Molecular Diagnostics in Pathology

Time for a Next-Generation Pathologist?

Matteo Fassan, MD, PhD

 Context.—Comprehensive molecular investigations of customized processing of biospecimens. Moreover, increased mainstream carcinogenic processes have led to the use of requests for molecular testing have paralleled the recent, effective molecular targeted agents in most cases of solid sharp decrease in tumor material to be analyzed—material tumors in clinical settings. that currently comprises cytology specimens or, at minimum, Objective.—To update readers regarding the evolving small biopsies in most cases of metastatic/advanced disease. role of the pathologist in the therapeutic decision-making Traditional diagnostic pathology has been completely revo- process and the introduction of next-generation technol- lutionized by the introduction of next-generation technolo- ogies into pathology practice. gies, which provide multigene, targeted mutational profiling, Data Sources.—Current literature on the topic, primar- even in the most complex of clinical cases. Combining ily sourced from the PubMed (National Center for traditional and molecular knowledge, pathologists integrate Biotechnology Information, Bethesda, Maryland) database, the morphological, clinical, and molecular dimensions of a were reviewed. disease, leading to a proper diagnosis and, therefore, the Conclusions.—Adequate evaluation of cytologic-based most-appropriate tailored therapy. and tissue-based predictive diagnostic biomarkers largely (Arch Pathol Lab Med. 2018;142:313–320; doi: 10.5858/ depends on both proper pathologic characterization and arpa.2017-0269-RA)

hree main paradigm shifts1 have radically changed the histochemical, immunohistochemical, and electron micros- T pathology world. In 1761, publishing De Sedibus et copy technologies. Causis Morborum per Anatomen Indagatis [On the Seats and However, many years passed from the interpretation of Causes of Diseases],2 Giovanni Battista Morgagni first raised the genomic code to the clinical translation of the molecular pathologic anatomy to an experimental science and data, engendering the birth of ‘‘personalized medicine.’’ For demonstrated that most illnesses have their origins in many decades, there was no significant change in the daily specific organs or tissues. Approximately a century later, in practices of surgical pathologists and cytopathologists, who 1858, Rudolf Virchow published Die Cellularpathologie in performed primarily microscopic evaluation in addition to ihrer Begrundung¨ auf Physiologische and Pathologische Gewe- clinicopathologic correlations. Indeed, only in recent years belehre [Cellular Pathology as Based Upon Physiological and has a complete morphomolecular approach had a central Pathological Histology],3 in which Virchow demonstrated that role in the therapeutic decision trail of most solid tumors, illnesses originate in the cells, promoting the introduction of sharply affecting pathologic diagnostic practices and labo- histopathology. Another century later, the discovery of the ratory workflows. DNA helix by Watson and Crick (1953)4 led to the birth of The actual challenges are (1) not only to ensure that molecular pathology. pathologists remain conscious of the clinical application of Throughout the past 3 centuries, the rigorous scientific molecular profiling but also to reassert that the value of method, first employed by Morgagni and later introduced traditional pathology has only been enriched by molecular into clinical practice, was first enriched by Virchow’s cyto/ profiling (similar to that of incorporating immunohisto- histologic dogma and subsequently further developed by the chemistry), (2) to integrate sophisticated molecular tech- nologies into traditional pathology laboratories by introducing new workflows, and (3) to disseminate knowl- Accepted for publication July 21, 2017. edge of molecular pathology among pathologists by From the Department of Medicine, Surgical Pathology and investing in postgraduate medical education programs for Cytopathology Unit, University of Padua, Padua, Italy. trained pathologists and by improving pathology residency The author has no relevant financial interest in the products or companies described in this article. training programs. Presented in part at the V Molecular Cytopathology Focus on Next Generation Sequencing in Cytopathology meeting; October 18, THE NOVEL CENTRAL ROLE OF PATHOLOGISTS IN THE 2016; Napoli, Italy. THERAPEUTIC DECISION-MAKING PROCESS Reprints: Matteo Fassan, MD, PhD, Department of Medicine, Surgical Pathology and Cytopathology Unit, University of Padua, Via Before the ‘‘molecular revolution,’’ the pathologist was Aristide Gabelli, 61, 35121 Padua, Italy (email: matteo.fassan@ broadly considered an ‘‘oracle,’’ far from daily clinical unipd.it). practice, who would confirm clinical and/or radiologic Arch Pathol Lab Med—Vol 142, March 2018 Next-Generation Pathologist—Fassan 313 suspicions. However, that notion is clearly no longer the biospecimens, significantly affecting their clinical indica- case for the following reasons: tion.19–25 This experience has pinpointed the compelling prerequisite for the increasing involvement of pathologists 1. A pathologist is a clinician who provides an interpreta- in biomarker research and development. In fact, the tion of the morphological (and molecular) features morphological and molecular