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American Molecular Society for Diagnostics Clinical Sheryl Alberta – No Disclosures 101 Laboratory April 10, 2019 Science - Sheryl Alberta, MT(ASCP)MB Michigan Spectrum Health Advanced Technology Laboratories 2019 Spring Meeting

Introduction – What is Methods: Molecular testing? 1. IHC - Immunohistochemistry -Techniques used to analyze an 2. HLA - Human Leukocyte Antigen Proteomics individual’s genetic code (genome) - Also known as histocompatibility and how their cells express their 3. Flow Cytometry genes as proteins (proteome) ______1. Cytogenetics -Personalized Medicine: when these 2. Molecular Diagnostics Genomics techniques are applied to medical testing

Why do Molecular Testing? A real life example case: 1. Diagnostic: What is the disease?

2. Prognostic: What is the predicted -Acute Promyelocytic Leukemia outcome of the disease?

3. Therapeutic: What are the potential treatments for the disease?

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A real life example case: Goal of Molecular Testing: -Flow Cytometry – initial diagnosis based on WBC markers -To provide a more complete -Cytogenetics – confirmatory testing for picture of a patient’s disease in PML/RARA translocation order to make better patient care decisions -Molecular – follow-up testing to follow treatment and to monitor MRD (minimal residual disease)

Molecular Biology is a relatively new field: Review -1980’s – started the use of applying knowledge and techniques to clinical applications of the -1990’s – a distinct field of molecular and genomic laboratory medicine emerged -1995 – formation of the Association for Basics Molecular Pathology (AMP)

23 pairs of chromosomes

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≈ 25,000 genes

3 billion base pairs in the human genome (2 meters long)

Genome = an individual’s complete set of DNA Adenine = Thymine Gene = basic functional unit of DNA Cytosine = Guanine -Inherited -Contains instructions to make proteins -Few hundred to 2 million+ base pairs -Contain exons and introns

Exons = coding sections of DNA and its corresponding RNA transcript that are translated into a protein

Introns = Introns are noncoding sections of DNA or its corresponding RNA transcript that are spliced out before the RNA molecule is translated into a protein

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Analogy of a Library Codon = A sequence of 3 nucleotides that corresponds with a specific amino acid or Genome = All of the books stop signal Chromosome = Different sections -The full set of codons (64) is called the Gene = Individual books genetic code Exons = The text of the book; the story -The genetic code is described as redundant Introns = What holds the book because a single amino acid may be coded together; non-story parts for by more than one codon

Amino Acids = Are the building blocks of proteins

-There are 20 amino acids

-The sequence of the amino acids of a specific protein is determined by the sequence of the bases in the gene that encodes that protein

-The chemical properties of the amino acids of proteins determine the biological activity of the protein

Function Description Example

Protein = Are large, complex molecules Antibody Antibodies bind to specific foreign particles, such Immunoglobulin G that play many critical roles in the body as viruses and bacteria, to help protect the body. (IgG) Enzyme Enzymes carry out almost all of the thousands of Phenylalanine -They do most of the work in cells and are chemical reactions that take place in cells. They hydroxylase also assist with the formation of new molecules by required for the structure, function, and reading the genetic information stored in DNA. regulation of the body’s tissues and organs Messenger Messenger proteins, such as some types of Growth hormones, transmit signals to coordinate biological Hormone processes between different cells, tissues, and -The sequence of amino acids determines each organs. protein’s unique 3-dimensional structure and Structural These proteins provide structure and support for Actin Component cells. On a larger scale, they also allow the body to its specific function move. Transport/ These proteins bind and carry atoms and small Ferritin Storage molecules within cells and throughout the body.

