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