a strong desire to have additional clinical decision-support tools to help with interpretation of pharmacogenomic results. We developed and tested a novel disease-drug association tool that enables pharmacogenomic-based prescribing to treat common diseases. First, 324 drugs were mapped to 484 distinct diseases (mean number of drugs treating each disease was 4.9 [range 1-37]). Then, the disease-drug association tool was pharmacogenomically annotated, with an average of 1.8 pharmacogenomically-annotated drugs associated/disease. Applying this tool to a prospectively-enrolled >1,000 patient cohort from a tertiary medical center showed that 90% of the top approximately 20 diseases in this population and >/=93% of patients could appropriately be treated with >/=1 medication with actionable pharmacogenomic information. When combined with clinical patient genotypes, this tool permits delivery of patient-specific pharmacogenomically-informed disease treatment recommendations to inform the treatment of many medical conditions of the U.S. population, a key initial step towards implementation of precision medicine.

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C) Implementation of Pharmacogenomics: Evidence Needs Relling MV, Veenstra DL. Implementation of Pharmacogenomics: Evidence Needs. Institute of Medicine (Perspective Series). February 26, 2015.

Summary: The evidentiary needs for genomic technologies are a significant barrier to the translation of genomic testing into clinical use. However, in many instances there is sufficient evidence to justify the use of genetic testing to information choice or dosage of medications. This discussion

paper, the last of seven individually authored commentaries that explores the evidence needed to support the use of genome sequencing in the clinic, examines the policy issues and evidence needs for implementing pharmacogenomics testing.

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D) Comparison of Delivery Strategies for Pharmacogenetic Testing Services Haga SB, Moaddeb J. Comparison of delivery strategies for pharmacogenetic testing services. Pharmacogenet Genomics. 2014; 24(3): 139-145.

Summary: The number and use of pharmacogenetic tests to assess a patient's likelihood of response or risk of an adverse event is expanding across medical specialties and becoming more prevalent. During this period of development and translation, different approaches are being investigated to optimize delivery of pharmacogenetic services. In this paper, we review pre-emptive and point-of-care delivery approaches currently implemented or being investigated and discuss the advantages and disadvantages of each approach. The continued growth in knowledge about the genetic basis of drug response combined with development of new and less expensive testing technologies and electronic medical records will impact future delivery systems. Regardless of delivery approach, the currently limited knowledge of health professionals about genetics generally or PGx specifically will remain a major obstacle to utilization.

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E) Clinical Pharmacogenetics Implementation: Approaches, Successes, and Challenges Weitzel KW, Elsey AR, Langaee TY, et al. Clinical pharmacogenetics implementation: approaches, successes, and challenges. Am J Med Genet C Semin Med Genet. 2014; 166C(1): 56-67.

Summary: Current challenges exist to widespread clinical implementation of genomic medicine and pharmacogenetics. The University of Florida (UF) Health Personalized Medicine Program (PMP) is a pharmacist-led, multidisciplinary initiative created in 2011 within the UF Clinical Translational Science Institute. Initial efforts focused on pharmacogenetics, with long-term goals to include expansion to disease-risk prediction and disease stratification. Herein we describe the processes for development of the

program, the challenges that were encountered and the clinical acceptance by clinicians of the genomic medicine implementation. The initial clinical implementation of the UF PMP began in June 2012 and targeted clopidogrel use and the CYP2C19 genotype in patients undergoing left heart catheterization and percutaneous-coronary intervention (PCI). After 1 year, 1,097 patients undergoing left heart catheterization were genotyped preemptively, and 291 of those underwent subsequent PCI. Genotype results were reported to the medical record for 100% of genotyped patients. Eighty patients who underwent PCI had an actionable genotype, with drug therapy changes implemented in 56 individuals. Average turnaround time from blood draw to genotype result entry in the medical record was 3.5 business days. Seven different third party payors, including Medicare, reimbursed for the test during the first month of billing, with an 85% reimbursement rate for outpatient claims that were submitted in the first month. These data highlight multiple levels of success in clinical implementation of genomic medicine.

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F) Predicting the Cost and Pace of Pharmacogenomic Advances: An Evidence-Based Study Arnaout R, Buck TP, Roulette P, Sukhatme VP. Predicting the cost and pace of pharmacogenomic advances: an evidence-based study. Clin Chem. 2013; 59(4): 649-57.

Summary: Adverse outcomes associated with prescription drug use are common and costly. Many adverse outcomes can be avoided through pharmacogenomics: choosing and dosing of existing drugs according to a person's genomic variants. Finding and validating associations between outcomes and genomic variants and developing guidelines for avoiding drug- related adverse outcomes will require further research; however, no data- driven estimates yet exist for the time or money required for completing this research. METHODS: We identified examples of associations between adverse outcomes and genomic variants. We used these examples to estimate the time and money required to identify and confirm other associations, including the cost of failures, and to develop and validate pharmacogenomic dosing guidelines for them. We built a Monte Carlo model to estimate the time and financial costs required to cut the overall rate of drug-related adverse outcomes by meaningful amounts. We analyzed the model's predictions for a broad range of assumptions. RESULTS AND CONCLUSIONS: Our model projected that the development of guidelines capable of cutting overall drug-related adverse outcomes by 25%-50% with current approaches will require investment of single-digit billions of dollars

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and take 20 years. The model forecasts a pump-priming phase of 5-7 years, which would require expenditures of hundreds of millions of dollars, with little apparent return on investment. The single most important parameter was the extent to which genomic variants cause adverse outcomes. The size of the labor force was not a limiting factor. A "50 000 Pharmacogenomes Project" could speed progress. Our approach provides a template for other areas of genomic research.

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5.2 Cardiovascular Pharmacogenomics

A) The Clinical Pharmacogenetics Implementation Consortium Guideline for SLCO1B1 and Simvastatin-Induced Myopathy: 2014 Update Ramsey LB, Johnson SG, Caudle KE, et al. The Clinical Pharmacogenetics Implementation Consortium Guideline for SLCO1B1 and Simvastatin- Induced Myopathy: 2014 Update. Clin Pharmacol Ther. 2014; 96(4): 423- 428.

Summary: Simvastatin is among the most commonly used prescription medications for cholesterol reduction. A single coding single-nucleotide polymorphism, rs4149056T>C, in SLCO1B1 increases systemic exposure to simvastatin and the risk of muscle toxicity. We summarize evidence from the literature supporting this association and provide therapeutic recommendations for simvastatin based on SLCO1B1 genotype. This article is an update to the 2012 Clinical Pharmacogenetics Implementation Consortium guideline for SLCO1B1 and simvastatin-induced myopathy.

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B) Cardiovascular Pharmacogenomics; State of Current Knowledge and Implementation in Practice Shahabi P, Dubé MP. Cardiovascular pharmacogenomics; state of current knowledge and implementation in practice. Int J Cardiol. 2015;184:772-795.

Summary: Pharmacogenomics (PGx) is the science that examines how an individual's genetic make-up affects the safety and efficacy of therapeutic drugs. PGx of response to cardiovascular (CV) medications is of the most successfully translated branches of PGx into the clinical workout. However,

the clinical implementation of PGx of CV drugs is yet far beyond the growth of our understanding of the role of genetics in drug therapy. A considerable amount of efforts have been devoted by the regulatory agents like the food and drug administration (FDA) as well as the expert-based networks such as the Clinical Pharmacogenetics Implementation Consortium (CPIC) to overcome the existing barriers. This has been done, at least in part, for some of the most widely prescribed CV drugs, including clopidogrel, warfarin and simvastatin for which the PGx knowledge have been satisfactorily robust to provoke the CPIC to issue the guidelines for these drugs and the FDA to update the drugs' labeling, both strongly recommended the use of genotype- guided dosing for these medications, provided that the genetic data are available. For other drugs, however, studies have produced contradictory results and further large and well-designed clinical trials are required to expand and confirm the clinical utility of their PGx data. This review paper presents the current state of knowledge in the field of PGx of CV medications and describes the facilities assisting to the translation of PGx data into the clinical practice. Afterward, the existing body of PGx literature of the most- commonly used CV medications is comprehensively discussed.

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C) A Prospective Randomized Evaluation of a Pharmacogenomic Approach to Antiplatelet Therapy Among Patients with ST-Elevation Myocardial Infarction: The RAPID STEMI Study So DY, Wells GA, McPherson R, et al. A prospective randomized evaluation of a pharmacogenomic approach to antiplatelet therapy among patients with ST-elevation myocardial infarction: the RAPID STEMI study. Pharmacogenomics J. 2016;16(1):71-8.

Summary: Treatment of carriers of the CYP2C19*2 allele and ABCB1 TT genotype with clopidogrel is associated with increased ischemic complications after percutaneous coronary intervention (PCI). We sought to evaluate a pharmacogenomic strategy among patients undergoing PCI for ST-elevation myocardial infarction (STEMI), by performing a randomized trial, enrolling 102 patients. Point-of-care genetic testing for CYP2C19*2, ABCB1 TT and CYP2C19*17 was performed with carriers of either the CYP2C19*2 allele or ABCB1 TT genotype randomly assigned to a strategy of prasugrel 10 mg daily or an augmented dosing strategy of clopidogrel (150 mg daily for 6 days then 75 mg daily). The primary end point was the proportion of at-risk carriers exhibiting high on-treatment platelet reactivity (HPR), a marker associated with increased adverse cardiovascular events, after 1 month. Fifty-nine subjects (57.8%) were identified as carriers of at

least one at-risk variant. Treatment with prasugrel significantly reduced HPR compared with clopidogrel by P2Y12 reaction unit (PRU) thresholds of >234 (0 vs 24.1%, P=0.0046) and PRU>208 (3.3 vs 34.5%, P=0.0025). The sensitivity of point-of-care testing was 100% (95% CI 88.0-100), 100% (86.3- 100) and 96.9% (82.0-99.8) and specificity was 97.0% (88.5-99.5), 97.1% (89.0-99.5) and 98.5% (90.9-99.9) for identifying CYP2C19*2, ABCB1 TT and CYP2C19*17, respectively. Logistic regression confirmed carriers as a strong predictor of HPR (OR=6.58, 95% CI 1.24-34.92; P=0.03). We confirmed that concurrent identification of three separate genetic variants in patients with STEMI receiving PCI is feasible at the bedside. Among carriers of at-risk genotypes, treatment with prasugrel was superior to an augmented dosing strategy of clopidogrel in reducing HPR.

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D) Genetics and the Clinical Response to Warfarin and Edoxaban: Findings from the Randomised, Double-blind ENGAGE AF-TIMI 48 Trial Mega JL, Walker JR, Ruff CT, et al. Genetics and the clinical response to warfarin and edoxaban: findings from the randomised, double-blind ENGAGE AF-TIMI 48 trial. Lancet. 2015; 385(9984): 2280-2287

Summary: BACKGROUND: Warfarin is the most widely used oral anticoagulant worldwide, but serious bleeding complications are common. We tested whether genetic variants can identify patients who are at increased risk of bleeding with warfarin and, consequently, those who would derive a greater safety benefit with a direct oral anticoagulant rather than warfarin. METHODS: ENGAGE AF-TIMI 48 was a randomised, double-blind trial in which patients with atrial fibrillation were assigned to warfarin to achieve a target international normalised ratio of 2.0-3.0, or to higher-dose (60 mg) or lower-dose (30 mg) edoxaban once daily. A subgroup of patients was included in a prespecified genetic analysis and genotyped for variants in CYP2C9 and VKORC1. The results were used to create three genotype functional bins (normal, sensitive, and highly sensitive responders to warfarin). This trial is registered with ClinicalTrials.gov, number NCT00781391. FINDINGS: 14 348 patients were included in the genetic analysis. Of 4833 taking warfarin, 2982 (61.7%) were classified as normal responders, 1711 (35.4%) as sensitive responders, and 140 (2.9%) as highly sensitive responders. Compared with normal responders, sensitive and highly sensitive responders spent greater proportions of time over- anticoagulated in the first 90 days of treatment (median 2.2%, IQR 0-20.2; 8.4%, 0-25.8; and 18.3%, 0-32.6; ptrend<0.0001) and had increased risks of bleeding with warfarin (sensitive responders hazard ratio 1.31, 95% CI 1.05-

1.64, p=0.0179; highly sensitive responders 2.66, 1.69-4.19, p<0.0001). Genotype added independent information beyond clinical risk scoring. During the first 90 days, when compared with warfarin, treatment with edoxaban reduced bleeding more so in sensitive and highly sensitive responders than in normal responders (higher-dose edoxaban pinteraction=0.0066; lower- dose edoxaban pinteraction=0.0036). After 90 days, the reduction in bleeding risk with edoxaban versus warfarin was similarly beneficial across genotypes. INTERPRETATION: CYP2C9 and VKORC1 genotypes identify patients who are more likely to experience early bleeding with warfarin and who derive a greater early safety benefit from edoxaban compared with warfarin.

Full text available from The Lancet (USD 31.50) PMID: 25769357

E) Limited Clinical Utility of Genotype-Guided Warfarin Initiation Dosing Algorithms Versus Standard Therapy: A Meta-Analysis and Trial Sequential Analysis of 11 Randomized Controlled Trials Tang HL, Shi WL, Li XG, Zhang T, Zhai SD, Xie HG. Limited clinical utility of genotype-guided warfarin initiation dosing algorithms versus standard therapy: a meta-analysis and trial sequential analysis of 11 randomized controlled trials. Pharmacogenomics J. 2015;15(6):496-504.

Summary: In terms of inconsistent conclusions across all relevant randomized controlled trials (RCTs) and available meta-analyses, we aimed to use a meta-analysis and trial sequential analysis (TSA) to evaluate whether clinical utility of a genotype-guided warfarin initiation dosing algorithm could be better than that of a standard therapy regimen, and whether currently relevant evidence could be reliable and conclusive. Overall, 11 eligible RCTs involving 2677 patients were included for further analyses. Compared with fixed dose or clinically adjusted warfarin initiation dosing regimens, genotype-guided algorithms significantly increased time in therapeutic range, shortened time to first therapeutic international normalized ratio (INR) and time to stable doses, but did not show any marked improvements in excessive anticoagulation, bleeding events, thromboembolism, or all-cause mortality. Subgroup analyses revealed that, genotype-guided algorithms showed better control in the outcomes of time in therapeutic range or excessive anticoagulation than fixed-dose regimens rather than clinically adjusted regimens. Except for excessive anticoagulation, currently available evidence of all other outcomes was unreliable and inconclusive as determined with TSA. Our findings suggest that genotype-guided warfarin initiation dosing algorithms have superiority in the improvement of surrogate quality markers for anticoagulation control, but that this does not translate into statistically significant differences in clinical

outcomes, which is largely because of the insufficient sample size in the RCTs analyzed.

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F) Poor Warfarin Dose Prediction with Pharmacogenetic Algorithms that Exclude Genotypes Important for African Americans Drozda K, Wong S, Patel SR, et al. Poor warfarin dose prediction with pharmacogenetic algorithms that exclude genotypes important for African Americans. Pharmacogenet Genomics. 2015; 25(2): 73-81.

Summary: OBJECTIVES: Recent clinical trial data cast doubt on the utility of genotype-guided warfarin dosing, specifically showing worse dosing with a pharmacogenetic versus clinical dosing algorithm in African Americans. However, many genotypes important in African Americans were not accounted for. We aimed to determine whether omission of the CYP2C9*5, CYP2C9*6, CYP2C9*8, CYP2C9*11 alleles and rs12777823 G>A genotype affects performance of dosing algorithms in African Americans. METHODS: In a cohort of 274 warfarin-treated African Americans, we examined the association between the CYP2C9*5, CYP2C9*6, CYP2C9*8, CYP2C9*11 alleles and rs12777823 G>A genotype and warfarin dose prediction error with pharmacogenetic algorithms used in clinical trials. RESULTS: The http://www.warfarindosing.org algorithm overestimated doses by a median (interquartile range) of 1.2 (0.02-2.6) mg/day in rs12777823 heterozygotes (P<0.001 for predicted vs. observed dose), 2.0 (0.6-2.8) mg/day in rs12777823 variant homozygotes (P=0.004), and 2.2 (0.5-2.9) mg/day in carriers of a CYP2C9 variant (P<0.001). The International Warfarin Pharmacogenetics Consortium (IWPC) algorithm underdosed warfarin by 0.8 (-2.3 to 0.4) mg/day for patients with the rs12777823 GG genotype (P<0.001) and overdosed warfarin by 0.7 (-0.4 to 1.9) mg/day in carriers of a variant CYP2C9 allele (P=0.04). Modifying the http://www.warfarindosing.org algorithm to adjust for variants important in African Americans led to better dose prediction than either the original http://www.warfarindosing.org (P<0.01) or IWPC (P<0.01) algorithm. CONCLUSION: These data suggest that, when providing genotype-guided warfarin dosing, failure to account for variants important in African Americans leads to significant dosing error in this population.

Full text available from Pharmacogenomics and Genomics (subscription required) PMID: 25461246

G) Outcomes of Pharmacogenetics-Guided Dosing of Warfarin: A Systematic Review and Meta-Analysis Tang Q, Zou H, Guo C, Liu Z. Outcomes of pharmacogenetics-guided dosing of warfarin: a systematic review and meta-analysis. Int J Cardiol. 2014; 175(3): 587-591.

No summary available.

Full text available from International Journal of Cardiology (USD 31.50) PMID: 25005340

H) Clinical Pharmacogenetics Implementation Consortium Guidelines for CYP2C19 Genotype and Clopidogrel Therapy: 2013 Update Scott SA, Sangkuhl K, Stein CM, et al. Clinical Pharmacogenetics Implementation Consortium Guidelines for CYP2C19 Genotype and Clopidogrel Therapy: 2013 Update. Clin Pharmacol Ther. 2013; 94(3): 317- 323.