evaluations of a biospecimen consistent with the clinical, radiologic, therapeutic, and are not mutually exclusive, but complementary,26 and laboratory-collected data. A typical example is the pathologists are the only people capable of piecing together evaluation of a thyroid aspirate purely composed of the morphological, molecular, and clinical features of each Hurthle¨ cells; in that setting, the decision to follow a case of disease. patient, rather than to perform a lobectomy, is based on This notion is also true for the introduction of liquid clinical, sonographic, and laboratory correlations. biopsies into clinical practice27: it is impossible to contem- 2. A pathologic diagnosis relies on a subjective evaluation. plate treating a patient without first determining a cytologic/ Indeed, pathologic features often overlap among various histologic diagnosis.28 diseases, and tissue sampling may be insufficient.5 All these factors may preclude a definitive diagnosis and The Selection of the Most Adequate Sample may, therefore, encourage the direct involvement of the One typical example of the dichotomy between the pathologist in multidisciplinary meetings. pathologist and the primary care clinician is the single request of a mutational profile of a biospecimen with no (or The Introduction of Biomarkers into Clinical Practice incomplete) morphologic assessment because the clinician The ‘‘solitude of the pathologist’’ ended with the may think the latter is not worth pursuing (eg, pleural introduction into clinical practice of a new generation of effusions obtained in stage IV lung adenocarcinoma patients drugs targeting specific genes and molecular pathways, or biopsy samples obtained to test eligibility for innovative combined with cellular/tissue biomarkers, which would clinical trials). In any case, it is common in pathology that allow caregivers to foresee the patient’s response to those some of those biospecimens will not contain sufficient expensive drugs.6,7 The introduction of new genetic thera- material to conduct an adequate molecular survey. pies has demanded a reevaluation of pathologic diagnoses Thus, a morphologic assessment should be performed on as the backbone of therapeutic decision-making and not every biospecimen for the following reasons: (1) to verify merely confirmation of clinical hypotheses. that the sample is the best representation of the disease (ie, For example, advanced patients with colorectal cancer, the presence of tumor necrosis, sampling errors, strong who are eligible for anti-epidermal growth factor receptor inflammatory component), (2) to confirm the histotype of (anti-EGFR) therapy, should undergo ‘‘extended’’ RAS the lesion, (3) to select the most proper area for the mutational testing.8 Moreover, DNA mismatch repair status molecular survey, (4) to choose the correct analysis has been recommended for all patients with colorectal according to the technical sensitivity of the molecular device cancer to investigate for possible Lynch syndrome. Notably, used, (5) to identify artifacts because of inaccurate preana- the pathologic diagnostic report should provide all such lytic steps that can dramatically affect molecular profiling, information.8–10 In a similar manner, the EGFR/ALK and (6) to assess the tumor’s morphological heterogeneity, (anaplastic lymphoma kinase)/ROS1 (and in some instances, which can introduce analytic biases (Figure 1).23 MET and KRAS) status may be assessed in lung adenocar- Moreover, the pathologist’s simultaneous evaluation of cinomas at diagnosis of an advanced disease, at recurrence, the morphology of the sample and of the molecular profiling or in patients with an early stage (ie, stages I–III) disease to be performed is the most effective option of ensuring who undergo surgical resection.11 The BRAF (NRAS and avoidance of useless (and expensive) molecular surveys.29 KIT) status may be tested in metastatic malignant melano- Pathologists should proceed according to the proper choice ma,12 the KIT/PDGFRA mutations in gastrointestinal stromal of the required molecular survey, according to published tumors,13 the somatic BRCA1/2 status in ovarian cancers,14 molecular testing guidelines8,30–32 and to the application of and the 1p/19q codeletion, MGMT promoter methylation, the molecular test for every patient who needs it (even if and mutation in IDH1/2 genes in brain tumors.15 Further- that could potentially clash with some public health- more, the targeting of immune checkpoints has led to the spending review plans). use of novel biomarkers (primarily the programmed death Notably, reflex testing by pathologists has previously been ligand-1 [PD-L1] immunohistochemical assessment) in conducted in numerous pathologic fields (ie, thyroid many different tumors.16 nodules, lymphoproliferative disorders, and soft-tissue Although traditional pathologists remain wary of this sarcomas, in particular) in which molecular information is complex molecular background, several years ago, morpho- an essential diagnostic tool.33–37 For example, the assess- logical classification was completely replaced by molecular ment of BRAF status may play a central role in distinguish- classification in breast cancers.17 Following initial difficulties ing the diagnosis of hairy cell leukemia from other B-cell in standardizing the assessment of the new biomarkers,18 lymphoproliferative neoplasms exhibiting similar