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Analogy of a Library

Nucleotides = Alphabet/Letters

Codons = Words/Syllables

Amino Acids = Translated Words/Syllables

Proteins = Translated books

Somatic = The cells of the body except Germline = is inherited in the lineage for the germ cells of germ cells, namely sperm and ovum -Somatic mutations are genetic alterations -When a mutation occurs within germ acquired by a cell that can be passed to cells, it is called a germline mutation the progeny of the mutated cell in the -Germline mutations affect every cell in course of cell division an organism and are passed on to -Somatic mutations can occur in any of offspring the cells of the body except the germ cells and therefore are not passed on to children

Methods: IHC – Immunohistochemistry: -Used by Pathologists to help determine 1. IHC - Immunohistochemistry cell origin and cell expression 2. HLA - Human Leukocyte Antigen - Also known as histocompatibility -Uses antibodies to test for certain 3. Flow Cytometry antigens (markers) in a sample of tissue 4. Cytogenetics 5. Molecular Diagnostics

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IHC – Immunohistochemistry: -The antibodies are usually linked to an enzyme or a fluorescent dye. When the antibodies bind to the antigen in the tissue sample, the enzyme or dye is activated, and the antigen can then be seen under a microscope -Example would be PDL-1 or PD-1 which, if positive, indicates the use of a targeted checkpoint inhibitor therapy.

HLA - Human Leukocyte Antigen: HLA - Human Leukocyte Antigen: -The HLA gene family provides instructions for making a group of related proteins known as the -The HLA complex consists of more than human leukocyte antigen (HLA) complex 200 genes located close together on chromosome 6 -The HLA complex helps the immune system distinguish the body's own proteins from proteins -Genes in this complex are categorized made by foreign invaders such as viruses and bacteria into three basic groups: class I, class II, and class III

-HLA typing is used to match patients Flow Cytometry: and donors for organ, bone marrow or cord blood transplants -Is a laser, or impedance-based, technology -Used for cell counting, cell sorting, and -The best transplant outcomes biomarker detection by suspending cells in a happen when donor and recipient stream of fluid and passing them single file HLA types closely match through an electronic detection apparatus

-A close HLA match will reduce the -Uses fluorescent labeled antibodies to detect incidence of GVHD specific antigens on the cell surface

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White Blood Cell Surface Markers

Flow Cytometry Testing:

CD19+ CD10+ -Tests can be performed on blood, bone marrow, lymph nodes and other tissues -Leukemia and lymphoma immunophenotyping for diagnosis

-Cell sorting for further testing of specific B-ALL identified by abnormal CD19+ and CD10+ populations cell types

Cytogenetics:

1. Karyotyping

2. FISH

3. Microarray

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Karyotyping: Karyotyping: -A picture of a person’s chromosomes -Attention is paid to their length, the position of the centromeres, banding pattern, any -The chromosomes are isolated, stained, differences between the sex chromosomes, and examined under the microscope and any other physical characteristics

-A picture of the chromosomes is cut up -For cytogenetic testing, cancer cells are and rearranged by the chromosome’s grown in culture for up to 2 weeks before size their chromosomes can be looked at under the microscope

Karyotyping: -Translocation - part of one chromosome has broken off and is now located on another chromosome -Inversion - part of a chromosome is upside down (now in reverse order) but still attached to the right chromosome -Deletion - indicates part of a chromosome has been lost -Duplication happens when part of a chromosome has been copied, and too many copies of it are found in the cell

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FISH Testing

Fluorescent in-situ hybridization: -Uses fluorescent probes that bind to specific DNA sequences on chromosomes

-Fluorescence microscopy is used to find out where the fluorescent probe is bound to the chromosomes

-Can be used on cells grown in culture as well as tissue slides from FFPE samples

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Microarray (DNA chip or Biochip): -Microscope slides that are printed with thousands of tiny spots in defined positions, with each spot containing a known DNA sequence or gene -The DNA molecules attached to each slide act as probes to detect gene expression -The tumor DNA is labeled with green dye and the control, (normal) DNA is labeled with red dye. Both sets of labeled DNA are then inserted into the chip and allowed to hybridize, or bind, to the synthetic DNA on the chip