Summary: Cytochrome P450 (CYP)2C19 catalyzes the bioactivation of the antiplatelet prodrug clopidogrel, and CYP2C19 loss-of-function alleles impair formation of active metabolites, resulting in reduced platelet inhibition. In addition, CYP2C19 loss-of-function alleles confer increased risks for serious adverse cardiovascular (CV) events among clopidogrel-treated patients with acute coronary syndromes (ACSs) undergoing percutaneous coronary intervention (PCI). Guideline updates include emphasis on appropriate indication for CYP2C19 genotype-directed antiplatelet therapy, refined recommendations for specific CYP2C19 alleles, and additional evidence from an expanded literature review (updates at http://www.pharmgkb.org).

Free full text available from PubMed PMID: 23698643

5.3 Pharmacogenomics in Cancer

Hertz DL, Rae J212. Pharmacogenetics of cancer drugs. Annu Rev Med. 2015;66:65-81.

A) Pharmacogenetics of Cancer Drugs Hertz DL, Rae J. Pharmacogenetics of cancer drugs. Annu Rev Med. 2015;66:65-81.

Summary: The variability in treatment outcomes among patients receiving the same therapy for seemingly similar tumors can be attributed in part to genetics. The tumor's (somatic) genome largely dictates the effectiveness of the therapy, and the patient's (germline) genome influences drug exposure and the patient's sensitivity to toxicity. Many potentially clinically useful associations have been discovered between common germline genetic polymorphisms and outcomes of cancer treatment. This review highlights the germline pharmacogenetic associations that are currently being used to guide cancer treatment decisions, those that are most likely to someday be clinically useful, and associations that are well known but their roles in clinical management are not yet certain. In the future, germline genetic information will likely be available from tumor genetic analyses, creating an efficient opportunity to integrate the two genomes to optimize treatment outcomes for each individual cancer patient.

Full text available from Annual Reviews (USD 32.00 for 24 hour access) PMID: 25386932

B) Pharmacogenetics and Pharmacogenomics: A Bridge to Individualized Cancer Therapy Weng L, Zhang L, Peng Y, Huang RS. Pharmacogenetics and pharmacogenomics: a bridge to individualized cancer therapy. Pharmacogenomics. 2013; 14(3): 315-324.

Summary: In the past decade, advances in pharmacogenetics and pharmacogenomics (PGx) have gradually unveiled the genetic basis of interindividual differences in drug responses. A large portion of these advances have been made in the field of anticancer therapy. Currently, the US FDA has updated the package inserts of approximately 30 anticancer agents to include PGx information. Given the complexity of this genetic information (e.g., tumor mutation and gene overexpression, chromosomal translocation and germline variations), as well as the variable level of scientific evidence, the FDA recommendation and potential action needed varies among drugs. In this review, we have highlighted some of these PGx discoveries for their scientific values and utility in improving therapeutic efficacy and reducing side effects. Furthermore, examples are also provided for the role of PGx in new anticancer drug development by revealing novel druggable targets.

Free full text available from PubMed PMID: 23394393

C) Dose-Finding and Pharmacokinetic Study to Optimize the Dosing of Irinotecan According to the UGT1A1 Genotype of Patients with Cancer Innocenti F, Schilsky RL, Ramirez J, et al. Dose-finding and pharmacokinetic study to optimize the dosing of irinotecan according to the UGT1A1 genotype of patients with cancer. J Clin Oncol. 2014; 32(22): 2328-2334.

Summary: PURPOSE: The risk of severe neutropenia from treatment with irinotecan is related in part to UGT1A1*28, a variant that reduces the elimination of SN-38, the active metabolite of irinotecan. We aimed to identify the maximum-tolerated dose (MTD) and dose-limiting toxicity (DLT) of irinotecan in patients with advanced solid tumors stratified by the *1/*1, *1/*28, and *28/*28 genotypes. PATIENTS AND METHODS: Sixty-eight patients received an intravenous flat dose of irinotecan every 3 weeks. Forty- six percent of the patients had the *1/*1 genotype, 41% had the *1/*28 genotype, and 13% had the *28/*28 genotype. The starting dose of irinotecan was 700 mg in patients with the *1/*1 and *1/*28 genotypes and 500 mg in patients with the *28/*28 genotype. Pharmacokinetic evaluation was performed at cycle 1. RESULTS: In patients with the *1/*1 genotype, the MTD was 850 mg (four DLTs per 16 patients), and 1,000 mg was not

tolerated (two DLTs per six patients). In patients with the *1/*28 genotype, the MTD was 700 mg (five DLTs per 22 patients), and 850 mg was not tolerated (four DLTs per six patients). In patients with the *28/*28 genotype, the MTD was 400 mg (one DLT per six patients), and 500 mg was not tolerated (three DLTs per three patients). The DLTs were mainly myelosuppression and diarrhea. Irinotecan clearance followed linear kinetics. At the MTD for each genotype, dosing by genotype resulted in similar SN-38 areas under the curve (AUCs; r(2) = 0.0003; P = .97), but the irinotecan AUC was correlated with the actual dose (r(2) = 0.39; P < .001). Four of 48 patients with disease known to be responsive to irinotecan achieved partial response. CONCLUSION: The UGT1A1*28 genotype can be used to individualize dosing of irinotecan. Additional studies should evaluate the effect of genotype-guided dosing on efficacy in patients receiving irinotecan.

Full text available from Journal of Clinical Oncology (subscription required) PMID: 24958824

D) Pharmacogenomics of Breast Cancer Therapy: An Update Westbrook K, Stearns V. Pharmacogenomics of breast cancer therapy: an update. Pharmacol Ther. 2013; 139(1): 1-11.

Summary: Clinical and histopathologic characteristics of breast cancer have long played an important role in treatment decision-making. Well-recognized prognostic factors include tumor size, node status, presence or absence of metastases, tumor grade, and hormone receptor expression. High tumor grade, presence of hormone receptors, and HER2-positivity are a few predictive markers of response to chemotherapy, endocrine manipulations, and anti-HER2 agents, respectively. However, there is much heterogeneity of outcomes in patients with similar clinical and pathologic features despite equivalent treatment regimens. Some of the differences in response to specific therapies can be attributed to somatic tumor characteristics, such as degree of estrogen receptor expression and HER2 status. In recent years, there has been great interest in evaluating the role that pharmacogenetics/pharmacogenomics, or variations in germline DNA, play in alteration of drug metabolism and activity, thus leading to disparate outcomes among patients with similar tumor characteristics. The utility of these variations in treatment decision-making remains debated. Here we review the data available to date on genomic variants that may influence response to drugs commonly used to treat breast cancer. While none of the variants reported to date have demonstrated clinical utility, ongoing prospective studies and increasing understanding of pharmacogenetics will allow us to better predict risk of toxicity or likelihood of response to specific treatments and to provide a more personalized therapy.

Free full text available from PubMed PMID: 23500718 NOTE: Also cited in Section 2.2.1

E) CYP2D6 Genotype and Adjuvant Tamoxifen: Meta-Analysis of Heterogeneous Study Populations Province MA, Goetz MP, Brauch H, et al. CYP2D6 genotype and adjuvant tamoxifen: meta-analysis of heterogeneous study populations. Clin Pharmacol Ther. 2014; 95(2): 216-227.

Summary: The International Tamoxifen Pharmacogenomics Consortium was established to address the controversy regarding cytochrome P450 2D6 (CYP2D6) status and clinical outcomes in tamoxifen therapy. We performed a meta-analysis on data from 4,973 tamoxifen-treated patients (12 globally distributed sites). Using strict eligibility requirements (postmenopausal women with estrogen receptor-positive breast cancer, receiving 20 mg/day tamoxifen for 5 years, criterion 1); CYP2D6 poor metabolizer status was associated with poorer invasive disease-free survival (IDFS: hazard ratio = 1.25; 95% confidence interval = 1.06, 1.47; P = 0.009). However, CYP2D6 status was not statistically significant when tamoxifen duration, menopausal status, and annual follow-up were not specified (criterion 2, n = 2,443; P = 0.25) or when no exclusions were applied (criterion 3, n = 4,935; P = 0.38). Although CYP2D6 is a strong predictor of IDFS using strict inclusion criteria, because the results are not robust to inclusion criteria (these were not defined a priori), prospective studies are necessary to fully establish the value of CYP2D6 genotyping in tamoxifen therapy.

Free full text available from PubMed PMID: 24060820

F) Pharmacokinetics and Pharmacogenetics of Gemcitabine as a Mainstay in Adult and Pediatric Oncology: An EORTC-PAMM Perspective Ciccolini J, Serdjebi C, Peters GJ, Giovannetti E. Pharmacokinetics and Pharmacogenetics of Gemcitabine as a Mainstay in Adult and Pediatric Oncology: An EORTC-PAMM Perspective. Cancer Chemother Pharmacol. 2016. [Epub ahead of print]

Summary: Gemcitabine is an antimetabolite ranking among the most prescribed anticancer drugs worldwide. This nucleoside analog exerts its antiproliferative action after tumoral conversion into active triphosphorylated nucleotides interfering with DNA synthesis and targeting ribonucleotide reductase. Gemcitabine is a mainstay for treating pancreatic and lung

cancers, alone or in combination with several cytotoxic drugs (nab-paclitaxel, cisplatin and oxaliplatin), and is an option in a variety of other solid or hematological cancers. Several determinants of response have been identified with gemcitabine, i.e., membrane transporters, activating and inactivating enzymes at the tumor level, or Hedgehog signaling pathway. More recent studies have investigated how germinal genetic polymorphisms affecting cytidine deaminase, the enzyme responsible for the liver disposition of gemcitabine, could act as well as a marker for clinical outcome (i.e., toxicity, efficacy) at the bedside. Besides, constant efforts have been made to develop alternative chemical derivatives or encapsulated forms of gemcitabine, as an attempt to improve its metabolism and pharmacokinetics profile. Overall, gemcitabine is a drug paradigmatic for constant searches of the scientific community to improve its administration through the development of personalized medicine in oncology.

Free full text available from Cancer Chemotherapy and Pharmacology PMID: 27007129

5.4 Pharmacogenomics in Anesthesia

A) Pharmacogenomic Variability and Anaesthesia Searle R, Hopkins PM. Pharmacogenomic variability and anaesthesia. Br J Anaesth. 2009; 103(1): 14-25.

Summary: The concept of 'personalized medicine' in which a knowledge of genetic factors guides prescribing tailored to the individual is popularly considered to be an inevitable consequence of completion of the International Human Genome Project. We should not forget, however, that a personal or family history of one of several uncommon pharmacogenetic conditions has influenced the use of the implicated drug(s) during anaesthesia for the past 50 yr. Although this has been important for those affected, pharmacogenomics heralds the prospect of an individual's genetic profile informing every prescription. Progress has been rapid in some areas, notably cancer chemotherapy where response to treatment can be predicted on the basis of the genetic profile of the tumour cells. The situation is different for most currently available drugs, including those used by anaesthetists, where genetic variability to drug response is presumed to be the result of a complex interaction of multiple factors. We review the nature and investigation of pharmacogenomic variability and contrast the progress made with research into opioid variability with the more limited literature concerning i.v. and inhalation anaesthetics.

Free full text available from British Journal of Anaesthesia PMID: 19482856

B) The Pharmacogenomics of Pain Management: Prospects for Personalized Medicine Ting S, Schug S. The Pharmacogenomics of Pain Management: Prospects for Personalized Medicine. J Pain Res. 2016; 9:49-56.

Summary: Pain is a common symptom that can be complex to treat. Analgesic medications are the mainstay treatment, but there is wide interindividual variability in analgesic response and adverse effects. Pharmacogenomics is the study of inherited genetic traits that result in these individual responses to drugs. This narrative review will attempt to cover the current understanding of the pharmacogenomics of pain, examining common genes affecting metabolism of analgesic medications, their distribution throughout the body, and end organ effects.

Free full text available from PubMed PMID: 26929662

5.5 Pharmacogenomics of Infectious Diseases and Interactions with the Immune System

A) First External Quality Assurance Program of the Italian HLA-B*57:01 Network Assessing the Performance of Clinical Virology Laboratories in HLA-B*57:01 Testing Meini G, Russo CD, Allice T, et al. First External Quality Assurance Program of the Italian HLA-B*57:01 Network Assessing the Performance of Clinical Virology Laboratories in HLA-B*57:01 Testing. J Clin Virol. 2016; 78:1-3.

Summary: BACKGROUND: Since the HLA-B*57:01 allele is strongly associated with abacavir hypersensitivity reaction, testing for the presence of HLA-B*57:01 is mandatory before administration of abacavir. While HLA- B*57:01 testing is usually provided by pharmacogenetics, genetics or blood transfusion services, clinical virology laboratories can be an optimal opportunity for HLA-B*57:01 testing since they receive blood samples for routine HIV monitoring and have the expertise for convenient and less expensive PCR-based point mutation assays. OBJECTIVES: The Italian HLA-B*57:01 Network gathers accredited clinical virology laboratories offering HLA-B*57:01 testing in Italy with the aim to share protocols, test new

methods, develop and maintain external quality assurance (EQA) programs. STUDY DESIGN: A panel of 9HLA-B*57:01-positive and 16HLA-B*57:01- negative frozen blood samples were blindly distributed to 10 units including 9 clinical virology laboratories and one reference pharmacology laboratory. Each laboratory was free to use its own routine method for DNA extraction and HLA-B*57:01 testing. RESULTS: DNA was extracted by automated workstations in 6 units and by manual spin columns in 4. Eight units used the Duplicalpha Real Time HLA-B*57:01 kit by Euroclone and two units used two different PCR homemade protocols. All the 10 units correctly identified all the 25 samples. CONCLUSIONS: The first HLA-B*57:01 EQA program run in Italy showed that clinical virology units are equipped and proficient for providing HLA-B*57:01 testing by inexpensive assays easy to integrate into their routine.

Full text available from Journal of Clinical Virology (USD 31.50 for 24 hour access) PMID: 26946153

B) Pharmacogenomics of Antimicrobial Agents Aung AK, Haas DW, Hulgan T, Phillips EJ. Pharmacogenomics of antimicrobial agents. Pharmacogenomics. 2014; 15(15): 1903-1930.

Summary: Antimicrobial efficacy and toxicity varies between individuals owing to multiple factors. Genetic variants that affect drug-metabolizing enzymes may influence antimicrobial pharmacokinetics and pharmacodynamics, thereby determining efficacy and/or toxicity. In addition, many severe immune-mediated reactions have been associated with HLA class I and class II genes. In the last two decades, understanding of pharmacogenomic factors that influence antimicrobial efficacy and toxicity has rapidly evolved, leading to translational success such as the routine use of HLA-B*57:01 screening to prevent abacavir hypersensitivity reactions. This article examines recent advances in the field of antimicrobial pharmacogenomics that potentially affect treatment efficacy and toxicity, and challenges that exist between pharmacogenomic discovery and translation into clinical use.

Full text available from Pharmacogenomics (USD 60.00 for 24 hour access) PMID: 25495412

C) Drug Hypersensitivity and Human Leukocyte Antigens of the Major Histocompatibility Complex Bharadwaj M, Illing P, Theodossis A, Purcell AW, Rossjohn J, McCluskey J. Drug hypersensitivity and human leukocyte antigens of the major histocompatibility complex. Annu Rev Pharmacol Toxicol. 2012;52:401-431.

Summary: The human leukocyte antigen (HLA) genes are the most polymorphic in the human genome and are critical in regulating specific immunity, hence their historical discovery as "immune response" genes. HLA allotypes are also implicated in unwanted immune reactions, including drug hypersensitivity syndrome, in which small therapeutic drugs interact with antigenic peptides to drive T cell responses restricted by host HLA. Abacavir, allo-purinol, and carbamazepine are three commonly used drugs that cause a T cell-mediated hypersensitivity that is HLA linked, with each drug exhibiting striking specificity for presentation by defined HLA allotypes. Recent findings have begun to unearth the mechanistic basis for these HLA associations, and here we review recent advances in the field of HLA- associated drug hypersensitivities.

Full text available from Annual Reviews Pharmacology and Toxicology (USD 32.00) PMID: 22017685

D) Pharmacogenetic Aspects of the Use of Tacrolimus in Renal Transplantation: Recent Developments and Ethnic Considerations Tang JT, Andrews LM, van Gelder T, et al. Pharmacogenetic Aspects of the Use of Tacrolimus in Renal Transplantation: Recent Developments and Ethnic Considerations. Expert Opin Drug Metab Toxicol. 2016;12(5):555-65.

Summary: Introduction: Tacrolimus (Tac) is effective in preventing acute rejection but has considerable toxicity and inter-individual variability in pharmacokinetics and pharmacodynamics. Part of this is explained by polymorphisms in genes encoding Tac-metabolizing enzymes and transporters. A better understanding of Tac pharmacokinetics and pharmacodynamics may help to minimize different outcomes amongst transplant recipients by personalizing immunosuppression. Areas covered: The pharmacogenetic contribution of Tac metabolism will be examined, with a focus on recent discoveries, new developments and ethnic considerations. Expert opinion: The strongest and most consistent association in pharmacogenetics is between the CYP3A5 genotype and Tac dose requirement, with CYP3A5 expressers having a ~40-50% higher dose requirement compared to non-expressers. Two recent randomized-controlled clinical trials using CYP3A5 genotype, however, did not show a decrease in

acute rejections nor reduced toxicity. CYP3A4*22, CYP3A4*26, and POR *28 are also associated with Tac dose requirements and may be included to provide the expected improvement of Tac therapy. Studies focusing on the intracellular drug concentrations and on calcineurin inhibitor-induced nephrotoxicity also seem promising. For all studies, however, the ethnic prevalence of genotypes should be taken into account, as this may significantly impact the effect of pre-emptive genotyping.