clinical the proliferation index MIB1, estrogen/progesterone recep- and morphological features.38 Theimportanceofthe tor status, and HER2 (human epidermal growth factor molecular survey in routine diagnosis is highlighted by the receptor 2) became mandatory in a breast cancer pathologic introduction of molecular profiling in the classification of report. many tumors (particularly in distinguishing different malig- In short, the molecular revolution has barely begun, and nant central nervous system tumors).39 the necessity of morphomolecular diagnoses will continue Conversely, only referencing molecular information may to increase. often be misleading if that information is not supported by Notably, many promising biomarkers have not been the histopathologic features, emphasizing the importance of implemented into routine pathology because of inaccurate a matched morphomolecular diagnosis. This scenario occurs assessment and preservation of the cytologic/histologic in cases of Ig or TCR gene clonality, which can not be 314 Arch Pathol Lab Med—Vol 142, March 2018 Next-Generation Pathologist—Fassan Figure 1. Difficult samples to be characterized molecularly. A, Fine-needle aspiration (FNA) cytology smear obtained from a Hashimoto thyroiditis. Note the prevalent lymphocytic component. B, An FNA smear characterized by an evident necroinflammatory component. C, An FNA smear characterized by significant hypocellularity. D, Cancer heterogeneity for MLH1 (MutL homolog 1) expression in a high-grade colorectal adenocarcinoma showing a positive immunoreaction in the mucoid, differentiated portion (upper left, above the dotted line, which separates the 2 components) and a negative immunoreaction in the other neoplastic cells, marked by a solid-type growth pattern. E, A case of rectal adenocarcinoma presenting as a Mandard tumor regression grade 2 after neoadjuvant therapy. Neoplastic glands (arrowheads) are seldom still preserved, and their microdissection may be complex. F, The marginal portion of a medullary subtype colorectal adenocarcinoma marked by a huge intraepithelial and peritumoral inflammatory cell infiltration (hematoxylin-eosin, original magnifications 340 [A and B], 35 [C and E], and 310 [F]; original magnification 320 [D]). considered proof of B-cell or T-cell malignancy without thermore, improvement in molecular technologies has been jointly assessing the histopathologic, flow cytometry, and largely encouraged to respond to both the growing demand immunophenotypic features.40,41 for molecular surveys in clinical practice and the simulta- neous decrease in the amount of neoplastic samples in NEXT-GENERATION TECHNOLOGIES nearly every metastatic/advanced case.49,50 IN OLD-GENERATION LABORATORIES Preanalytic Phase in Cytology High-quality microscopy is the primary prerequisite for remarkable molecular pathology.23 However, most of the Cytopathologic specimens have the primary role in the technologies employed in cytologic and histopathologic molecular profiling of several neoplastic lesions.51–56 For evaluations were designed more than a century ago and rely example, in lung cancers, cytology may represent the only on morphology. Therefore, ‘‘traditional’’ cyto/histopatholo- way to obtain diagnostic samples in patients with advanced gy technologies could have a dramatic effect on molecular diseases or with low performance status who are unable to pathology.42 In addition, the preanalytic phase, which undergo an open biopsy.57,58 However, the DNA/RNA includes the management and processing of biospecimens, supplied by most cytologic specimens is limited (even can affect the quality of proteins and nucleic acids present in though enriched in neoplastic cells) and is thus inadequate the samples (Figure 2, A through E).29,43,44 for sequential, single-gene diagnostic tests.59,60 This problem Contributing to the conceptual confusion in the diagnostic has recently been overcome by the introduction of NGS routine, the concept of ‘‘a single biomarker for a specific applications into clinical practice.59,60 drug’’ has recently become outdated, engendering a multi- The combination of rapid on-site evaluation,61 the almost marker diagnostic routine that necessitates the use of a set complete absence of formalin-based fixation, and the of different markers (for example, next-generation sequenc- potential use of smears immediately in combination with ing [NGS] and multiplex genotyping platforms).45–48 Fur- cell blocks represent the best situation in the extraction of Arch Pathol Lab Med—Vol 142, March 2018 Next-Generation Pathologist—Fassan 315 Figure 2. Examples of cross-contamination during sample processing and section prepa- ration. A, A messy pathology hood after the resection of a fresh specimen. Gauze was placed under the specimen to demonstrate how the amount of blood and microscopic fragments of the specimens can accumulate after a single resection. This image demon- strates that the waste of the hood can significantly affect the following molecular surveys. B and C, Preparation of histology slices. The knife should be replaced regularly, and the microtome must be cleaned with every change of formalin-fixed, paraffin-em- bedded sample. This process is true for both histopathology and cell block specimens. D and E, Typical example of a ‘‘shared’’ water bath showing the increase in small debris because of the accumulation of previous cut samples.