Microarray (DNA chip or Biochip):

-Chromosomal microarray testing is designed to detect copy number changes of chromosomal material

-Balanced chromosomal rearrangements, such as inversions or translocations, are not detected by this assay

PCR = Polymerase Chain Reaction: Molecular Diagnostics: -Polymerase chain reaction (PCR) is a -Consists of many different ways to technique used to amplify a single copy or a evaluate DNA and RNA few copies of a segment of DNA across several orders of magnitude, generating -Gets down to the base pair level thousands to millions of copies of a particular DNA sequence

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Real Time PCR: -Also known as quantitative PCR (qPCR) -It monitors the amplification of a targeted DNA molecule during the PCR (i.e. in real- time, and not at its end, as in conventional PCR) -Sequence-specific DNA probes consisting of primers that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence.

Chimerism Testing: -Also known as Bone Marrow Engraftment -For Allogeneic Hematopoietic Stem Cell Transplantation -Testing to see if the donor cells have fully engrafted into the recipient -Looks at several different markers that are different between donor and recipient -Is also used to track double donor transplants

Partial Partial T-cell 100% Donor Engraftment Engraftment

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Partial Partial T-cell Partial Partial T-cell 100% Donor Engraftment Engraftment 100% Donor Engraftment Engraftment

Microsatellite Instability (MSI) Testing: Microsatellite Instability (MSI) Testing: -Microsatellites are tandem repeats of short -The FDA has granted an accelerated approval to sequences of DNA pembrolizumab (Keytruda) for the treatment of -Occurs due to errors during mitosis adult and pediatric patients with unresectable or metastatic, microsatellite instability-high (MSI-H) -Usually repaired by the MMR genes or mismatch repair deficient (dMMR) solid tumors -If the MMR genes are not functioning properly, that have progressed after prior treatment and many microsatellites are formed who have no satisfactory alternative treatment options, as well as for patients with MSI-H or -MSI-High results are associated with HNPCC or dMMR colorectal cancer following progression on Lynch syndrome a fluoropyrimidine, oxaliplatin, and irinotecan.

Methylation Testing:

-DNA methylation is a process by which methyl groups (CH3) are added to the DNA molecule

-Methylation can change the activity of a DNA segment without changing the sequence -When located in a gene promoter, DNA methylation typically acts to repress gene transcription, in essence “turns it off”

-Epigenetics

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Methylation Testing: DNA Sequencing: -MGMT methylation evaluation in Glioblastoma helps determine the use of temozolomide as a -Determining the exact order of the treatment option base pairs in a segment of DNA

-MLH1 methylation testing is used to rule out -Looking for single-nucleotide Lynch Syndrome in Colorectal Cancer (CRC) substitutions, small insertions and deletions -SEPT9 methylation is associated with CRC and can be used as a screening method for those who refuse a colonoscopy

SNP = Single Nucleotide Polymorphism: -Germline DNA sequence variations that occur when a single nucleotide (A, T, C, or G) in the genome sequence is altered -Benign

SNV = Single Nucleotide Variant: - DNA variation in a single nucleotide without any limitations of frequency and may arise in somatic cells - Pathogenic or benign

Pyrosequencing Different sequencing methods: KRAS codons 12 & 13 -Pyrosequencing -Looks at small areas of a gene (a few codons) -Good sensitivity (5%) -Sanger sequencing -Looks at a larger area of a gene (whole exon) -Medium sensitivity (20%)

-Next Generation sequencing (NGS) Normal sequence is : GGT GGC GTA GG -Can look at multiple genes at the same time codon: 12 13 14 15 -Good sensitivity (3-5%)

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Pyrosequencing Pyrosequencing KRAS codons 12 & 13 KRAS codons 12 & 13