Full text available from Expert Opinion on Drug Metabolism & Toxicology (USD 89.00) PMID: 27010623

5.6 Psychiatric Pharmacogenomics

A) Pharmacogenomics in Psychiatry: The Relevance of Receptor and Transporter Polymorphisms Reynolds GP, McGowan OO, Dalton CF. Pharmacogenomics in psychiatry: the relevance of receptor and transporter polymorphisms. Br J Clin Pharmacol. 2014; 77(4): 654-672.

Summary: The treatment of severe mental illness, and of psychiatric disorders in general, is limited in its efficacy and tolerability. There appear to be substantial interindividual differences in response to psychiatric drug treatments that are generally far greater than the differences between individual drugs; likewise, the occurrence of adverse effects also varies profoundly between individuals. These differences are thought to reflect, at least in part, genetic variability. The action of psychiatric drugs primarily involves effects on synaptic neurotransmission; the genes for neurotransmitter receptors and transporters have provided strong candidates in pharmacogenetic research in psychiatry. This paper reviews some aspects of the pharmacogenetics of neurotransmitter receptors and transporters in the treatment of psychiatric disorders. A focus on serotonin, catecholamines and amino acid transmitter systems reflects the direction of research efforts, while relevant results from some genome-wide association studies are also presented. There are many inconsistencies, particularly between candidate gene and genome-wide association studies. However, some consistency is seen in candidate gene studies supporting established pharmacological mechanisms of antipsychotic and antidepressant response with associations of functional genetic polymorphisms in, respectively, the dopamine D2 receptor and serotonin transporter and receptors. More recently identified effects of genes related to amino acid neurotransmission on the outcome of

treatment of schizophrenia, bipolar illness or depression reflect the growing understanding of the roles of glutamate and gamma-aminobutyric acid dysfunction in severe mental illness. A complete understanding of psychiatric pharmacogenomics will also need to take into account epigenetic factors, such as DNA methylation, that influence individual responses to drugs.

Free full text available from PubMed PMID: 24354796

B) Clinical Pharmacogenetics Implementation Consortium Guideline for CYP2D6 and CYP2C19 Genotypes and Dosing of Tricyclic Antidepressants Hicks JK, Swen JJ, Thorn CF, et al. Clinical Pharmacogenetics Implementation Consortium guideline for CYP2D6 and CYP2C19 genotypes and dosing of tricyclic antidepressants. Clin Pharmacol Ther. 2013; 93(5): 402-408.

Summary: Polymorphisms in CYP2D6 and CYP2C19 affect the efficacy and safety of tricyclics, with some drugs being affected by CYP2D6 only, and others by both polymorphic enzymes. Amitriptyline, clomipramine, doxepin, imipramine, and trimipramine are demethylated by CYP2C19 to pharmacologically active metabolites. These drugs and their metabolites, along with desipramine and nortriptyline, undergo hydroxylation by CYP2D6 to less active metabolites. Evidence from published literature is presented for CYP2D6 and CYP2C19 genotype-directed dosing of tricyclic antidepressants.

Free full text available from PubMed PMID: 23486447 NOTE: Also cited in Section 5

5.7 Other Pharmacogenomics

A) Relevance of UDP-Glucuronosyltransferase Polymorphisms for Drug Dosing: A Quantitative Systematic Review Stingl JC, Bartels H, Viviani R, Lehmann ML, Brockmoller J. Relevance of UDP-glucuronosyltransferase polymorphisms for drug dosing: A quantitative systematic review. Pharmacol Ther. 2014; 141(1): 92-116.

Summary: UDP-glucuronosyltransferases (UGT) catalyze the biotransformation of many endobiotics and xenobiotics, and are coded by

polymorphic genes. However, knowledge about the effects of these polymorphisms is rarely used for the individualization of drug therapy. Here, we present a quantitative systematic review of clinical studies on the impact of UGT variants on drug metabolism to clarify the potential for genotype- adjusted therapy recommendations. Data on UGT polymorphisms and dose- related pharmacokinetic parameters in man were retrieved by a systematic search in public databases. Mean estimates of pharmacokinetic parameters were extracted for each group of carriers of UGT variants to assess their effect size. Pooled estimates and relative confidence bounds were computed with a random-effects meta-analytic approach whenever multiple studies on the same variant, ethnic group, and substrate were available. Information was retrieved on 30 polymorphic metabolic pathways involving 10 UGT enzymes. For irinotecan and mycophenolic acid a wealth of data was available for assessing the impact of genetic polymorphisms on pharmacokinetics under different dosages, between ethnicities, under comedication, and under toxicity. Evidence for effects of potential clinical relevance exists for 19 drugs, but the data are not sufficient to assess effect size with the precision required to issue dose recommendations. In conclusion, compared to other drug metabolizing enzymes much less systematic research has been conducted on the polymorphisms of UGT enzymes. However, there is evidence of the existence of large monogenetic functional polymorphisms affecting pharmacokinetics and suggesting a potential use of UGT polymorphisms for the individualization of drug therapy.

Full text available from Pharmacology & Therapeutics (USD 35.95) PMID: 24076267

Selected analysis tools for NGS of cancer genomes. Ding et al91, Analysis of next-generation genomic data in cancer: accomplishments and challenges, Hum Mol Genet, 2010 19(R2):R189, by permission of Oxford University Press.

This section includes the following topics:

• Analysis Pipeline and Software (Section 6.1); • Clinical Informatics and Reporting (Section 6.2); • Databases (Section 6.3); • Considerations of Depth of Coverage (Section 6.4); and • Bioinformatics for Pathogens (Section 6.5).

6.1 Analysis Pipeline and Software

A) Good Laboratory Good Practice for Clinical Next-Generation Sequencing Informatics Pipelines Gargis AS, Kalman L, Bick DP, et al. Good Laboratory Practice for Clinical Next-Generation Sequencing Informatics Pipelines. Nat Biotechnol. 2015; 33(7): 689-693.

Summary: We report principles and guidelines that were developed by the Next-Generation Sequencing: Standardization of Clinical Testing II (Nex- StoCT II) informatics workgroup, which was first convened on October 11– 12, 2012, in Atlanta, Georgia, by the US Centers for Disease Control and Prevention (CDC; Atlanta, GA). We present…

Full text available from Nature Biotechnology (USD 18.00) PMID: 26154004

B) Assuring the Quality of Next-Generation Sequencing in Clinical Laboratory Practice Gargis AS, Kalman L, Berry MW, et al. Assuring the Quality of Next- Generation Sequencing in Clinical Laboratory Practice. Nat Biotechnol. 2012; 30(11): 1033-1036.

Summary: (first 100 words) We direct your readers’ attention to the principles and guidelines (Supplementary Guidelines) developed by the Next- generation Sequencing: Standardization of Clinical Testing (Nex-StoCT) workgroup. These guidelines represent initial steps to ensure that results from tests based on next-generation sequencing (NGS) are reliable and useful for clinical decision making. The US Centers for Disease Control and Prevention (CDC) convened this national workgroup, which collaborated to define platform-independent approaches for establishing technical process elements of a quality management system (QMS) to assure the analytical validity and compliance of NGS tests with existing regulatory and professional quality standards. The workgroup identified…

Free full text available from PubMed PMID: 23138292

Reprinted by permission from Macmillan Publishers Ltd: Nature Biotechnology, Gargis AS et al. Assuring the quality of next-generation sequencing in clinical laboratory practice. Nat Biotechnol. 30 (11); 1033-6, copyright 2012.

C) Developing Genome and Exome Sequencing for Candidate Gene Identification in Inherited Disorders: An Integrated Technical and Bioinformatics Approach Coonrod EM, Durtschi JD, Margraf RL,Voelkerding KV. Developing genome and exome sequencing for candidate gene identification in inherited disorders: an integrated technical and bioinformatics approach. Arch Pathol Lab Med. 2013; 137(3): 415-433.

of NGS has fostered the concurrent development of bioinformatics approaches to expeditiously analyze the large data sets generated. Next- generation sequencing has been used for important discoveries in the research setting and is now being implemented into the clinical diagnostic arena. OBJECTIVE: To review the current literature on technical and bioinformatics approaches for exome and genome sequencing and highlight examples of successful disease gene discovery in inherited disorders. To discuss the challenges for implementing NGS in the clinical research and diagnostic arenas. DATA SOURCES: Literature review and authors' experience. CONCLUSIONS: Next-generation sequencing approaches are powerful and require an investment in infrastructure and personnel expertise for effective use; however, the potential for improvement of patient care through faster and more accurate molecular diagnoses is high.

Free full text available from the CAP’s Archives PMID: 22770468 NOTE: Also cited in Section 3.1

D) Expanding the Computational Toolbox for Mining Cancer Genomes Ding L, Wendl MC, McMichael JF, Raphael BJ. Expanding the computational toolbox for mining cancer genomes. Nat Rev Genet. 2014; 15(8): 556-570.

Summary: High-throughput DNA sequencing has revolutionized the study of cancer genomics with numerous discoveries that are relevant to cancer diagnosis and treatment. The latest sequencing and analysis methods have successfully identified somatic alterations, including single-nucleotide variants, insertions and deletions, copy-number aberrations, structural variants and gene fusions. Additional computational techniques have proved useful for defining the mutations, genes and molecular networks that drive diverse cancer phenotypes and that determine clonal architectures in tumour samples. Collectively, these tools have advanced the study of genomic, transcriptomic and epigenomic alterations in cancer, and their association to clinical properties. Here, we review cancer genomics software and the insights that have been gained from their application.

Free full text available from PubMed PMID: 25001846

E) A Brief Introduction to Web-Based Genome Browsers Wang J, Kong L, Gao G, Luo J. A brief introduction to web-based genome browsers. Brief Bioinform. 2013; 14(2): 131-143.

Summary: Genome browser provides a graphical interface for users to browse, search, retrieve and analyze genomic sequence and annotation data. Web-based genome browsers can be classified into general genome browsers with multiple species and species-specific genome browsers. In this review, we attempt to give an overview for the main functions and features of web-based genome browsers, covering data visualization, retrieval, analysis and customization. To give a brief introduction to the multiple-species genome browser, we describe the user interface and main functions of the Ensembl and UCSC genome browsers using the human alpha-globin gene cluster as an example. We further use the MSU and the Rice-Map genome browsers to show some special features of species- specific genome browser, taking a rice transcription factor gene OsSPL14 as an example.

Free full text available from Briefings in Bioinformatics PMID: 22764121

6.2 Clinical Informatics and Reporting

A) Annotation of Sequence Variants in Cancer Samples: Processes and Pitfalls for Routine Assays in the Clinical Laboratory Lee LA, Arvai KJ,Jones D. Annotation of Sequence Variants in Cancer Samples: Processes and Pitfalls for Routine Assays in the Clinical Laboratory. J Mol Diagn. 2015; 17(4): 339-351.

Summary: As DNA sequencing of multigene panels becomes routine for cancer samples in the clinical laboratory, an efficient process for classifying variants has become more critical. Determining which germline variants are significant for cancer disposition and which somatic mutations are integral to cancer development or therapy response remains difficult, even for well- studied genes such as BRCA1 and TP53. We compare and contrast the general principles and lines of evidence commonly used to distinguish the significance of cancer-associated germline and somatic genetic variants. The factors important in each step of the analysis pipeline are reviewed, as are some of the publicly available annotation tools. Given the range of indications and uses of cancer sequencing assays, including diagnosis, staging, prognostication, theranostics, and residual disease detection, the need for flexible methods for scoring of variants is discussed. The usefulness of protein prediction tools and multimodal risk-based or Bayesian approaches are highlighted. Using TET2 variants encountered in hematologic neoplasms, several examples of this multifactorial approach to classifying sequence

variants of unknown significance are presented. Although there are still significant gaps in the publicly available data for many cancer genes that limit the broad application of explicit algorithms for variant scoring, the elements of a more rigorous model are outlined.

Free full text available from Journal of Molecular Diagnostics PMID: 25977238

B) Communicating New Knowledge on Previously Reported Genetic Variants Aronson SJ, Clark EH, Varugheese M, Baxter S, Babb LJ, Rehm HL. Communicating new knowledge on previously reported genetic variants. Genet Med. 2012. doi: 10.1038/gim.2012.19.

Summary: Genetic tests often identify variants whose significance cannot be determined at the time they are reported. In many situations, it is critical that clinicians be informed when new information emerges on these variants. It is already extremely challenging for laboratories to provide these updates. These challenges will grow rapidly as an increasing number of clinical genetic tests are ordered and as the amount of patient DNA assayed per test expands; the challenges will need to be addressed before whole-genome sequencing is used on a widespread basis. Information technology infrastructure can be useful in this context. We have deployed an infrastructure enabling clinicians to receive knowledge updates when a laboratory changes the classification of a variant. We have gathered statistics from this deployment regarding the frequency of both variant classification changes and the effects of these classification changes on patients. We report on the system's functionality as well as the statistics derived from its use.

Free full text available from Genetics in Medicine PMID: 22481129

C) Informatics and Clinical Genome Sequencing: Opening the Black Box Moorthie S, Hall A, Wright CF. Informatics and clinical genome sequencing: opening the black box. Genet Med. 2013; 15(3): 165-171

Summary: Adoption of whole-genome sequencing as a routine biomedical tool is dependent not only on the availability of new high-throughput sequencing technologies, but also on the concomitant development of methods and tools for data collection, analysis, and interpretation. It would also be enormously facilitated by the development of decision support

systems for clinicians and consideration of how such information can best be incorporated into care pathways. Here we present an overview of the data analysis and interpretation pipeline, the wider informatics needs, and some of the relevant ethical and legal issues.

Free full text available from Genetics in Medicine PMID: 22975759

D) Standardized Decision Support in Next Generation Sequencing Reports of Somatic Cancer Variants Dienstmann R, Dong F, Borger D, et al. Standardized decision support in next generation sequencing reports of somatic cancer variants. Mol Oncol. 2014; 8(5): 859-873.

Summary: Of hundreds to thousands of somatic mutations that exist in each cancer genome, a large number are unique and non-recurrent variants. Prioritizing genetic variants identified via next generation sequencing technologies remains a major challenge. Many such variants occur in tumor genes that have well-established biological and clinical relevance and are putative targets of molecular therapy, however, most variants are still of unknown significance. With large amounts of data being generated as high throughput sequencing assays enter the clinical realm, there is a growing need to better communicate relevant findings in a timely manner while remaining cognizant of the potential consequences of misuse or overinterpretation of genomic information. Herein we describe a systematic framework for variant annotation and prioritization, and we propose a structured molecular pathology report using standardized terminology in order to best inform oncology clinical practice. We hope that our experience developing a comprehensive knowledge database of emerging predictive markers matched to targeted therapies will help other institutions implement similar programs.

Full text available from Molecular Oncology (USD 31.50) PMID: 24768039

E) NGS Informatics Catching Up to Clinical Demands Check W. NGS informatics catching up to clinical demands. CAP TODAY. 2014 November.

Summary: November 2014—When Birgit H. Funke, PhD, gave a talk earlier this year on incorporating bioinformatic tools and pipelines into medical NGS, at Molecular Medicine Tri-Con 2014, one of her slides showed the main bioinformatics activities needed to support sequencing. Among them were

designing and building pipelines to manage genetic data, writing scripts for data analysis pipelines, and building custom applications. But the point she emphasized most was “Clinical add-on: documentation + validation.” Dr. Funke, who is assistant professor of pathology at Massachusetts General Hospital/Harvard Medical School and director of clinical research and development for the Laboratory for Molecular Medicine at Partners HealthCare, said that working with an awareness of clinical application is new for bioinformaticians. In an interview with CAP TODAY, she illustrates this with a brief dialogue she had experienced: Lab director: “Where did you store that script?” Bioinformatician: “I don’t know, but I can rewrite it for you.” While this attitude is all right for research work, it just won’t cut it in the world of CAP-accredited, CLIA-certified laboratory testing. “We have to validate everything, and that includes scripts,” Dr. Funke says. “Building a team of clinical bioinformaticians is a painful process. It is almost a new discipline.” It takes about a year, she estimates, for a bioinformatician to get on the same page with the rest of the laboratory.

Free full text available from CAP TODAY

Reprinted by permission from Macmillan Publishers Ltd: Nature Biotechnology, Gargis AS et al. Good laboratory practice for clinical next-generation sequencing informatics pipelines. Nat Biotechnol. 30 (7): 689-93, copyright 2015.

6.3 Databases

 ClinGen https://www.clinicalgenome.org/  COSMIC http://cancer.sanger.ac.uk/cosmic/  HGMD https://portal.biobase-international.com/cgi-bin/portal/login.cgi  OMIM http://www.ncbi.nlm.nih.gov/omim  PharmGKB https://www.pharmgkb.org/  IMGT/HLA http://www.ebi.ac.uk/ipd/imgt/hla/  PATRIC https://www.patricbrc.org/portal/portal/patric/Home

A) DNA-Mutation Inventory to Refine and Enhance Cancer Treatment (Direct): A Catalog of Clinically Relevant Cancer Mutations to Enable Genome-Directed Anticancer Therapy Yeh P, Chen H, Andrews J, Naser R, Pao W, Horn L. DNA-Mutation Inventory to Refine and Enhance Cancer Treatment (Direct): A Catalog of Clinically Relevant Cancer Mutations to Enable Genome-Directed Anticancer Therapy. Clin Cancer Res. 2013; 19(7): 1894-1901.