high-quality DNA/RNA, which is nearly impossible in remaining aspirates and needle rinses are generally recov- traditional histology.62 Furthermore, cytology is a rapid, ered in preservatives or in cell blocks for further analyses in low-cost, minimally invasive, and a well-tolerated proce- current clinical practices.66,67 dure that is widely employed to achieve a definitive A considerable methodological problem is that nearly all diagnosis in many laboratories.63 However, a more-cus- NGS diagnostic assays have been designed and validated tomized approach is required for the management of specimens when compared with histology. Indeed, all the only on formalin-fixed, paraffin-embedded (FFPE) biospeci- international recommendations for good practices in lung mens; thus, it is also an important challenge for the cancer clearly state the crucial need for an accurate cytologist to validate the NGS results during cytologic acquisition and preservation of all residual cytologic material preparations. Thus, an alternative validation strategy could for molecular investigations.29,64,65 Consequently, all the focus on key elements of analytic sensitivity and specificity 316 Arch Pathol Lab Med—Vol 142, March 2018 Next-Generation Pathologist—Fassan and limit the detection to relevant genes using known A specific problem in cytology is establishing the reference standards.62 minimum cellular cutoff required for NGS analyses.59,74 Adequate preanalytic management of cytologic specimens Most published studies chose samples with at least 20% has become the leading component of the subsequent neoplastic cells,29,59 which may not reflect daily diagnostic molecular assessment of lesions.62 Preanalytic variability practices. ‘‘The magic number’’ depends on both the target strongly depends on applied cytologic preparations (eg, capture and the various platform types, which range direct smears, cytospin preparations, cell blocks, and liquid- between 100 and 15 000 cells.59,62 Moreover, even the based cytology samples), which include typical processing DNA input can significantly range from 10 ng for the Ion techniques, fixatives, and stains. Torrent (Thermo Fisher Scientific, Waltham, Massachusetts) Among several cytologic preparations, the cell block is sequencing of PCR products to the 170 ng required for the similar to typical surgical pathologic processing. However, Illumina NGS hybridization capture (Illumina, San Diego, similar to histology, the process of paraffin embedding California).59 generally comprises several formalin-fixation steps, and use In summary, these results indicate that an adequate of the same histologic processors, even though samples are molecular survey requires an acceptable morphological collected in nonformalin-based solution.67 Moreover, cell analysis derived from a neoplastic cell–rich FNA biospeci- block preparation methods can vary widely at different men.75 institutions.68 A remarkable advantage of using FFPE cell block samples The Preanalytic Phase in Histopathology is the opportunity to have a better selection of the material Every pathology department processes biospecimens from to be molecularly analyzed; an inadequate qualification of various health clinics and surgeries and, occasionally, from the sample can influence the results more than the technical different hospitals. Therefore, DNA/RNA degradation may inconsistencies from the brittleness of the FFPE specimen.57 result because of a delay in transporting those specimens Conversely, an unwarranted cell block preanalytic step is and/or incorrect sample preservation.76 To solve that the section preparation of the molecular analysis, which problem, biospecimens should be stored using a chemical may be the primary source of cross-contamination (Figure 2, fixative or be vacuum packaged. However, an important B). This risk must be considered after the implementation of preanalytic variable is the poor fixation of a biospecimen, sensitive molecular technologies that are capable of particularly in surgical samples. However, tissue formalin- analyzing biospecimens containing less than 1% mutated fixation causes nucleic acid fragmentation.77 Fixation in alleles. Technicians may prevent contamination with (1) the neutral-buffered formalin for 12 to 24 hours (8 hours for regular replacement of the knife and the use of disposable small tissue specimens) is the best option for preserving plastic ware, and (2) by preventing the use of ‘‘shared’’ nucleic acids and maintaining good morphology, whereas water baths.50 overfixation may considerably hamper the next molecular An easier option than the cell block for extracting high- analysis. Moreover, something equally remarkable is that quality DNA is the use of precise, small amounts of most pathology laboratories do not operate every day of the cytologic sample in suspension, which has been shown week. Therefore, overfixation of biospecimens is a daily well in the molecular characterization of human papilloma- occurrence because of laboratory closures. virus testing and thyroid nodules.69 Moreover, the NGS Cross-contamination is another important