Normal sequence is : GGT GGC GTA GG Normal sequence is : GGT GGC GTA GG codon: 12 13 14 15 codon: 12 13 14 15 This sequence is: GG/TT GGC GTA GG This sequence is: A/GGT GGC GTA GG GGT>GTT G12V GGT>AGT G12S

Sanger Sequencing Sanger Sequencing

G>A

Next Generation Sequencing Next Generation Sequencing

EGFR L858R ERBB2 (HER2) A771_M774dup

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Next Generation Sequencing Advantages of NGS: -Ability to look at more areas of a gene -exons vs. codons -finding mutations missed before

-Ability to look at multiple genes at the same time -Lung panel, Heme panel, etc. EGFR E746_S752delinsQ -Quicker TAT by reducing reflex testing

Advantages of NGS – a case study: Advantages of NGS – a case study:

Patient with AML -8 years ago Patient with AML -3 years ago

-Tested for FLT3-ITD, FLT3-TKD, NPM1 -Tested for FLT3-ITD, FLT3-TKD, NPM1, CEBPA, KIT, IDH1 & IDH2 -Patient is NPM1 positive, negative for FLT3. -puts them in the favorable risk category -Patient is NPM1 positive and IDH1 R132H positive; negative for FLT3, CEBPA, KIT and IDH2. -IDH1 mutations are associated with reduced overall survival in favorable risk AML patients with mutated NPM1 and normal FLT3-ITD

Advantages of NGS – a case study: What does Molecular testing help provide? Patient with AML – today -Better defined/more accurate diagnosis -Tested with Heme Panel (44 genes) -Ability to follow NCCN Guidelines and -Patient has NPM1, IDH1 R132H, TET2 V1064fs, and recommendations TP53 C275S mutations -Targeted therapeutics, including resistance -NCCN Guidelines (v.1.2017) states that patients with testing TP53 mutations, but with an otherwise normal karyotype, should be considered for appropriate clinical -Opportunities for clinical trials trials or early allogeneic hematopoietic cell transplantation (HCT) -MRD testing

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How big a panel is necessary? What’s next? -Depends on the patient -Testing Plasma -Depends on the diagnosis or suspected diagnosis -Circulating Tumor Cells (CTC) -MPN -Cell free DNA (cfDNA) -AML -Standard of care vs. no standard of care -Pharmocogenomics -Germline testing to determine a person’s -Should follow current NCCN Guidelines response to drugs/medications -Metastatic CRC -NSCLC -Larger Panels – Tumor Mutation Burden (TMB) -Breast

What’s next? Goal of Molecular Testing: -Updated Guidelines and Recommendations -To provide a more complete -Joint consensus recommendations picture of a patient’s disease in -American Society of Clinical Pathologists (ASCP) order to make better patient care -College of American Pathologists (CAP) -Association for Molecular Pathology (AMP) decisions -American Society of Clinical Oncology (ASCO)

Thank you Flow Cytometry Dr. Jennifer Stumph Juli Allaire Kara Winczkowski Dr. Melissa Bombery Stephanie Charkowski Molly Catalina Dr. Mihaela Chiselite Robert Humes II Rebecca Klein Dr. Elena Ivan Susan Padilla Sandra Vanvels Dr. Frances Rosario-Quinones Pamela Scofield

Cytogenetics Dr. Timothy Drumheller Denise Kowalko Nicole Pierce Questions? Rachel Eernisse Kathleen Mansfield Seneca Russo Anne Hampton Jonathan McCourt Lee Ann Sharp Tyler Hoffman Karen Meilinger Kirsten Stevens Julie Kieft Daniel O’Malley Lynn Vander Laan Scott Kossoris Christine Yax [email protected] Molecular Diagnostics Dr. Cong Liu Stephanie Shaffer Shelby Wood Yoleidis Besse Natalie Sluja Carly Mercier Patricia Botma Sandi Talbott Nathan Clifford Julie Colvin Kelly Teneyck Jim Dobrenski Devyn McMahan Leah Thompson Suzanne Snider

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