Summary: PURPOSE: Tumor gene mutation status is becoming increasingly important in the treatment of patients with cancer. A comprehensive catalog of tumor gene-response outcomes from individual patients is needed, especially for actionable mutations and rare variants. We created a proof-of-principle database [DNA-mutation Inventory to Refine and Enhance Cancer Treatment (DIRECT)], starting with lung cancer-associated EGF receptor (EGFR) mutations, to provide a resource for clinicians to prioritize treatment decisions based on a patient's tumor mutations at the point of care. METHODS: A systematic search of literature published between June 2005 and May 2011 was conducted through PubMed to identify patient-level, mutation-drug response in patients with non-small cell lung cancer (NSCLC) with EGFR mutant tumors. Minimum inclusion criteria included patient's EGFR mutation, corresponding treatment, and an associated radiographic outcome. RESULTS: A total of 1,021 patients with 1,070 separate EGFR tyrosine kinase inhibitor therapy responses from 116 different publications were included. About 188 unique EGFR mutations occurring in 207 different combinations were identified: 149 different mutation combinations were associated with disease control and 42 were associated with disease progression. Four secondary mutations, in 16 different combinations, were associated with acquired resistance. CONCLUSIONS: As tumor sequencing becomes more common in oncology, this comprehensive electronic catalog can enable genome-directed anticancer

therapy. DIRECT will eventually encompass all tumor mutations associated with clinical outcomes on targeted therapies. Users can make specific queries at http://www.mycancergenome.org/about/direct to obtain clinically relevant data associated with various mutations.

Free full text available from PubMed PMID: 23344264

B) Towards Precision Medicine: Advances in Computational Approaches for the Analysis of Human Variants Peterson TA, Doughty E, Kann MG. Towards precision medicine: advances in computational approaches for the analysis of human variants. J Mol Biol. 2013; 425(21): 4047-4063.

Summary: Variations and similarities in our individual genomes are part of our history, our heritage, and our identity. Some human genomic variants are associated with common traits such as hair and eye color, while others are associated with susceptibility to disease or response to drug treatment. Identifying the human variations producing clinically relevant phenotypic changes is critical for providing accurate and personalized diagnosis, prognosis, and treatment for diseases. Furthermore, a better understanding of the molecular underpinning of disease can lead to development of new drug targets for precision medicine. Several resources have been designed for collecting and storing human genomic variations in highly structured, easily accessible databases. Unfortunately, a vast amount of information about these genetic variants and their functional and phenotypic associations is currently buried in the literature, only accessible by manual curation or sophisticated text text-mining technology to extract the relevant information. In addition, the low cost of sequencing technologies coupled with increasing computational power has enabled the development of numerous computational methodologies to predict the pathogenicity of human variants. This review provides a detailed comparison of current human variant resources, including HGMD, OMIM, ClinVar, and UniProt/Swiss-Prot, followed by an overview of the computational methods and techniques used to leverage the available data to predict novel deleterious variants. We expect these resources and tools to become the foundation for understanding the molecular details of genomic variants leading to disease, which in turn will enable the promise of precision medicine.

Free full text available from PubMed PMID: 23962656

C) DNA Variant Databases: Current State and Future Directions Plazzer JP, Macrae F. DNA variant databases: current state and future directions. Methods Mol Biol. 2014; 1168:263-73. doi: 10.1007/978-1-4939- 0847-9_15.

Summary: In this chapter we aim to provide an overview of DNA variant databases, commonly known as Locus-Specific Databases (LSDBs), or Gene-Disease Specific Databases (GDSDBs), but the term variant database will be used for simplicity. We restrict this overview to germ-line variants, particularly as related to Mendelian diseases, which are diseases caused by a variant in a single gene. Common difficulties associated with variant databases and some proposed solutions are reviewed. Finally, systems where technical solutions have been implemented are discussed. This work will be useful for anyone wishing to establish their own variant database, or to learn about the global picture of variant databases, and the technical challenges to be overcome.

Full text available from Clinical Bioinformatics (USD 49.95) PMID: 24870141

D) Bioinformatics in Bacterial Molecular Epidemiology and Public Health: Databases, Tools and the Next-Generation Sequencing Revolution Carrico JA, Sabat AJ, Friedrich AW, Ramirez M, ESCMID Study Group for Epidemiological Markers (ESGEM). Bioinformatics in bacterial molecular epidemiology and public health: databases, tools and the next-generation sequencing revolution. Euro Surveill. 2013; 18(4): 20382.

Summary: Advances in typing methodologies have been the driving force in the field of molecular epidemiology of pathogens. The development of molecular methodologies, and more recently of DNA sequencing methods to complement and improve phenotypic identification methods, was accompanied by the generation of large amounts of data and the need to develop ways of storing and analysing them. Simultaneously, advances in computing allowed the development of specialised algorithms for image analysis, data sharing and integration, and for mining the ever larger amounts of accumulated data. In this review, we will discuss how bioinformatics accompanied the changes in bacterial molecular epidemiology. We will discuss the benefits for public health of specialised online typing databases and algorithms allowing for real-time data analysis and visualisation. The impact of the new and disruptive next-generation sequencing methodologies will be evaluated, and we will look ahead into these novel challenges.

Return to Table of Contents Section 6

Free full text available from Eurosurveillance PMID: 23369390

E) Power in Numbers: Meta-Analysis to Identify Inhibitor-Sensitive Tumor Genotypes Oxnard GR, Janne PA. Power in Numbers: Meta-Analysis to Identify Inhibitor-Sensitive Tumor Genotypes. Clin Cancer Res. 2013; 19(7): 1634- 1636.

Summary: Widespread tumor genotyping has increased the complexity of lung cancer care, often identifying mutations of uncertain clinical significance. In the accompanying article, the authors carried out a meta-analysis of the published literature on EGF receptor (EGFR) genotype and erlotinib/gefitinib sensitivity to develop a publicly accessible database to inform patient care.

Free full text available from PubMed PMID: 23403632

6.4 Considerations of Depth of Coverage

A) Sequencing Depth and Coverage: Key Considerations in Genomic Analyses Sims D, Sudbery I, Ilott NE, Heger A, Ponting CP. Sequencing depth and coverage: key considerations in genomic analyses. Nat Rev Genet. 2014; 15(2): 121-132.

Summary: Sequencing technologies have placed a wide range of genomic analyses within the capabilities of many laboratories. However, sequencing costs often set limits to the amount of sequences that can be generated and, consequently, the biological outcomes that can be achieved from an experimental design. In this Review, we discuss the issue of sequencing depth in the design of next-generation sequencing experiments. We review current guidelines and precedents on the issue of coverage, as well as their underlying considerations, for four major study designs, which include de novo genome sequencing, genome resequencing, transcriptome sequencing and genomic location analyses (for example, chromatin immunoprecipitation followed by sequencing (ChIP-seq) and chromosome conformation capture (3C)).

Full text available from Nature Reviews Genetics (USD 32.00 for 24 hour access)

PMID: 24434847

B) Impact of the Next-Generation Sequencing Data Depth on Various Biological Result Inferences Hou R, Yang Z, Li M, Xiao H. Impact of the next-generation sequencing data depth on various biological result inferences. Sci China Life Sci. 2013; 56(2): 104-109.

Summary: Next-generation sequencing (NGS) technologies have revolutionized the field of genomics and provided unprecedented opportunities for high-throughput analysis at the levels of genomics, transcriptomics and epigenetics. However, the cost of NGS is still prohibitive for many laboratories. It is imperative to address the trade-off between the sequencing depth and cost. In this review, we will discuss the effects of sequencing depth on the detection of genes, quantification of gene expression and discovering of gene structural variants. This will provide readers information on choosing appropriate sequencing depth that best meet the needs of their particular project.

Free full text available from Science China Life Science PMID: 23393025

6.5 Bioinformatics for Pathogens

A) Bioinformatics of Antimicrobial Resistance in the Age of Molecular Epidemiology McArthur AG,Wright GD. Bioinformatics of Antimicrobial Resistance in the Age of Molecular Epidemiology. Curr Opin Microbiol. 2015; 27:45-50.

Summary: Antimicrobial resistance is a global health challenge and has an evolutionary trajectory ranging from proto-resistance in the environment to untreatable clinical pathogens. Resistance is not static, as pathogenic strains can move among patient populations and individual resistance genes can move among pathogens. Effective treatment of resistant infections, antimicrobial stewardship, and new drug discovery increasingly rely upon genotype information, powered by decreasing costs of DNA sequencing. These new approaches will require advances in microbial informatics, particularly in development of reference databases of molecular determinants such as our Comprehensive Antibiotic Resistance Database and clinical metadata, new algorithms for prediction of resistome and resistance

phenotype from genotype, and new protocols for global collection and sharing of high-throughput molecular epidemiology data.

Full text available from Current Opinion in Microbiology (USD 41.95) PMID: 26241506

B) Web Resources for Microbial Data Sun Q, Liu L, Wu L, et al. Web Resources for Microbial Data. Genomics Proteomics Bioinformatics. 2015; 13(1): 69-72.

Summary: There are multitudes of web resources that are quite useful for the microbial scientific research community. Here, we provide a brief introduction on some of the most notable microbial web resources and an evaluation of them based upon our own user experience.

Free full text available from PubMed PMID: 25721609

C) PATRIC: The Comprehensive Bacterial Bioinformatics Resource with a Focus on Human Pathogenic Species Gillespie JJ, Wattam AR, Cammer SA, et al. PATRIC: the comprehensive bacterial bioinformatics resource with a focus on human pathogenic species. Infect Immun. 2011; 79(11): 4286-4298.

Summary: Funded by the National Institute of Allergy and Infectious Diseases, the Pathosystems Resource Integration Center (PATRIC) is a genomics-centric relational database and bioinformatics resource designed to assist scientists in infectious-disease research. Specifically, PATRIC provides scientists with (i) a comprehensive bacterial genomics database, (ii) a plethora of associated data relevant to genomic analysis, and (iii) an extensive suite of computational tools and platforms for bioinformatics analysis. While the primary aim of PATRIC is to advance the knowledge underlying the biology of human pathogens, all publicly available genome- scale data for bacteria are compiled and continually updated, thereby enabling comparative analyses to reveal the basis for differences between infectious free-living and commensal species. Herein we summarize the major features available at PATRIC, dividing the resources into two major categories: (i) organisms, genomes, and comparative genomics and (ii) recurrent integration of community-derived associated data. Additionally, we present two experimental designs typical of bacterial genomics research and report on the execution of both projects using only PATRIC data and tools. These applications encompass a broad range of the data and analysis tools available, illustrating practical uses of PATRIC for the biologist. Finally, a

summary of PATRIC's outreach activities, collaborative endeavors, and future research directions is provided.

Free full text available from PubMed PMID: 21896772

D) Pathogen Genome Bioinformatics Sintchenko V, Roper MP. Pathogen genome bioinformatics. Methods Mol Biol. 2014; 1168: 173-193.

Summary: Recent advances in DNA sequencing technology have made the whole-genome sequencing of pathogens in a clinically relevant turn-around time both technically and economically feasible. The DNA sequencing of pathogens with epidemic potential offers new and exciting opportunities for high-resolution public health surveillance. This chapter outlines major methods and bioinformatics tools for pathogen genome characterization, the identification of infectious disease clusters, as well as for genomics-guided biosurveillance. Existing challenges are also considered.

Full text available from Clinical Bioinformatics (USD 49.95) PMID: 24870136

Return to Table of Contents 376 Section 7 Issues for the Practicing Pathologist

It is an exciting time in genomic medicine. Opportunities to obtain genomic information are increasing at a seemingly exponential pace. With these opportunities come new challenges that must be overcome. Genomics yields massive amounts of data. It must now be determined how this data will be presented clearly and concisely in reports. How will variants be classified? What incidental findings will be reported? How will clinicians be educated so they, in turn, can discuss these results with their patients? In what way will reports be updated to reflect new information?

As genomic information is obtained for patients and research participants, a new era of patient privacy and ethical decision-making also enters. In what ways can genomics be discussed with patients to obtain a true informed consent? How will patient privacy be protected in databases and electronic medical records? What measures will be taken to ensure equitable access to genomics for all patient populations? How will medical necessity and ethical decision-making be balanced in newborn and minor children populations?

Landmark changes are also being made in the legal, regulatory and billing environments. How will patents relate to genomics in the future? What changes will be seen with billing and reimbursement for testing? How will the Food and Drug Administration (FDA) regulate genomic testing? As patients take greater initiative for their own genomic testing, how will direct-to-consumer (DTC) testing proceed?

Many have already embraced these challenges and many are now laying the path to ensure quality, ethical, and meaningful testing.

This section includes the following topics:

• Insights from Adopters (Section 7.1); • Pathology Reports in the Era of Genomics (Section 7.2); and • Patient Consent Forms, Privacy and Ethics (Section 7.3);

Return to Table of Contents Section 7

7.1 Insights from Adopters

This section includes insights from the following adopters:

• Pranil K. Chandra (Section 7.1.1); • Jordan S. Laser (Section 7.1.2); and • Samuel K. Caughron (Section 7.1.3).

7.1.1 Insights from Adopter - Pranil K. Chandra, DO, FCAP

Pranil K. Chandra, DO, FCAP, is board certified in anatomic and clinical pathology, hematopathology and molecular genetic pathology. He completed his AP/CP training and fellowships in hematopathology, oncologic pathology and molecular pathology at NYU Langone Medical Center and the University of Texas-MD Anderson Cancer Center. While at MD Anderson, Dr. Chandra served as Chief Fellow.

Dr. Chandra is a member of the American Society of Clinical Pathology, Association of Molecular Pathology (AMP), and the College of American Pranil K. Chandra, DO, FCAP Pathologists (CAP) as well a medical consultant in molecular pathology, cancer genomics, and personalized/precision medicine. He was the 2015 chair of the genomics education working group of the CAP’s Personalized Healthcare Committee and is also an active member of the AMP-based Economic Affairs Committee.

Dr. Chandra is a molecular oncologic pathologist in a large community practice composed of 80+ pathologists. As Assistant Vice President Medical Director of Molecular Pathology and Genomic Medicine Services at PathGroup, in addition to clinical sign-out responsibilities, he is responsible for clinical and administrative oversight of molecular and genomic testing services as well as implementation and standardization of testing throughout the network of more than 100 hospitals in integrated regional health care systems.

Dr. Chandra likes to add value to the health care system by functioning as a consultant for genomics testing.

Dr. Chandra’s insights for the next wave of adopters (May 2016):

1 Learn about basic In order to critically evaluate genomics tests molecular biology, that are currently available in the marketplace, PCR, and genomics it is imperative to have a solid basic techniques. understanding of molecular biology and related techniques such as PCR, next generation sequencing, and other promising high- throughput technologies such as SNP-based microarray. 2 Become a content Clinicians, especially oncologists, look to expert in genomics pathologists to lead efforts in responsible testing by keeping utilization of genomic testing. This means abreast of the having good knowledge of what represents literature. standard of care genomics testing based on NCCN and/or other guidelines. It’s also important be an avaricious reader and keep abreast of the constantly changing genomic testing landscape and latest promising biomarkers that are published in high-impact journals. 3 Identify genomic These are tests that improve outcomes or tests that add value decrease health-care costs by informing any to the health care combination of diagnosis, prognosis, and/or system. therapy decisions. 4 Reach out to your Cancer genomics testing is expanding rapidly oncologyReturn clinicians, to Table ofand Contents the pathologist has a significant hospital leadership, opportunity to be involved in test selection, and/or other utilization, and even interpretation of results. stakeholders and Both academic and community based practices initiate thoughtful are expanding “Precision Medicine” initiatives dialogue regarding and pathologists need to have a seat at that genomics testing. table or be prepared to be on the menu!!! 5 Be a champion of Be proud of the fact that laboratory testing pathology and drives greater than 2/3 of clinical management genomics testing decisions but accounts for less than 5% of total and speak up. health care costs.

6 Find innovative ways When I first started as a pathologist in private to add value as an practice, I was fortunate to work with the Vice- integral member of President of Cancer Services in the health care the health care system. The project was large and required delivery team. extensive literature review. Together, we developed a standardized algorithm for molecular work-up of malignancies which was operationalized and incorporated throughout 14 hospitals in the network. This was a very valuable experience that was outside the realm of my day to day responsibilities as a pathologist but instrumental in demonstrating to hospital leadership the value I could bring by leveraging my genomic pathology expertise. 7 Educate, educate, Giving presentations and/or our perspectives and educate. during tumor board, regional pathology meetings, oncology symposiums, nursing society meetings, cancer registrar events, and other multi-disciplinary venues is one way we can increase awareness of what we do as pathologists. The PHC has created numerous educational tools including a PPT presentation on the basics of next generation sequencing. These are available through the CAP website. 8 If your practice does There is a general underutilization of not allow building a molecular/genomics tests that should be molecular or offered as standard of care, especially in genomics laboratory, community practice. Pathologists need to take find a send-out the lead in making sure that appropriate testing laboratory to ensure is done. Take for example KRAS testing. It that cancer patients has been estimated that if every metastatic receive standard of colon cancer was genotyped for KRAS when care. making a decision to place the patient on anti- EGFR monoclonal therapy, that would result in a cost savings of more than $500 million dollars to the health care system. This is why expanded RAS testing in metastatic colorectal cancer is considered standard of care by CAP and ASCO.