preanalytic approaches can also be applied successfully in DNA step to be checked before molecular profiling. As for cell retrieved from fine-needle aspiration (FNA) needle rinses block samples, section preparation may represent a signif- and effusion fluids,70 whereas the use of diagnostic smears icant source of cross-contamination. Another important (and their definite loss) is reserved for cases in which no cause is represented by inadequate sampling room clean- other material is available.67,71 liness, particularly in high-throughput laboratories. In the Specimens must be examined to verify the amount of larger sections of molecular laboratories, investigations are neoplastic cells before molecular profiling, and doing so, also conducted daily on specimens sent from external occasionally, requires great effort to achieve the proper laboratories, which makes it impossible to provide evidence microdissection to remove the nonneoplastic portion of cross-contamination that may have occurred before (mainly inflammatory cells and necrotic debris) and enrich paraffin inclusion. the tumor cell portion. Moreover, it should be clear that if Despite the preanalytic steps, insufficient or inadequate the cytology is a good representation of the lesion, the samples can lead to misguided selection of the neoplastic cytologic samples are also affected by intratumor molecular area. Indeed, most biospecimens lack an adequate tumor heterogeneity. In addition, FNAs frequently have a higher cell component, impairing the accuracy of the molecular test neoplastic to healthy-cell ratio compared with the core (ie, tumor component under the technical sensitivity of the needle biopsies.67 molecular method)78; therefore, in most cases, a macro/ Ideally, in the case of indeterminate samples, a (cyto)pa- microdissection or an eventual 1-mm core sampling must thologist skilled in molecular pathology who is aware of the be performed to enrich the neoplastic cellularity.79–81 The analytic sensitivities of the different molecular devices72 laser-captured microdissection is generally reserved for should always offer a point of view toward choosing the research.29 most appropriate technology.10,73 In addition, it is common Since the first reports regarding the accuracy of the FFPE- for traditional (cyto)pathologists to overstate neoplastic derived DNA for NGS-based analyses were published,82 cellularity because they often tend to analyze the extension several (primarily) amplicon-based NGS assays have been of the neoplastic areas, rather than the neoplastic to approved clinically, even if most of them employed a set of healthy-cell ratio in the same area (a large, diploid cancer the most-frequent clinically actionable genomic alterations, cell nucleus has nearly the same genomic content as the shortening the turnaround time required to provide small nuclei of tiny healthy lymphocytes!), which is the molecular profiling.50,71,83–85 Moreover, following the correct assessment before conducting molecular profiling. ‘‘FFPE-friendly’’ approach, NGS is now easier to perform, Arch Pathol Lab Med—Vol 142, March 2018 Next-Generation Pathologist—Fassan 317 including on both clinically and pathologically well-profiled throughput technologies in their daily practices. Although, FFPE blocks retrieved from pathology archives. However, today, we take next-generation technology for granted, can one of the most important challenges for the molecular we, therefore, assume that there is already a next-generation pathologist is the standardization of NGS assays because pathologist? NGS combines several devices, reagents, and bioinformatics The molecular revolution that has occurred in pathology pipelines.50 diagnostics has not been joined by the updating of training Bioinformatics has been poorly applied in pathology programs, allowing access to high-throughput technology because of its complexity in analytic methods.60 In addition, information to only a few people. This impasse and its bioinformaticians must consider that both the preanalytic implications for both the diagnostic and academic/research processing and the preservation of FFPE biospecimens can dimensions have been recognized by the pathology introduce artifacts into DNA, such as the deamination of community,93–96 and integration of traditional pathology cytosine bases, resulting in C:G.T:A substitutions during training by molecular diagnostics is widely suggested. amplification.86 These types of artifacts are because of both This suggestion does not mean that all pathologists formalin fixation and the degradation of the biospecimen should develop a particular expertise in genomic medicine that occurs naturally (which is intrinsically associated with and genome-based research. However, all pathologists (in hydrolytic deamination).87 Notably, pretreatment of the addition to their imperative training in morphology) should DNA sample with uracil-DNA glycosylase has been shown be able to address molecular information, know how to to sharply decrease the artifactual deamination, with no select the most adequate sample for analysis, understand