9 Be willing to take on The field of pathology is evolving rapidly. We new roles and have seen a departure of what was traditionally responsibilities. a histologic based classification of cancer to one that integrates changes on a genomic level. As the “doctor’s doctor”, we have to continue to expand our consultative guidance to physicians to include diagnostic, prognostic, and therapeutic information. This will require us learning more about the significance of genomic changes in predicting response (or lack thereof) to oncology therapeutics including targeted therapies and immuno-oncology agents. 10 Get involved in The field of pathology continues to experience genomics and significant reimbursement and regulatory pathology advocacy pressures. We need our pathology community efforts. to be actively involved in demonstrating how extremely valuable our clinical efforts are so that the laboratory community is not shortchanged in being able to deliver the highest quality of care to our patients.

Dr. Chandra’s suggested articles:

A) Precision Oncology: An Overview Garraway LA, Verweij J, Ballman KV. Precision oncology: an overview. J Clin Oncol. 2013; 31(15): 1803-1805.

No summary available

Free full text available from Journal of Clinical Oncology PMID: 23589545

B) Prognostic Relevance of Integrated Genetic Profiling in Acute Myeloid Leukemia Patel JP, Gonen M, Figueroa ME, et al. Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N Engl J Med. 2012; 366(12): 1079-1089.

Summary: BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous disease with respect to presentation and clinical outcome. The prognostic value of recently identified somatic mutations has not been systematically evaluated in a phase 3 trial of treatment for AML. METHODS:

We performed a mutational analysis of 18 genes in 398 patients younger than 60 years of age who had AML and who were randomly assigned to receive induction therapy with high-dose or standard-dose daunorubicin. We validated our prognostic findings in an independent set of 104 patients. RESULTS: We identified at least one somatic alteration in 97.3% of the patients. We found that internal tandem duplication in FLT3 (FLT3-ITD), partial tandem duplication in MLL (MLL-PTD), and mutations in ASXL1 and PHF6 were associated with reduced overall survival (P=0.001 for FLT3-ITD, P=0.009 for MLL-PTD, P=0.05 for ASXL1, and P=0.006 for PHF6); CEBPA and IDH2 mutations were associated with improved overall survival (P=0.05 for CEBPA and P=0.01 for IDH2). The favorable effect of NPM1 mutations was restricted to patients with co-occurring NPM1 and IDH1 or IDH2 mutations. We identified genetic predictors of outcome that improved risk stratification among patients with AML, independently of age, white-cell count, induction dose, and post-remission therapy, and validated the significance of these predictors in an independent cohort. High-dose daunorubicin, as compared with standard-dose daunorubicin, improved the rate of survival among patients with DNMT3A or NPM1 mutations or MLL translocations (P=0.001) but not among patients with wild-type DNMT3A, NPM1, and MLL (P=0.67). CONCLUSIONS: We found that DNMT3A and NPM1 mutations and MLL translocations predicted an improved outcome with high-dose induction chemotherapy in patients with AML. These findings suggest that mutational profiling could potentially be used for risk stratification and to inform prognostic and therapeutic decisions regarding patients with AML. (Funded by the National Cancer Institute and others.).

Free full text available from PubMed PMID: 22417203 NOTE: Also cited in Section 2.12.1

C) Personalized Medicine in a Phase I Clinical Trials Program: The MD Anderson Cancer Center Initiative Tsimberidou AM, Iskander NG, Hong DS, et al. Personalized medicine in a phase I clinical trials program: the MD Anderson Cancer Center initiative. Clin Cancer Res. 2012; 18(22): 6373-6383.

Summary: PURPOSE: We initiated a personalized medicine program in the context of early clinical trials, using targeted agents matched with tumor molecular aberrations. Herein, we report our observations. PATIENT AND METHODS: Patients with advanced cancer were treated in the Clinical Center for Targeted Therapy. Molecular analysis was conducted in the MD Anderson Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory. Patients whose tumors had an aberration were treated with

matched targeted therapy, when available. Treatment assignment was not randomized. The clinical outcomes of patients with molecular aberrations treated with matched targeted therapy were compared with those of consecutive patients who were not treated with matched targeted therapy. RESULTS: Of 1,144 patients analyzed, 460 (40.2%) had 1 or more aberration. In patients with 1 molecular aberration, matched therapy (n = 175) compared with treatment without matching (n = 116) was associated with a higher overall response rate (27% vs. 5%; P < 0.0001), longer time-to- treatment failure (TTF; median, 5.2 vs. 2.2 months; P < 0.0001), and longer survival (median, 13.4 vs. 9.0 months; P = 0.017). Matched targeted therapy was associated with longer TTF compared with their prior systemic therapy in patients with 1 mutation (5.2 vs. 3.1 months, respectively; P < 0.0001). In multivariate analysis in patients with 1 molecular aberration, matched therapy was an independent factor predicting response (P = 0.001) and TTF (P = 0.0001). CONCLUSION: Keeping in mind that the study was not randomized and patients had diverse tumor types and a median of 5 prior therapies, our results suggest that identifying specific molecular abnormalities and choosing therapy based on these abnormalities is relevant in phase I clinical trials.

Free full text available from Clinical Cancer Research PMID: 22966018

D) Personalized Genomic Analyses for Cancer Mutation Discovery and Interpretation Jones S, Anagnostou V, Lytle K, et al. Personalized genomic analyses for cancer mutation discovery and interpretation. Sci Transl Med. 2015; 7(283): 283ra253.

predisposing genes in 3% of patients with apparently sporadic cancers. In contrast, a tumor-only sequencing approach could not definitively identify germline changes in cancer-predisposing genes and led to additional false- positive findings comprising 31% and 65% of alterations identified in targeted and exome analyses, respectively, including in potentially actionable genes. These data suggest that matched tumor-normal sequencing analyses are essential for precise identification and interpretation of somatic and germline alterations and have important implications for the diagnostic and therapeutic management of cancer patients.

Full text available from Science Translational Medicine (USD 20.00) PMID: 25877891 NOTE: Also cited in Section 1.6.2

E) A Molecular Diagnostic Perfect Storm: The Convergence of Regulatory & Reimbursement Forces that Threaten Patient Access to Innovations in Genomic Medicine Pratt PM, Aisner DL, Day SP, et al. A Molecular Diagnostic Perfect Storm: The Convergence of Regulatory & Reimbursement Forces that Threaten Patient Access to Innovations in Genomic Medicine. Association for Molecular Pathology. 2015.

No summary available.

Free full text available from Association for Molecular Pathology

7.1.2 Insights from Adopter—Jordan S. Laser, MD, FCAP

Jordan S. Laser, MD, FCAP, is a board certified anatomic, clinical, and molecular genetic pathologist. Currently employed at Northwell in New York, Dr. Laser serves the Department of Pathology and Laboratory Medicine in the following roles: Medical Director, LIJ Laboratories, Senior Director, Division of Cytogenetics and Molecular Pathology; and Director, Division of Near Patient Testing. His expertise includes, molecular and genomic medicine, laboratory management, health care finance, and standards and regulations.

The Division of Cytogenenetics and Molecular Pathology is responsible for all Jordan S. Laser, MD, FCAP testing for the North Shore LIJ Health System. The Division focuses largely on oncologic and constitutional disorders with a full spectrum of diagnostics to support the clinical needs. As it serves a broad spectrum of patients and covers a large geographical area, it generally focuses on high volume tests with

technology that supports a high throughput. Accordingly, the service is considered a large traditional molecular diagnostic laboratory in the sense that it focus on single gene analytes.

As the national healthcare environment changes, health systems, hospitals and private labs must focus on providing not only quality laboratory results, but deliver value. Value may be delivered as an in-house assay or best delivered by sending to a reference lab. It is becoming more and more important for laboratory directors to make this critical assessment to balance care and cost.

Dr. Laser’s insights for the next wave of adopters (April 2015):

1 Keep the patient in When determining what tests to develop, your mind at all always consider the value it has to the times management of the patient first. If it doesn’t add value…don’t develop it. 2 Understand ‘who’ the As with all projects, it is critical to understand test impacts who it impacts (ie. Stakeholders) and to assess their opinions on the test. Consider those beyond the obvious (patient, provider, lab) and consider administrators, finance directors etc. Some will be supporters, some will not. Identifying your stakeholders will ensure success of what you are trying to accomplish 3 Take a course in Even a simple day course in finance can have basic financial significant impact on your understanding and concepts ability to perform a sound financial analysis of a particular test. This will help you make a much more informed decision on what tests to develop and what tests to send to a reference laboratory 4 Leave your ego at Remember….patient first. Your responsibility the door to the patient is to deliver care in both the quality and cost perspectives (ie value). If quality is equal, and the cost is less to send a test to a reference lab…don’t be afraid to send it out. 5 Prepare to advocate Again, patient first. Even if it costs more to for a financial loss perform it in your laboratory, if you provide higher quality or meet critical turnaround times that a reference lab cannot, you must be prepared to advocate for the test even though it will be a financial burden to the lab.

6 ‘Phone a friend’ Many of us have made similar decisions regarding sending a test to a reference lab or to develop it in house. Reach out to a colleague; post a question on a listserv. We can always learn from each other’s experiences. 7 Think outside of the Health care delivery is complex and technology box brings both opportunities and challenges. Always challenge assumptions and don’t be afraid to propose creative solutions to the challenges you face.

Dr. Laser’s suggested articles and resources:

A) Precision Medicine: The Future or Simply Politics? Rubin R. Precision medicine: the future or simply politics? JAMA. 2015; 313(11): 1089-1091.

Summary: Barrett Rollins, MD, PhD, chief scientific officer at the Dana- Farber Cancer Institute, tells an anecdote to illustrate the promise of “precision medicine.” A 35-year-old patient at Dana-Farber was dying of myeloid sarcoma, a rare tumor consisting of a solid collection of leukemic cells outside of the bone marrow. Although the man was about to enter hospice care, he consented to having his tumor’s genome sequenced.

Unexpectedly, sequencing revealed a mutation in a platelet-derived growth factor receptor gene, giving rise to a constitutively active kinase targeted by the kinase inhibitor imatinib mesylate, marketed as Gleevec. Myeloid sarcoma is not among Gleevec’s indications, but the man’s tumor shrank after he began taking the pill. Months later, he is still alive, no longer a candidate for hospice care, Rollins said.

Full text available from JAMA (USD 30.00 for 24 hour access) PMID: 25781428

B) Improving the Value of Costly Genetic Reference Laboratory Testing with Active Utilization Management Dickerson JA, Cole B, Conta JH, et al. Improving the value of costly genetic reference laboratory testing with active utilization management. Arch Pathol Lab Med. 2014; 138(1): 110-113.

Summary: CONTEXT: Tests that are performed outside of the ordering institution, send-out tests, represent an area of risk to patients because of

complexity associated with sending tests out. Risks related to send-out tests include increased number of handoffs, ordering the wrong or unnecessary test, specimen delays, data entry errors, preventable delays in reporting and acknowledging results, and excess financial liability. Many of the most expensive and most misunderstood tests are send-out genetic tests. OBJECTIVE: To design and develop an active utilization management program to reduce the risk to patients and improve value of genetic send-out tests. DESIGN: Send-out test requests that met defined criteria were reviewed by a rotating team of doctoral-level consultants and a genetic counselor in a pediatric tertiary care center. RESULTS: Two hundred fifty- one cases were reviewed during an 8-month period. After review, nearly one- quarter of genetic test requests were modified in the downward direction, saving a total of 2% of the entire send-out bill and 19% of the test requests under management. Ultimately, these savings were passed on to patients. CONCLUSIONS: Implementing an active utilization strategy for expensive send-out tests can be achieved with minimal technical resources and results in improved value of testing to patients.

Free full text available from the CAP’s Archives PMID: 24377818

C) The Business of Genomic Testing: A Survey of Early Adopters Crawford JM, Bry L, Pfeifer J, et al. The business of genomic testing: a survey of early adopters. Genet Med. 2014; 16(12): 954-961.

Summary: PURPOSE: The practice of "genomic" (or "personalized") medicine requires the availability of appropriate diagnostic testing. Our study objective was to identify the reasons for health systems to bring next- generation sequencing into their clinical laboratories and to understand the process by which such decisions were made. Such information may be of value to other health systems seeking to provide next-generation sequencing testing to their patient populations. METHODS: A standardized open-ended interview was conducted with the laboratory medical directors and/or department of pathology chairs of 13 different academic institutions in 10 different states. RESULTS: Genomic testing for cancer dominated the institutional decision making, with three primary reasons: more effective delivery of cancer care, the perceived need for institutional leadership in the field of genomics, and the premise that genomics will eventually be cost- effective. Barriers to implementation included implementation cost; the time and effort needed to maintain this newer testing; challenges in interpreting genetic variants; establishing the bioinformatics infrastructure; and curating data from medical, ethical, and legal standpoints. Ultimate success depended on alignment with institutional strengths and priorities and working

closely with institutional clinical programs. CONCLUSION: These early adopters uniformly viewed genomic analysis as an imperative for developing their expertise in the implementation and practice of genomic medicine.

Free full text available from PubMed PMID: 25010053

D) Costs and Effectiveness of Genomic Testing in the Management of Colorectal Cancer Goldstein DA, Shaib WL, Flowers CR. Costs and effectiveness of genomic testing in the management of colorectal cancer. Oncology (Williston Park). 2015; 29(3): 175-183.

Summary: Numerous genomic tests are available for use in colorectal cancer, with a widely variable evidence base for their effectiveness and cost- effectiveness. In this review, we highlight many of these tests, with a focus on their proposed role, the evidence base to support that role, and the associated costs and risks. The tests with the strongest evidence base are KRAS genetic testing in the metastatic setting and microsatellite instability testing in selected patients and in stage II disease. There also may be a role for delineating recurrence-risk signatures for selected patients with stage II disease. The evidence to support broad utilization of uridine 5'-diphospho- glucuronosyltransferase 1A1 (UGT1A1) and dihydropyrimidine dehydrogenase (DPD) testing to guide irinotecan and fluorouracil dosing remains limited. There is much anticipation that next-generation sequencing will herald a new era of targeted therapy for patients with colorectal cancer; however, currently there are no data to support the introduction of widespread testing.

Free full text available from Oncology Journal PMID: 25783977

7.1.3 Insights from Adopter—Samuel K. Caughron, MD, FCAP

Samuel K. Caughron, MD, FCAP, is a member of the MAWD Pathology Group in Kansas City. In 2009, Dr. Caughron joined MAWD Pathology, a 12 pathologist group, to establish a community based molecular pathology lab. He currently is Director of the MAWD Molecular Lab in addition to practicing routine anatomic and clinical pathology at a 450 bed community hospital. Dr. Caughron had previously practiced in Billings, Montana where he helped establish a molecular Samuel K. Caughron, lab for a 7 member pathology group. MD, FCAP

Dr. Caughron serves as a member of the College of American Pathologists’ (CAP) House of Delegates as well as being a past member of the Board of Governors.

Dr. Caughron received his medical degree from Creighton University School of Medicine in Omaha, Nebraska; he also completed his residency training in Anatomic and Clinical Pathology there. After residency, he completed a fellowship in Molecular Genetic Pathology at Vanderbilt University Medical Center in Nashville, Tennessee. He is board certified in Anatomic and Clinical Pathology, as well as Molecular Genetic Pathology.

Dr. Caughron’s insights for the next adopters (September 2012):

1 Move beyond your Molecular pathology is a new area for many comfort zone pathologists, especially those who have been in practice since before it was a part of pathology training. But even for those who are trained in molecular pathology, the field is moving so quickly that yesterday's conclusions may not be true today. Adoption of molecular testing requires practicing in an area where things can change rapidly, where the basis for decisions will not be as established - an area that is uncomfortable for many pathologists. Learn to accept and live with it to help patients the best you can. 2 Find a way to stay As molecular testing advances, there will be current with new tremendous benefits possible for patient care. advances Today there are several examples like imatinib (Gleevec®) for CML, crizotinib (Xalkori®) for non-small cell lung cancer, and vemurafenib (Zelboraf®) for advanced melanoma. If you are going to get into molecular testing, even if you are not performing all of the testing, you have to stay current so that you know the possibilities for your patients. 3 You don't have to In fact, no lab does it all. Even the largest "do it all" to adopt reference labs send specimens for some tests molecular pathology to other reference labs. Pick a focus for your testing lab based on your patient mix, requests from other specialties, or target areas for growth.

4 Take care of your Don't give up that job to another doctor or lab. own patients This does not mean you have to perform all the testing for your patients. You cannot. But if there are tests being sent out to other labs, find out what the testing it being used for and what its real value is to the patient. If it does not make sense, add value and establish expertise by intervening. Do not allow yourself to be taken out of the loop for lab testing on your patients. 5 Be a visible resource Take, or make, opportunities to present on to your colleagues topics relevant to your molecular lab. The CAP for molecular testing offers the Short Presentations on Emerging Concepts (SPECs) for exactly this purpose. After you have set up a test, you are a local expert on that test. Explain what you are doing, and help your colleagues make sense of the results. Pathology is not the only specialty struggling to keep pace with the advances. But we are the only specialty with the training to properly evaluate, implement, and interpret the testing. 6 Take advantage of As mentioned above, the CAP offers the the national SPECs to jump start a presentation you are organizations asked to give about some topics. The offerings to support Association of Molecular Pathology has a molecular pathology tremendous number of resources available to help understand and implement molecular testing. And there are others. If you seek educational opportunities, you will find them in abundance. 7 Invest in This is a challenging, and rapidly changing understanding the area. However, the final success of your lab coverage, coding will probably depend on being able to make it and reimbursement financially viable. Coding is how you describe for molecular testing to payers what you did in your lab. Reimbursement is what you get paid. Coverage is whether you get paid. All of these crucial ingredients have to be in the proper mix to achieve success. Get to know them well.

8 Understand the big Molecular testing is really in its infancy or early picture and true childhood. There is a lot of growth yet to value of the testing happen. Adopting molecular testing today will you offer yield fruit down the road, but only if it fits with the delivery of healthcare in your area. Bringing valuable testing online may not immediately generate a revenue stream, but may be compelling for its cost savings in other areas of care. Find the opportunity and real value. You may have to give it time. You may have to sell it to colleagues, to payers, to administration. But it is there or will come. 9 Work with good My father once told me, "You can never go people wrong hiring good people." The success of your molecular efforts will depend entirely on the people who are involved, from the medical technologist, to the billing department, to the support staff. Find the best possible people you can for each job and treat them well. You will never regret it. 10 Think like a doctor As pathologists, we can get caught up in our analyses and lose sight of what the information we provide really means to the care and life of the patient. Molecular pathology is an opportunity for pathologists to claim a new and vital role in helping to take care of patients, by providing critical new kinds of information. When adopting or performing a test, keep asking: What is the clinical value to a test being performed? How will it impact treatment?

Dr. Caughron’s suggested articles and resources:

A) PubMed.com and Google.com While not traditional resources, online search capability is essential to staying current. I could not do my job without rapid access to information. Take a few minutes to become familiar with optimizing searching on PubMed and Google. It will yield tremendous returns.

B) CAP Accreditation Checklist for Molecular Labs

C) Oncology journals

7.2 Pathology Reports in the Era of Genomics

A) Cancer Biomarkers: The Role of Structured Data Reporting Simpson RW, Berman MA, Foulis PR, et al. Cancer Biomarkers: The Role of Structured Data Reporting. Arch Pathol Lab Med. 2015;139 (5): 587-593

Summary: Context .- The College of American Pathologists has been producing cancer protocols since 1986 to aid pathologists in the diagnosis and reporting of cancer cases. Many pathologists use the included cancer case summaries as templates for dictation/data entry into the final pathology report. These summaries are now available in a computer-readable format with structured data elements for interoperability, packaged as "electronic cancer checklists." Most major vendors of anatomic pathology reporting software support this model. Objectives .- To outline the development and advantages of structured electronic cancer reporting using the electronic cancer checklist model, and to describe its extension to cancer biomarkers and other aspects of cancer reporting. Data Sources .- Peer-reviewed literature and internal records of the College of American Pathologists. Conclusions .- Accurate and usable cancer biomarker data reporting will increasingly depend on initial capture of this information as structured data. This process will support the standardization of data elements and biomarker terminology, enabling the meaningful use of these datasets by pathologists, clinicians, tumor registries, and patients.

Free full text available from the CAP’s Archives PMID: 25275812

B) Clinical Analysis and Interpretation of Cancer Genome Data Van Allen EM, Wagle N, Levy MA. Clinical analysis and interpretation of cancer genome data. J Clin Oncol. 2013; 31(15): 1825-1833.

Summary: The scale of tumor genomic profiling is rapidly outpacing human cognitive capacity to make clinical decisions without the aid of tools. New frameworks are needed to help researchers and clinicians process the information emerging from the explosive growth in both the number of tumor genetic variants routinely tested and the respective knowledge to interpret their clinical significance. We review the current state, limitations, and future trends in methods to support the clinical analysis and interpretation of cancer genomes. This includes the processes of genome-scale variant identification, including tools for sequence alignment, tumor-germline comparison, and molecular annotation of variants. The process of clinical interpretation of tumor variants includes classification of the effect of the variant, reporting the results to clinicians, and enabling the clinician to make a clinical decision

based on the genomic information integrated with other clinical features. We describe existing knowledge bases, databases, algorithms, and tools for identification and visualization of tumor variants and their actionable subsets. With the decreasing cost of tumor gene mutation testing and the increasing number of actionable therapeutics, we expect the methods for analysis and interpretation of cancer genomes to continue to evolve to meet the needs of patient-centered clinical decision making. The science of computational cancer medicine is still in its infancy; however, there is a clear need to continue the development of knowledge bases, best practices, tools, and validation experiments for successful clinical implementation in oncology.

Full text available from Journal of Clinical Oncology (subscription required) PMID: 23589549

C) Return of Anticipated and Incidental Results from Next-Generation Sequencing: Implications From Providers and Patients Williams JK, Cashion AK, Brooks PJ. Return of Anticipated and Incidental Results from Next-Generation Sequencing: Implications from providers and patients. Institute of Medicine (Perspective Series). February 19, 2015.

Summary: Next-generation sequencing is currently being used clinically to identify the best treatment course for cancer patients, to provide patients with a diagnosis for a previously undiagnosed disease, and to diagnose common conditions such as heart disease. In the process of generating anticipated information related to any of these conditions, sequencing can also generate additional information unrelated to the condition for which a patient has sought treatment. These incidental findings can pose significant challenges for both the patient and provider. This discussion paper, the sixth of seven individually authored commentaries that explores the evidence needed to support the use of genome sequencing in the clinic, examines the implications for clinicians and patients related to returning and receiving, respectively, incidental next-generation sequencing results.

Free full text available from Institute of Medicine

D) The Unintended Implications of Blurring the Line Between Research and Clinical Care in a Genomic Age Berkman BE, Hull SC, Eckstein L. The unintended implications of blurring the line between research and clinical care in a genomic age. Per Med. 2014. 11(3): 285-295.

Summary: While the development of next-generation sequencing technology has had a paradigm-changing impact on biomedical research, there is likely

to be a gap between discovery of therapeutic benefits in research and actual adoption of the new technology into clinical practice. This gap can create pressure on the research enterprise to provide individualized care more typical of the clinic setting because it is uniquely accessible in research. This blurring of the line between research and clinical care is understandable, and perhaps even inevitable. But even if the gap is only transitory, such a blurring can have lasting implications, both by expanding obligations imposed on researchers, but also by challenging long-held ethical views. We explore this idea, focusing on how the dissolving distinction between research and clinical care has influenced the vigorous debate around how researchers should manage genetic findings (sometimes separated into primary and incidental or secondary findings) resulting from research.

Free full text available from PubMed PMID: 25506378

E) ACMG Recommendations for Reporting of Incidental Findings in Clinical Exome and Genome Sequencing Green RC, Berg JS, Grody WW, et al. ACMG recommendations for reporting of incidental findings in clinical exome and genome sequencing. Genet Med. 2013; 15(7): 565-574.

Summary: In clinical exome and genome sequencing, there is a potential for the recognition and reporting of incidental or secondary findings unrelated to the indication for ordering the sequencing but of medical value for patient care. The American College of Medical Genetics and Genomics (ACMG) recently published a policy statement on clinical sequencing that emphasized the importance of alerting the patient to the possibility of such results in pretest patient discussions, clinical testing, and reporting of results. The ACMG appointed a Working Group on Incidental Findings in Clinical Exome and Genome Sequencing to make recommendations about responsible management of incidental findings when patients undergo exome or genome sequencing. This Working Group conducted a year-long consensus process, including an open forum at the 2012 Annual Meeting and review by outside experts, and produced recommendations that have been approved by the ACMG Board. Specific and detailed recommendations, and the background and rationale for these recommendations, are described herein. The ACMG recommends that laboratories performing clinical sequencing seek and report mutations of the specified classes or types in the genes listed here. This evaluation and reporting should be performed for all clinical germline (constitutional) exome and genome sequencing, including the "normal" of tumor-normal subtractive analyses in all subjects, irrespective of age but excluding fetal samples. We recognize that there are insufficient data on

penetrance and clinical utility to fully support these recommendations, and we encourage the creation of an ongoing process for updating these recommendations at least annually as further data are collected.

Free full text available from PubMed PMID: 23788249 NOTE: Also cited in Section 3.9.2

7.3 Patient Consent Forms, Privacy and Ethics

A) Clinical Integration of Next Generation Sequencing: A Policy Analysis Kaufman D, Curnutte M, McGuire AL. Clinical Integration of Next Generation Sequencing: A Policy Analysis. J Law Med Ethics. 2014;42 Suppl 1:5-8. doi: 10.1111/jlme.12158.

Summary: In 1996, President Clinton offered a promissory vision for human genetics when he said: "I think it won't be too many years before parents will be able to go home from the hospital with their newborn babies with a genetic map in their hands that will tell them, here's what your child's future will likely be like."

Full text available from Journal of Law, Medicine & Ethics (USD 6.00-38.00) PMID: 25298287

B) Finding Fault? Exploring Legal Duties to Return Incidental Findings in Genomic Research Pike ER, Rothenberg KH,Berkman BE. Finding Fault? Exploring Legal Duties to Return Incidental Findings in Genomic Research. Georgetown Law J. 2014;102:795-843.

liability in circumstances in which notification could have potentially prevented injury. Although it is clear that ethical guidance should not be automatically codified as law and that crafting ethical obligations around legal duties can be inappropriate, the ethical debate should not proceed unaware of the potential legal ramifications of advancing and implementing an ethical obligation to return IFs. This Article assesses the legal claims that could be brought for a researcher's failure to return IFs. The potential for researchers to be held liable in tort is still uncertain and turns largely on a number of factors-including customary practice and guidance documents-that are still in flux. Unlike medical care, which has a well-defined duty into which evolving scientific knowledge about genetics and genomics can readily be incorporated, a researcher's duty to return IFs is less well defined, making it difficult to determine at the outset whether and when legal liability will attach. This Article advocates for a clearer, ethically sound standard of requiring that researchers disclose in the informed consent document which approach to offering IFs will be taken. This approach enables participants to know at the outset which findings, if any, will be returned, allows researchers to ascertain when their failure to appropriately return incidental findings will give rise to liability, and enables courts to make determinations that will produce more consistent legal guidance.

Free full text available from PubMed PMID: 25346543 NOTE: Also cited in Section 3.9.2

C) Finding Risks, Not Answers, in Gene Tests Grady D, Pollack A. Finding Risks, Not Answers, in Gene Tests. The New York Times. Sept. 22, 2014.

Summary: Jennifer was 39 and perfectly healthy, but her grandmother had died young from breast cancer, so she decided to be tested for mutations in two genes known to increase risk for the disease. When a genetic counselor offered additional tests for 20 other genes linked to various cancers, Jennifer said yes. The more information, the better, she thought.

Free full text available from The New York Times

D) The First Healthy Baby to Have His Genome Sequenced Before Birth; But Is It Ethical? Rivas A. The first healthy baby to have his genome sequenced before birth; but is it ethical? Medical Daily. July 17, 2014.

Summary: Imagine growing up and knowing exactly what diseases you were genetically predisposed to. If it was Alzheimer’s, would you take all possible measures to maintain your mental health? What if you were never actually predisposed to the disease and your parents had just interpreted your genome sequence wrong? These are questions many scientists are asking when it comes to sequencing the DNA of soon-to-be-born kids. And in part, they’re asking them because of people like Razib Khan, the father of the first healthy baby to be born with his entire genome sequenced before birth.

Free full text available from Medical Daily

E) Return of Results: Ethical and Legal Distinctions Between Research and Clinical Care Burke W, Evans BJ,Jarvik GP. Return of results: ethical and legal distinctions between research and clinical care. Am J Med Genet C Semin Med Genet. 2014; 166C(1): 105-111.

Summary: The return of individual results to research participants has been vigorously debated. Consensus statements indicate that researchers and bioethicists consider the return of research results most appropriate when the findings are clinically relevant. Even when clinical utility is the motivator, however, the return of individual research results is not equivalent to clinical care. There are important differences in the domains of research and medical care, both from a legal standpoint and in terms of the ethical responsibilities of clinicians and researchers. As a corollary, researchers risk promoting a therapeutic misconception if they create quasi-clinical settings for return of clinically relevant research results. Rather, efforts should be focused on clarity in the provision of research results, appropriate caveats and, most important, appropriate referrals when the results may be helpful to consider in medical care.

Free full text available from American Journal of Medical Genetics Part C: Seminars in Medical Genetics PMID: 24616381

F) Ethical and Legal Aspects of Noninvasive Prenatal Genetic Diagnosis Dickens BM. Ethical and legal aspects of noninvasive prenatal genetic diagnosis. Int J Gynaecol Obstet. 2014; 124(2): 181-184.

testing by amniocentesis or chorionic villus sampling, which risks causing spontaneous abortion. Ethical and legal issues of cffDNA testing will become more acute if testing expands to fetal whole-genome sequencing. Critical issues include the state of the science or diagnostic art; the appropriateness of offering the test; the implications of denying the test when it is available and appropriate; disclosure and counseling following test results; and management of patients' choices on acquiring test results. A challenge will be providing patients with appropriate counseling based on up-to-date genetic knowledge, and accommodating informed patients' legal choices.

Full text available from International Journal of Gynecology & Obstetrics (USD 31.50) PMID: 24299974 NOTE: Also cited in Section 3.10.1

G) Prenatal Whole-Genome Sequencing—Is the Quest to Know a Fetus's Future Ethical? Yurkiewicz IR, Korf BR, Lehmann LS. Prenatal whole-genome sequencing-- is the quest to know a fetus's future ethical? N Engl J Med. 2014; 370(3): 195-197.

Summary: Researchers have reported sequencing a fetal genome from cell-free fetal DNA in a pregnant woman's blood, heralding the possibility of performing whole-genome sequencing as early as the first trimester of pregnancy. This innovation raises ethical and policy questions.

Full text available from New England Journal of Medicine (subscription required) PMID: 24428465 NOTE: Also cited in Section 4.2.3

H) What to Consider Before Undergoing a DNA Test: A Columbia University Bioethicist Discusses Medical and Ethical Issues Raised by Testing Tergesen A. What to Consider Before Undergoing a DNA Test: A Columbia University bioethicist discusses medical and ethical issues raised by testing. The Wall Street Journal. Dec. 8, 2013.

Summary: As the price of sequencing a person's entire DNA has tumbled from $1 billion a decade ago to a few thousand dollars today, many adults— and their physicians—are turning to genetic tests to better understand and manage their health.

Free full text available from Wall Street Journal

I) Anticipate and Communicate: Ethical Management of Incidental and Secondary Findings in the Clinical, Research, and Direct-to-Consumer Contexts Presidential Commission for the Study of Bioethical Issues. Anticipate and Communicate: Ethical Management of Incidental and Secondary Findings in the Clinical, Research, and Direct-to-Consumer Contexts, 2013. Washington, DC: Presidential Commission for the Study of Bioethical Issues; 2013. Available at: http://bioethics.gov/sites/default/files/FINALAnticipateCommunicate_PCSBI_ 0.pdf

Summary: Anticipate and Communicate is the Bioethics Commission’s sixth major report. In this report the Bioethics Commission offers specific recommendations for the management of incidental and secondary findings in clinical, research and direct-to-consumer settings. Emerging medical technologies, changing cost structures, and evolving medical practice make the likelihood of discovering incidental and secondary findings across contexts a growing certainty. Such findings can be lifesaving, but also can lead to uncertainty and distress if they are unexpected or identify conditions for which no effective treatment is available.

The Bioethics Commission recommends that all practitioners anticipate and plan for incidental findings and communicate that plan to patients, research participants, and consumers so they are informed ahead of time about what to expect. By anticipating and communicating, shared decision-making between practitioners and potential recipients can ensure the best way forward should incidental findings be found.

Full text available from Presidential Commission for the Study of Bioethical Issues website

J) Ethical and Legal Implications of Whole Genome and Whole Exome Sequencing in African Populations Wright GE, Koornhof PG, Adeyemo AA,Tiffin N. Ethical and legal implications of whole genome and whole exome sequencing in African populations. BMC Med Ethics. 2013; 14: 21.

Summary: BACKGROUND: Rapid advances in high throughput genomic technologies and next generation sequencing are making medical genomic research more readily accessible and affordable, including the sequencing of patient and control whole genomes and exomes in order to elucidate genetic

factors underlying disease. Over the next five years, the Human Heredity and Health in Africa (H3Africa) Initiative, funded by the Wellcome Trust (United Kingdom) and the National Institutes of Health (United States of America), will contribute greatly towards sequencing of numerous African samples for biomedical research. DISCUSSION: Funding agencies and journals often require submission of genomic data from research participants to databases that allow open or controlled data access for all investigators. Access to such genotype-phenotype and pedigree data, however, needs careful control in order to prevent identification of individuals or families. This is particularly the case in Africa, where many researchers and their patients are inexperienced in the ethical issues accompanying whole genome and exome research; and where an historical unidirectional flow of samples and data out of Africa has created a sense of exploitation and distrust. In the current study, we analysed the implications of the anticipated surge of next generation sequencing data in Africa and the subsequent data sharing concepts on the protection of privacy of research subjects. We performed a retrospective analysis of the informed consent process for the continent and the rest-of- the-world and examined relevant legislation, both current and proposed. We investigated the following issues: (i) informed consent, including guidelines for performing culturally-sensitive next generation sequencing research in Africa and availability of suitable informed consent documents; (ii) data security and subject privacy whilst practicing data sharing; (iii) conveying the implications of such concepts to research participants in resource limited settings. SUMMARY: We conclude that, in order to meet the unique requirements of performing next generation sequencing-related research in African populations, novel approaches to the informed consent process are required. This will help to avoid infringement of privacy of individual subjects as well as to ensure that informed consent adheres to acceptable data protection levels with regard to use and transfer of such information.