changes to the DNA helix itself.87 the molecular performance of such tests, and incorporate the morphological and molecular data into an integrated The Next-Generation Working Group diagnostic report. For many years, the organization of pathology laborato- For example, a general cytopathologist should be aware of ries was based on the close collaboration among techni- how to manage a thyroid cytology specimen for both an cians, biologists, and pathologists, working together on both adequate cytology diagnosis and RAS/BRAF testing.37 the improvement of cytologic/histologic processing and the Moreover, the general cytopathologist must know how to achievement of the most-sophisticated morphological diag- interpret the molecular data compared with phenotypic noses. findings and to provide an appropriate diagnostic message During the past few years, because of new impetus, the to clinicians/surgeons, even though the pathologist may not modern pathology laboratory has been enriched by the be required to have an in-depth knowledge of ‘‘coverage,’’ novel expertise of molecular biologists/biotechnologists (and bioinformatics analysis, and qualification of the obtained laboratory technicians with a molecular background) who DNAs/RNAs. However, every pathology department should have a basic knowledge of cytology and surgical patholo- have subspecialized pathologists in molecular diagnostics gy.88 who should be familiar with high-throughput technologies This multidisciplinary team generally involves trained staff and will be the cultural bridge between ‘‘traditional’’ in complex bioinformatics data interpretation.60,89 Indeed, pathologists and the molecular pathology laboratory. Those pathology informatics has become increasingly relevant not people, however, should also be involved in routine only in the NGS context but also in some basic elements of cytopathology/histopathology diagnostics to maintain close traditional pathology, such as immunohistochemistry and in contact with the specimens and, therefore, the clinic. situ hybridization techniques. Traditional pathology core curriculum must be imple- These 2 methods continue to have a central role in both mented with basic molecular notions and the interpretation diagnosing illness—because of their excellent reproducibil- of the molecular data within the clinical context.97 However, ity and accuracy—and in detecting a genetic alteration in molecular pathologists should recognize that a BRAFV600E FFPE specimens. However, these analyses could be affected mutation is not a diagnosis, but merely a portion of one. by interpathologists’ and intrapathologists’ interpretative This goal of integrating traditional pathology training with biases, which are not consistent with the concept of molecular diagnostics can be reached by modifying resi- personalized medicine.90 dency education programs in pathology98,99 and by creating The recent introduction of digital pathology through the appropriate continuing medical education programs for employment of automated scoring of the immunohisto- trained pathologists. This latter point is central: we cannot chemical expression and the automated count of hybridiza- afford to wait to integrate new generations of pathologists tion signals has led to a more-quantitative interpretation of into our institutions. Moreover, the standardization and results.91,92 As has been observed for molecular profiling, the implementation of training programs is far from being a great challenge lies in having to address the large amount of forthcoming result. The molecular revolution is now, and data that requires interpretation with a comprehensive trained traditional pathologists are already principal actors algorithm capable of translating digital information into a in that paradigm. Pathologists should incorporate a simple quantitative score. clinically fitting molecular background rapidly, and they Therefore, modern pathology laboratories should invest must change the manner in which they practice cellular and more in informatics because analyses of these data will surgical pathology diagnostics. become the bottleneck of our future diagnostic workflows. CONCLUSIONS (CYTO)PATHOLOGIST, MOLECULAR Modern pathology practice has been suddenly and (CYTO)PATHOLOGIST, OR NEXT-GENERATION completely changed by the increased complexity of modern (CYTO)PATHOLOGIST? medicine, primarily regarding oncology, which this concise In the ‘‘next-generation sequencing era,’’ many pathology review has attempted to describe. Consequently, the laboratories have implemented NGS or multigene high- frameworks of our laboratories and pathology training 318 Arch Pathol Lab Med—Vol 142, March 2018 Next-Generation Pathologist—Fassan programs are experiencing a radical change in accordance 20. Simeon-Dubach D, Burt AD, Hall PA. Quality really matters: the need to improve specimen quality in biomedical research. J Pathol. 2012;228(4):431– with a pressing request for innovation. Currently, the 433. pathologist has a central role in the age of next-generation 21. Begley CG, Ellis LM. 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