Free full text available from PubMed PMID: 23714101

K) Ethical, Legal, and Counseling Challenges Surrounding the Return of Genetic Results in Oncology Lolkema MP, Gadellaa-van Hooijdonk CG, Bredenoord AL, et al. Ethical, legal, and counseling challenges surrounding the return of genetic results in oncology. J Clin Oncol. 2013; 31(15): 1842-1848.

Summary: In the last decade, an overwhelming number of genetic aberrations have been discovered and linked to the development of treatment for cancer. With the rapid advancement of next-generation sequencing (NGS) techniques, it is expected that large-scale DNA analyses

will increasingly be used to select patients for treatment with specific anticancer agents. Personalizing cancer treatment has many advantages, but sequencing germline DNA as reference material for interpreting cancer genetics may have consequences that extend beyond providing cancer care for an individual patient. In sequencing germline DNA, mutations may be encountered that are associated with increased susceptibility not only to hereditary cancer syndromes but also to other diseases; in those cases, disclosing germline data could be clinically relevant and even lifesaving. In the context of personal autonomy, it is necessary to develop an ethical and legal framework for how to deal with identified hereditary disease susceptibilities and how to return the data to patients and their families. Because clear legislation is lacking, we need to establish guidelines on disclosure of genetic information and, in the process, we need to balance privacy issues with the potential advantages and drawbacks of sharing genetic data with patients and their relatives. Importantly, a strong partnership with patients is critical for understanding how to maximize the translation of genetic information for the benefit of patients with cancer. This review discusses the ethical, legal, and counseling issues surrounding disclosure of genetic information generated by NGS to patients with cancer and their relatives. We also provide a framework for returning these genetic results by proposing a design for a qualified disclosure policy.

Full text available from Journal of Clinical Oncology (subscription required) PMID: 23589552

L) Policy Statement: Ethical and Policy Issues in Genetic Testing and Screening of Children Committee on Bioethics, Committee on Genetics, American College of Medical Genetics, Genomics Social, Ethical, Legal Issues Committee. Policy statement: Ethical and policy issues in genetic testing and screening of children. Pediatrics. 2013; 131(3): 620-622.

Summary: The genetic testing and genetic screening of children are commonplace. Decisions about whether to offer genetic testing and screening should be driven by the best interest of the child. The growing literature on the psychosocial and clinical effects of such testing and screening can help inform best practices. This policy statement represents recommendations developed collaboratively by the American Academy of Pediatrics and the American College of Medical Genetics and Genomics with respect to many of the scenarios in which genetic testing and screening can occur.

Free full text available from Pediatrics

PMID: 23428972

The following sections highlight available CAP resources:

• CAP Guidelines (Section 8.1); • CAP Cancer Biomarker Reporting Templates (Section 8.2); • CAP Electronic Cancer Checklists (eCC) (Section 8.3); • CAP PT: Proficiency Testing (Section 8.4); and • CAP LAP: Accreditation (Section 8.5).

8.1 CAP Guidelines

The College of American Pathologists developed the Pathology and Laboratory Quality Center, or “the CAP Center,” as a forum to author and maintain evidence- based guidelines and consensus statements. For more information about the CAP Center and its process, please visit the CAP Guidelines website.

The CAP Center has several guidelines published and in development in the area of molecular testing. Please follow the links for more information about each of these Center guidelines and other related resources.

Published:  ASCO/CAP ER/PgR Guideline and Resources  ASCO/CAP HER2 Testing Guidelines and Resources (2013 Update)  Bone Marrow Synoptic Reporting for Hematologic Neoplasms  CAP/ADASP Interpretive Diagnostic Error Reduction  CAP/ASCCP Lower Anogenital Squamous Terminology (LAST) for HPV-Associated Lesions: Consensus Recommendations and Resources  CAP/IASLC/AMP Molecular Testing Guidelines for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors  CAP/NSH Uniform Labeling of Blocks and Slides in Surgical Pathology  Principles of Analytic Validation of Immunohistochemical Assays  Validating Whole Slide Imaging for Diagnostic Purposes in Pathology

Pending Approval (as of June 1, 2016):  CAP/ASH Algorithm for Initial Work-up of Acute Leukemia  HER2 Testing and Clinical Decision Making in Gastroesophageal Adenocarcinoma: Guideline from the CAP/ASCP/ASCO  Molecular Markers for the Evaluation of Colorectal Cancer: Guideline from the ASCP/CAP/AMP/ASCO

In Development:  Appropriate Collection and Handling of Thoracic Specimens for Laboratory Testing  ASCP/CAP/ASH Requirements for Laboratory Workup of Malignant Lymphoma Guideline  CAP/IASLC/AMP Update to Molecular Testing for Selection of Lung Cancer Patients (Updating 2013 publication)  Diffuse Glioma Testing  HPV Testing in Head and Neck Squamous Cell Carcinomas  PD-L1 Lung Tumors  Quantitative Image Analysis for Immunohistochemistry

For the latest status updates, please check the CAP Center Upcoming Guidelines page.

A) CAP/NSH Histotechnology Committee Guidelines for Pre-Microscopic Examination in Surgical Pathology CAP/NSH Histotechnology Committee. CAP/NSH Histotechnology Committee Guidelines for Pre-Microscopic Examination in Surgical Pathology. 2013.

Summary: In spite of the abundant guidelines and recommendations published for specimen handling and testing in a clinical pathology laboratory, relatively little literature is available for guidance of specimen handling in a surgical pathology laboratory. This document does not relate to cytologic or clinical pathology samples.

The comprehensive table (see below) is intended to serve as a general guideline for proper specimen handling from the time it is taken from the patient to the time a completed slide of the specimen is given to a pathologist for interpretation. This document was created by members of the CAP/NSH Histotechnology Committee and is intended to serve as a guideline and NOT absolute recommendations for specimen handling. Each laboratory is advised to use these guidelines as a starting point and modify certain parameters to fit local institutional needs, as appropriate. Whenever

appropriate, regulatory references for certain guidelines are provided in the table.

It is the intent of the CAP/NSH Histotechnology Committee to update this document every 2 years and have the updated version of the document available on the College of American Pathologists (CAP) and National Society for Histotechnology (NSH) websites.

Access full table

B) Is Molecular AP Testing in Sync With Guidelines? Paxton A. Is molecular AP testing in sync with guidelines? CAP TODAY. 2014 November.

Summary: Molecular testing, on a steep growth curve in anatomic pathology, is increasingly providing decisive guidance for treatment of cancer patients. But while guidelines on clinical relevance and performance of common molecular tests are available and widely used in theory, to date there has been limited information on how well requests for molecular testing in AP laboratories are adhering to the guidelines. The CAP Quality Practices Committee hopes to fill part of that gap with its “Molecular Testing in Anatomic Pathology” Q-Probes study. The authors believe it is the first multi-institutional study assessing the effectiveness of anatomic pathology molecular testing by examining adherence to guidelines of the National Comprehensive Cancer Network, or NCCN. “Recently there has been a significant upsurge in molecular testing. It seems like every time you turn around there is a new test,” says study coauthor Michael Idowu, MD, director of the molecular genetic pathology fellowship and anatomic pathology quality management at the Virginia Commonwealth University Health System. “Appropriate test utilization and test performance are important for usable test results. We decided on this study because of increasing use of molecular testing and its impact on therapy.” A study of 26 laboratories and 2,230 test requests, this Q‑Probes looked at three criteria for evaluating molecular testing quality in AP: the appropriateness of requests for molecular testing, the adequacy of test material, and turnaround time.

Free full text available from CAP TODAY

8.2 CAP Cancer Biomarker Reporting Templates

In an effort to improve consistency and completeness in reporting results of cancer biomarker testing, the College of American Pathologists (CAP) has produced standardized templates for the reporting of cancer biomarker testing. These templates were developed to respond to pathologist user feedback about timing of reporting and structural consistency of ancillary studies data elements in the CAP Cancer Protocols and to provide educational materials to help pathologists better understand emerging biomarkers. The development of these templates has been a collaborative effort between CAP, AMP, ASCO, CDC, AJCC and many other participating organizations.

A) Introducing New College of American Pathologists Reporting Templates for Cancer Biomarkers Fitzgibbons PL, Lazar AJ, Spencer S. Introducing New College of American Pathologists Reporting Templates for Cancer Biomarkers. Arch Pathol Lab Med. 2014;138(2):157-8.

No summary available

Free full text available from the CAP’s Archives PMID: 23808404

Access: All current CAP Cancer Biomarker Templates are publicly available in Microsoft Word and PDF format. They can be found on the www.cap.org website by navigating to the Cancer Reporting Tools link within the Resources and Publications tab, or directly at www.cap.org/cancerprotocols

Published as of May 2015:  Template for Reporting Results of Biomarker Testing of Specimens From Patients With Carcinoma of the Breast  Template for Reporting Results of Biomarker Testing of Specimens From Patients With Carcinoma of the Colon and Rectum  Template for Reporting Results of HER2 (ERBB2) Biomarker Testing of Specimens from Patients with Adenocarcinoma of the Stomach or Esophagogastric Junction

Biomarkers Template Breast Breast Biomarker Reporting CNS Central Nervous System Biomarker Reporting Endocrine Thyroid Biomarker Reporting Gastrointestinal Colon and Rectum Biomarker Reporting GIST Biomarker Reporting Gastric HER2 Biomarker Reporting Gynecologic Endometrium Biomarker Reporting Hematologic Chronic Lymphocytic Leukemia Biomarker Reporting Chronic Myelogenous Leukemia Biomarker Reporting Diffuse Large B-Cell Lymphoma Biomarker Reporting Myeloproliferative Neoplasms Biomarker Reporting Skin Melanoma Biomarker Reporting Thorax Lung Biomarker Reporting

In Development:  Template for Reporting Results of Biomarker Testing of Specimens From Patients With Tumors of the Head and Neck

8.3 CAP Electronic Cancer Checklists (eCC)

For pathologists that would like to implement the CAP Cancer Biomarker Templates into their daily workflow within their laboratory information systems, the CAP offers an electronic version of these data entry forms through the CAP electronic Cancer Checklists (eCC).

Summary The amount of information pathologists provide in their reports on cancer specimens has increased in recent years, due to the expansion of scientific knowledge about cancer and continued advances in health care, such as molecular diagnostics and personalized medicine. The CAP eCC, the electronic version of the CAP Cancer Protocols and Cancer Biomarker Templates, helps pathologists and other health care professionals manage information by offering a standardized way to report cancer data electronically.

The CAP eCC advances the management and interoperability of health information through its XML (Extensible Markup Language) format that can be integrated into existing pathology laboratory information and cancer registry systems, and also supports ever-evolving health IT platforms. The eCC is partially coded with SNOMED Clinical Terms® (SNOMED CT®) and ICD-O3,

enabling multiple interdisciplinary providers to accurately communicate and share patient information within an electronic health record (EHR) system. Data can also be transmitted in real time, improving the timeliness, accuracy, and completeness of cancer reporting.

In addition, the CAP eCC focuses directly on real-world medical content standards from physician experts on the CAP’s Cancer, Cancer Biomarker, and Pathology Electronic Reporting (PERT) Committees, illustrating how technology directly supports meaningful use of patient data in the cancer care setting.

Features •Produced in interoperable XML format, which is broadly adopted by health information technology (HIT) and endorsed by international standards organizations such as HL7, IHE, and the IHTSDO • Content includes elements from the latest edition of the AJCC Cancer Staging Manual • Regularly scheduled content releases and updates • Provides maps encoded with SNOMED CT and ICDO3 • Integration into existing pathology and cancer registry systems • Individual laboratory practice customization available

Content Update The CAP eCC releases contain elements from the AJCC Cancer Staging Manual. The CAP Cancer and Biomarker Reporting Committees update the content found within the CAP Cancer Protocols and Biomarker Templates online at www.cap.org/cancerprotocols. The CAP eCC releases are distributed to licensees with the addition of new or updated content, technical features, or mappings.

Licensing The CAP eCC files are downloadable with a CAP eCC license. Vendor integration licensing options are also available. For more information, visit our website at www.cap.org/capecc, or contact us at 847-832-7700 or [email protected].

Goals • Support and aid pathologists in the diagnostic process • Standardize the collection of pathology data to improve cancer reporting, patient care, and research initiatives • Advance the pathologist’s role as chief diagnostician • Aid pathologists and laboratories in ensuring report completeness, and in meeting accreditation compliance standards

• Improve information sharing, and support interoperability and data exchange to foster a more efficient cancer reporting process • Promote broad adoption and utilization of structured pathology reporting

Usability • Clinical practice • Tumor Board • Data mining • Data analysis • Epidemiological reporting • Public health • Quality improvement • Cancer research • Cancer surveillance • Reimbursement • Tissue banking

Content Updates The CAP eCC releases contain elements from the most up to date edition of the AJCC Cancer Staging Manual, as well as periodic WHO Blue Book updates. The CAP Cancer Committee updates the content found within the CAP Cancer Protocols online at www.cap.org/cancerprotocols.

The CAP eCC releases are distributed to licensees with the addition of new or updated content, technical features, or mappings. Please visit our webpage for the most up to date information at www.cap.org/capecc.

For more information or for a demonstration of the CAP eCC, please contact us at 847-832-7700 or [email protected].

A) Upcoming CAP Cancer Protocol & electronic Cancer Checklist (eCC) Release Updates: What’s New in 2016? Baker T, Foulis P. Upcoming CAP Cancer Protocol & electronic Cancer Checklist (eCC) Release Updates: What’s New in 2016? [webinar]. Hot Topics in Pathology Series. Presented on August 20, 2015. Available at http://www.cap.org/web/submenu/calendar/webinars/previous- webinars?_afrLoop=217145469478120#%40%3F_afrLoop%3D2171454694 78120%26_adf.ctrl-state%3Duya30j6kr_38. Accessed August 21, 2015.

Summary: The CAP Cancer Protocols are considered the gold standard for cancer pathology reporting in the US and Canada, and are used worldwide as a reference for best practices in standardized reporting as well as for

supporting CAP Laboratory Accreditation Program and American College of Surgeons - Commission on Cancer synoptic reporting requirements. The electronic versions of these protocols, the CAP electronic Cancer Checklists (eCC), can be integrated into pathologist AP-LIS workflow to allow for the convenience of automated updates of Cancer Protocol content when new material is released. A Major Release affecting most of the CAP Cancer Protocols and eCCs will be available on http://www.cap.org/cancerprotocols and www.cap.org/capecc in Q1 of 2016.

This webinar will review the planned 2016 Cancer Protocol and eCC revisions, discuss schedules and timelines for future content updates and releases, and give insight into the development process of the AJCC 8th edition. In addition, Cancer and Pathology Electronic Reporting (PERT) Committee staff will provide a brief introduction to the CAP eCC, how it incorporates Cancer Protocol content, and its value to the pathologist and health systems.

Archived presentation available NOTE: Also cited in Section 11.2

8.4 CAP PT: Proficiency Testing

The College has many Surveys for molecular proficiency testing. Survey products covering cytogenetics, infectious disease, molecular oncology (solid tumors and hematologic), and biochemical and molecular genetics can be found in the 2016 Surveys catalog.

Sample Exchange Registry for Alternative Assessment The Sample Exchange Registry is an Internet-based service designed to connect laboratories performing testing where no formal proficiency testing (PT) is available. This service now includes all clinical laboratory disciplines. Laboratories can participate in the registry service at any time. When at least three laboratories are identified as testing for the same analyte, the CAP will facilitate the sample exchange. Visit www.cap.org/sampleexchange for additional information and to register for an exchange.

For more information on the Sample Exchange Registry, please contact: Ross Owen, Technical Specialist, Surveys College of American Pathologists 325 Waukegan Road Northfield, IL 60093

1-800-323-4040 x 7584 [email protected]

A) Current Landscape and New Paradigms of Proficiency Testing and External Quality Assessment for Molecular Genetics Kalman LV, Lubin IM, Barker S, et al. Current landscape and new paradigms of proficiency testing and external quality assessment for molecular genetics. Arch Pathol Lab Med. 2013; 137(7): 983-988.

Summary: CONTEXT: Participation in proficiency testing (PT) or external quality assessment (EQA) programs allows the assessment and comparison of test performance among different clinical laboratories and technologies. In addition to the approximately 2300 tests for individual genetic disorders, recent advances in technology have enabled the development of clinical tests that quickly and economically analyze the entire human genome. New PT/EQA approaches are needed to ensure the continued quality of these complex tests. OBJECTIVES: To review the availability and scope of PT/EQA for molecular genetic testing for inherited conditions in Europe, Australasia, and the United States; to evaluate the successes and demonstrated value of available PT/EQA programs; and to examine the challenges to the provision of comprehensive PT/EQA posed by new laboratory practices and methodologies. DATA SOURCES: The available literature on this topic was reviewed and supplemented with personal experiences of several PT/EQA providers. CONCLUSIONS: Proficiency testing/EQA schemes are available for common genetic disorders tested in many clinical laboratories but are not available for most genetic tests offered by only one or a few laboratories. Provision of broad, method-based PT schemes, such as DNA sequencing, would allow assessment of many tests for which formal PT is not currently available. Participation in PT/EQA improves the quality of testing by identifying inaccuracies that laboratories can trace to errors in their testing processes. Areas of research and development to ensure that PT/EQA programs can meet the needs of new and evolving genetic tests and technologies are identified and discussed.

Free full text available from the CAP’s Archives PMID: 23808472

8.4.1 NGS Proficiency Testing

In line with CAP’s mission to foster the highest standards in the practice of pathology, to advance the science of pathology, and to improve medical

laboratory service, CAP’s Next Generation Sequencing (NGS) Project Team collaborated to enhance the CAP’s Accreditation Programs Checklists for NGS and developed a methods-based proficiency test entitled Next-Generation Sequencing (program code NGS), demonstrating CAP’s leadership in advancing standards of practice in genomic medicine.

 Designed for laboratories performing both the wet bench and bioinformatics components  For laboratories using gene panels, exome, and whole genome sequencing for germline variants  Customers will receive 10.0-µg extracted DNA specimen twice per year  Customers will be able to test up to 200 chromosomal positions in each sample

If customers are interested in further information on this program, please refer to the 2016 Surveys catalog. See listing below.

Next-Generation Sequencing – Solid Tumor Survey (NGSST) This is a methods-based proficiency challenge for laboratories performing targeted next-generation sequencing of cancer genes or mutation hotspots in solid tumors. Laboratories will be asked to identify somatic single nucleotide variants and small insertions or deletions in the following genes: AKT1, ALK, APC, ATM, BRAF, CDH1, CTNNB1, EGFR, ERBB2, FBXW7, FGFR2, GNAQ, GNAS, HRAS, IDH1, KIT, KRAS, MET, NRAS, PDGFRA, PIK3CA, PTEN, SMAD4, SMARCB1, SMO, SRC, STK11, TP53.

Next-Generation Sequencing – Hematologic Malignancies Survey (NGSHM) This is a methods-based proficiency challenge for laboratories performing targeted next-generation sequencing of genes or mutation hotspots in hematologic malignancies. Laboratories will be asked to identify somatic single nucleotide variants and small insertions or deletions in the following genes: ASXL1, ATM, BRAF, CALR, CEBPA, CREBBP, CSF3R, DNMT3A, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KMT2D, MPL, MYD88, NOTCH1, NPM1, SF3B1, SRSF2, TET2, TP53, U2AF1.

Next-Generation Sequencing Bioinformatics Survey (NGSB) This is a somatic gene-based sequencing challenge in which files (BAM and/or FASTQ) are downloaded into your laboratory bioinformatics pipeline for analysis. This Survey is specific for the Illumina TruSeq Amplicon Cancer Panel and the Ion Torrent AmpliSeq Cancer Hotspot v2 Panel. This in silico program augments wet bench NGS proficiency testing programs by testing a greater number of variants, at a greater range of variant frequencies.

8.5 CAP LAP: Accreditation

The CAP Laboratory Accreditation Program is an internationally recognized program and the only one of its kind that utilizes teams of practicing laboratory professionals as inspectors. Designed to go well beyond regulatory compliance, the program helps laboratories achieve the highest standards of excellence to positively impact patient care. The program is based on rigorous accreditation standards that are translated into detailed and focused checklist requirements. The checklists, which provide a quality practice blueprint for laboratories to follow, are used by the inspection teams as a guide to assess the overall management and operation of the laboratory.

The Molecular Pathology Checklist (description below) is used, along with the All Common and Laboratory General Checklists, for inspection of laboratories performing testing using molecular methods. The checklists are available for download from eLAB Solutions or may be purchased as a set on the CAP website.

CAP continues to be actively engaged in updating the Molecular Pathology Checklist to reflect new technologies and applications. In the 2012 edition, the CAP introduced requirements on the clinical application of genomic analysis, more specifically next-generation sequencing (NGS), to prepare its laboratory customers for advancements in genomic testing. The 2013 edition was further expanded to contain new requirements for non-invasive screening of maternal plasma to identify fetal aneuploidy using next-generation sequencing.

Molecular Pathology Checklist description: The Molecular Pathology Checklist covers clinical molecular genetic testing in the areas of oncology, hematology, inherited disease, HLA typing, forensics and parentage applications. The Molecular Pathology Checklist is used to inspect a variety of methodologies, including polymerase chain reaction, arrays, fluorescence and non-florescence in situ hybridization, electrophoresis, Sanger sequencing, and next-generation sequencing.

The inspection of laboratories performing such molecular testing requires the Molecular Pathology Checklist, with the following exceptions:  The Cytogenetics or Anatomic Pathology Checklist (as appropriate) may be used to inspect fluorescence in situ hybridization (FISH), when such testing is performed in the cytogenetics, cytopathology or anatomic pathology section.  The Anatomic Pathology Checklist may be used to inspect in situ hybridization (ISH), when ISH testing is performed in the anatomic pathology or cytopathology section.

 The Microbiology Checklist is used to inspect laboratories performing molecular testing for infectious diseases, including FDA- cleared/approved, modified FDA-cleared/approved, and laboratory- developed methods.  The Histocompatibility Checklist is used to inspect HLA antigen typing performed using molecular methods for the purposes of transplantation.

Questions about the Molecular Pathology Checklist? Gain insight from the experience of an inspector that performs numerous molecular laboratory inspections each year by listening to the 2014 CAP audio conference “Complying with Molecular Pathology Accreditation Requirements.” The discussion will focus on those standards from the Molecular Pathology Checklist that challenge laboratories.

The following sections highlight CAP educational resources:

• CAP Short Presentations on Emerging Concepts (SPECs) (Section 9.1); • CAP Webinars (Section 9.2); • CAP Learning (Section 9.3); and • Molecular Oncology Tumor Board Series (Section 9.4).

9.1 CAP Short Presentations on Emerging Concepts (SPECs)

Pathology Short Presentations in Emerging Concepts (SPECs) are: • PowerPoint presentations on selected diseases where molecular tests play a key role in patient management. • Focused on molecular tests that are actionable to patient care today.

Pathologists will find the SPECs an especially valuable resource as they facilitate discussion with tumor boards or other physician colleagues.

View all the available SPECs by going to the Short Presentations on Emerging Concepts page under Resources and Publications tab on CAP website. A printed compilation of all the current SPECs with speaker notes and selected references can be purchased with preferred pricing for members by going to this webpage.

Anatomic Pathology:  Molecular Genetics of Renal Tumors (NEW)  Liquid Biopsy: Circulating Tumor in cfDNA (NEW)  PD-L1: Immune Check Point Blockade in Cancer (NEW)  Molecular Testing for Inherited and Sporadic Colorectal Cancer  Next-Generation Sequencing and Cancer Genomics  Diagnosis and Workup of Thyroid Cancer  HER2 Testing in Gastric/GEJ Adenocarcinoma  HER2 Testing in Breast Cancer  Molecular Testing in Breast Cancer

 Therapeutic Guidance for Metastatic Melanoma  Molecular Testing in the Workup of Polycythemia and Thrombocythemia

Clinical Pathology:  Diagnosis of Respiratory Viruses  Prenatal Screening for Down Syndrome: Past, Present, and Emerging Practices

Watch a SPEC presentation

9.2 CAP Webinars

CAP has webinars focused on informing pathologists on key genomic and molecular medicine topics. Since 2009, the "Hot Topics in Pathology" webinar series presents webinars on 1) oncology molecular topics 2) getting started in molecular/next steps in molecular topics and 3) next-generation sequencing topics. These webinars are complimentary and do not have CME.

To view new or archived webinars, go to www.cap.org > Calendar > webinars

9.2.1 Hot Topics in Pathology Webinars in 2016

Date Topic Speaker(s)

October 19, 2016 Microbiome Applications in Lynn Bry, MD, 11-12 pm Central Pathology PhD, FCAP

Nov 3, 2016 PD-L1: Immune Check Point Kenneth J. Bloom, 11-12 pm Central Blockade in Cancer MD, FCAP

Dec 14, 2016 Specimen Standards for Precision Carolyn Compton, 11-12 pm Central Medicine: It’s About Time! MD, PhD, FCAP

9.2.2 Archived Webinars

Topic Speaker(s)

Molecular Tests and Breast Cancer. Presented David G. Hicks, MD, June 22, 2016. Archived webinar available for free; FCAP presentation slides available

Renal Cell Carcinoma. Presented May 25, 2016. Michelle S. Hirsch, MD, Archived webinar available for free; presentation PhD, FCAP slides available Next Generation Sequencing (NGS SPEC). Pranil Kumar Chandra, Presented Apr 27, 2016. Archived webinar available DO, FCAP for free; presentation slides available Immune Check Point Blockade in Cancer. Kenneth J. Bloom, MD, Presented Dec 15, 2015. Archived webinar FCAP available for free; presentation slides available

HER2 Testing in Gastric and Gastroesophageal Christa L. Whitney- Junction Cancers: A New Therapeutic Target Miller, MD, FCAP & and Diagnostic Challenge. Presented Oct 28, David G. Hicks, MD, 2015. Archived webinar available for free; FCAP presentation slides available

Upcoming CAP Cancer Protocol & Electronic Cancer Checklist (eCC) Release Updates: Thomas Baker, MD, What’s New in 2016? Presented Aug 20, 2015. FCAP & Phil Foulis, MD, Archived webinar available for free; presentation FCAP slides available

NGS for Diagnosis in Challenging Cases. Lynette M. Sholl, MD, Presented June 25, 2015. Archived webinar FCAP available for free; presentation slides available

NGS for Inherited Disorders: Targeted Sequencing for Constitutional/Congenital John D. Pfeifer, MD, Disease Analysis. Presented Apr 22, 2015. PhD, FCAP & Catherine Archived webinar available for free; presentation E. Cottrell, PhD, FACMG slides available Hereditary Cancer Next-Generation Sequencing Panels in Clinical Practice. Presented Feb 25, Colin Pritchard, MD, 2015. Archived webinar available for free; PhD, FCAP presentation slides available

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9.3 CAP Learning

CAP’s online learning site has online resources for education by the experts.

CAP SAMs (Self-Assessment Modules) Consistent with the high quality you expect from the College, CAP SAMs are more comprehensive than simple “memory tests.” CAP SAM topics are identified as critical areas of importance to pathologists, and they include a strong focus on both the interpretation of concepts and their direct application to patient care.

CAP SAMs include both educational content and a multiple-choice post-test. Immediate feedback is provided after each question, providing additional educational material on both correct and incorrect responses. Participants who earn a passing score may apply their earned credit(s) to the American Board of Pathology’s Maintenance of Certification SAM requirements.

Title/Description CME/SAM

The Critical First Steps: Specimen Acquisition and Handling for Cancer Genomics This activity addresses specimen acquisition and handling for 1.25 cancer genomic testing. It will cover important considerations to communicate with the clinical team to ensure adequate specimens are presented for pathology review. Cancer Genomics: Pathologists Adding Value as a Molecular Consultant This activity addresses genomic test selection and interpretation, as well as how to educate and guide others as a 1.25 molecular consultant. In addition, the activity prepares you to contribute and apply cancer genomic information in new ways to add value to the healthcare system.

Archives Applied The Archives Applied CME/SAM program is designed for pathologists and includes educational content and a multiple-choice post-test based on select articles from the Archives of Pathology & Laboratory Medicine journal. Immediate feedback is provided after each question, and a post-test score is provided upon completion. Participants who earn a passing score on the post-test may apply their earned credit to the American Board of Pathology’s (ABP) Maintenance of Certification (MOC) SAM requirements. All participants who complete the activity can claim CME credit.

Title/Description CME/SAM

Major Molecular Biomarkers in Gastrointestinal Tract, Lung, and Breast Tumors The number of biomarkers important to the diagnosis, treatment, and prognosis of patients is rapidly increasing; and cancer biomarker testing puts the pathologist at the forefront of patient selection for specific targeted therapies. To provide 3.0 direction and address practical issues in cancer biomarker testing for pathologists and other physicians involved in everyday care of cancer patients, the Department of Pathology and Genomic Medicine at Houston Methodist Hospital in Texas hosted the Cancer Biomarker Conference (CBC) in March 2014.

HER2/neu Expression in Gastroesophageal Adenocarcinoma This activity is based on an article that appeared in the June 2012 issue of Archives of Pathology & Laboratory Medicine 1.0 about the quantitation of HER2/neu expression in primary gastroesophageal adenocarcinomas using conventional light microscopy and quantitative image analysis. It was developed by an expert in the field, Lizette Vila Duckworth.

Molecular Pathology eLearning Developed by members of the CAP Molecular Oncology committee, these activities have a unique course design:  Models critical molecular oncology competencies – knowledge, skills and behaviors – and reinforces key messages through knowledge checks, discussions and self-reflection.  Desired performance behaviors are modeled by characters in the story, which demonstrate a highly diversified view of patient/pathologist interactions with respect to molecular oncology.

Title/Description CME/SAM Classification and Clinical Management of Myeloproliferative Neoplasms with Eosinophilia (SAM eligible) This course uses a case-based approach to illustrate the 1.25 utilization of the updated classification scheme, including new terminology, relevant clinical findings, peripheral blood smear morphology, bone marrow aspirate smears and histology, and laboratory/molecular testing.

Return to Table of Contents Section 9

HER2 FISH Test Interpretation in Breast Cancer (SAM eligible) 2.5 This SAM is designed for pathologists who interpret HER2 FISH tests in breast cancer cases.

ER IHC Test Interpretation in Breast Cancer (SAM eligible) This activity is intended for pathologists who interpret ER IHC 2.5 tests in breast cancer cases.

HER2 IHC Test Interpretation in Breast Cancer (SAM eligible) 2.5 This activity is intended for pathologists who interpret HER2 IHC tests in breast cancer cases.

HER2 Bright-field Test Interpretation in Breast Cancer This activity addresses HER2 test interpretation and reporting in breast cancer using Bright-field in situ hybridization (BRISH). The content is based on the ASCO/CAP HER2 Guideline recommendations. Five interactive case studies representing a 2.5 range of issues are used to increase knowledge and skill in both test interpretation and reporting. Participants have access to a series of short tutorials and printable take-aways to help solve the case studies and deepen their understanding of HER2 BRISH testing.

2016 CPIP-D Case 4: Molecular Pathology – Mutational Profiling by Next Generation Sequencing (NGS) in Lung Cancer This activity features a challenging Molecular Pathology (lung 1.25 cancer) case. You will be presented with information including an introduction, case presentation, laboratory findings, discussions, and a summary. The case includes a series of thought-provoking questions related to the case.

Reporting for Neoplastic Pathology Biomarkers This activity will provide an overview of best practices for reporting lung biomarker results including updated guidelines 1.5 and templates, as well as practical tips for handling issues in reporting lung biomarker results and how to integrate biomarker results into the complete report or medical record.

Non-Small Cell Lung Cancer (NSCLC) and Molecular Testing Guidelines 2.5 This activity focuses on updated molecular testing guidelines and their application in small biopsy and cytology specimens.

CAP Advanced Practical Pathology Programs CAP Advanced Practical Pathology Programs (AP3) offer pathologists the opportunity to develop, demonstrate and be recognized for knowledge and skills in areas not currently addressed by the American Board of Pathology (ABP).

To receive a CAP AP3 Certificate of Recognition, applicants need to meet four key requirements:  Board Certification in anatomic and/or clinical pathology from the ABP, the American Osteopathic Board of Pathology (AOBP), or the Royal College of Physicians and Surgeons of Canada (RCPS).  Completion of Continuing Medical Education in the program’s area of special expertise. This will vary with each AP3.  Successful completion of a cognitive assessment that objectively measures practical knowledge in the program area of expertise.  Successful completion of one or more practical assessments that measure performance in the program area.

Course Code Title/Description Multidisciplinary Breast Pathology Advanced Practical Pathology Program The MBP AP3 provides pathologists with the focused MBP AP3 training, skills and education to effectively deliver their critical expertise across increasingly integrated, multidisciplinary breast cancer care teams.

9.4 Molecular Oncology Tumor Board Series

The Molecular Oncology Tumor Board series is a monthly user-driven online resource designed to help cancer care providers with the interpretation and understanding of tumor molecular profiling tests and studies. The free series is an educational collaboration between the College of American Pathologists (CAP), the American Society of Clinical Oncology’s (ASCO) ASCO University, and the Association for Molecular Pathology (AMP), and can be accessed at ASCOconnection.org. Participants who complete and pass a short post-test are eligible to earn CME credit.

During each tumor board, participants interact with expert pathologists and medical oncologists to discuss actionable aberrations and treatment options and to share their experiences and approaches with other cancer care professionals. Faculty and participant questions, comments, and responses to cases are posted through the online threaded discussion.

Series topics have included colorectal cancer (KRAS testing and BRAF mutation), lung cancer (ROS1), breast cancer (PIK3CA), chronic lymphocytic leukemia (TP53), and ovarian cancer (BRCA1).

The series can also be accessed at http://university.asco.org/motb.

Feedback on this content is welcome, including suggestions for articles, webinars, or other resources. Please send comments, suggestions, and questions to [email protected].

Cushman-Vokoun AM, Anderson MW, Bismar T, Chandra PK, Coleman JF, Gandour-Edwards R, Gruver AM, Kaufman JH, Kiechle FL, Laser JS, Moore F, Moyer AM, Olsen RJ. CAP Pathology Resource Guide: Precision Medicine. Version 2.0(1). Northfield, IL: College of American Pathologists; 2016.

Precision Medicine Resource Guide Contributors

Contributors/Editors: Allison M Cushman-Vokoun, MD, PhD, FCAP – The University of Nebraska Medical Center Matthew W Anderson, MD, PhD, FCAP – BloodCenter of Wisconsin

CAP Staff Contributors: Jill Kaufman, PhD – Staff Project Lead

CAP Program Content Contributors: Fay Shamanski, PhD Jaimie Gestes Halley, MT(ASCP) Patricia Vasalos Samantha Spencer, MD

Doug Murphy Sophia Dimoulis Theresa Ironside

Graphics support: Jill Payne Document editor: Kelly Westfall Pathology Resource Guide Advisor: Jill Kaufman, PhD

The CAP Pathology Resource Guide: Precision Medicine was updated by CAP’s Personalized Health Care Committee.

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