PHENO. PhenoPath at the USCAP

Transcription

1 TM PHE Spring 2016 Volume 19 No.1 The pathologists and team at PhenoPath couldn t be more pleased that USCAP is being held in Seattle this year, PhenoPath s hometown! Seattle is a beautiful city and there are a lot of fun and exciting things to do. We are involved in many activities at USCAP, some of which are described below. In addition, see below for an exciting event being held at PhenoPath. PhenoPath at the USCAP March 12-18, 2016 Seattle, Washington Attendees of the United States & Canadian Academy of Pathology (USCAP) annual meeting might be interested in the following presentations by or involving PhenoPath pathologists: Sunday, March 13, 2016, 7:30 PM-10:30 PM, Room CC Hall 4E / International Society of Breast Pathology: Allen M. Gown, MD will speak on HER2 testing in breast cancer at the symposium entitled, Second Opinions and Diagnostic Concordance in Breast Pathology : Moderated by Aysegul Sahin, MD, The University of Texas MD Anderson Cancer Center, Houston, and Gelareh Farshid, MD, The University of Adelaide, Australia Monday, March 14, 2016, 9:30 AM-12:30 PM, Room CC Hall 4ABC / Poster Session I #289 Programmed Cell Death 1 Biomarker Testing: RNA and Protein Assessment (Abstract #1919), Presented by Brandon S Sheffield, MD, University of British Columbia Tuesday, March 15, 2016, 9:00 AM-12:00 PM, Room CC Hall 4ABC / Poster Session III #295 TFE3 Immunohistochemistry: A Comparison of Two Methods, with Discussion of the Implications for the Diagnosis of TFE-3- Rearranged Neoplasms (Abstract #2069), Presented by Rosalind Sandell, MD, Mayo Clinic Tuesday, March 15, 2016, 1:00-4:30 PM, Room CC Hall 4ABC / Poster Session IV #54 Increased HER2 FISH-IHC Discordance and Decreased FISH Equivocals Result from 2013 ASCO-CAP HER2 Scoring Guidelines: A Study of 11,813 Cases (Abstract #157), Presented by Regan Fulton, MD, PhD, PhenoPath Tuesday, March 15, 2016, 1:00-4:30 PM, Room CC Hall 4ABC / Poster Session IV #96 Use of a Novel Rabbit Monoclonal Phospho-Histone H3 (Ser10) Versus H&E Mitotic Count in Invasive Melanoma (Abstract #499), Presented by Regan Fulton, MD, PhD, PhenoPath Visit PhenoPath booth #1026 in the Exhibit Hall and meet our staff and pathologists. Visit PhenoPath booth #1026 in the Exhibit Hall and meet our staff and pathologists. PhenoPath Open House! Are you coming to Seattle for the USCAP 2016 Annual Meeting? Come to PhenoPath for an Open House event on Tuesday, March 15 from 6:30 to 8:00 pm Check out our state-of-the art lab and meet the team (pathologists, lab supervisors, & client services representatives) while you are visiting Seattle for the USCAP 2016 Annual Meeting! Learn about and view some of the exciting pathology technologies PhenoPath offers. View some interesting cases. Visit with your colleagues, enjoy music, light dinner, wine, and beer! RSVP required at marketing@phenopath.com. Contact us regarding transportation

2 Introducing Dr. David Ng PhenoPath is delighted to welcome Dr. David Ng to the hematopathology team. Dr. Ng received his B.S. in Electrical Engineering from the University of Illinois at Urbana-Champaign, and his M.D. from the University of Illinois at Chicago. He completed his residency in Anatomic and Clinical Pathology at Dartmouth Hitchcock Medical Center, and his fellowship in Hematopathology at the University of Washington. Prior to coming to PhenoPath, Dr. Ng was a Senior Hematopathology Fellow in the Department of Laboratory Medicine at UWMC, where he developed algorithms for automated analysis in flow cytometry, leading to first authorship on a recent publication in the American Journal of Clinical Pathology (see reference below). Dr. Ng is board certified in Anatomic Pathology, Clinical Pathology, and Hematopathology. Dr. Ng brings a passion for Hematopathology to PhenoPath, and participates in our full range of services, including flow cytometry, immunohistochemistry, FISH, PCR, cytogenetics, and full case consultation. Dr. Ng has already made significant contributions to PhenoPath s hematopathology service, taking a leading role in the optimization and validation of the CD138-based plasma cell enrichment procedure, and participating in the development and validation of several new flow cytometric and immunohistochemical assays. Dr. Ng is an excellent addition to the PhenoPath team! Reference: Ng DP, et al. Computer-Aided Detection of Rare Tumor Populations in Flow Cytometry: An Example With Classic Hodgkin Lymphoma. AJCP 144(3):517-24, 2015 PD-L1 Expression on Cell Membrane of Positive Control Cell Line for 28-8 Assay PhenoPath at the Forefront: PD-L1 Immunohistochemical Assays for Non-Small Cell Lung Cancer and Other Malignancies The approval by the Food and Drug Administration (FDA) of the anti-pd1 targeted monoclonal antibodies, pembroluzimab [KEYTRUDA] and nivolumab [OPDIVO] follows the publication of several studies documenting efficacy of these drugs in melanoma, non-small cell lung carcinoma (NSCLC), renal cell carcinoma, and others. To review, programmed cell death protein 1 (PD-1) is an immune inhibitory receptor expressed on several immune cells, particularly cytotoxic T cells. PD-1 interacts with two ligands, programmed cell death ligand 1 (PD-L1) (B7-H1, CD274) and PD-L2 (B7-DC). PD-L1 may be expressed on tumor cells in addition to immune cells. The interaction of these ligands with PD-1 inhibits T cell activation and cytokine production. In the context of infection or inflammation in normal tissues, the interaction of PD-1 with PD-L1/2 is critical for maintaining homeostasis of the immune response and preventing autoimmunity. In tumor microenvironments, however, the interaction of PD-1 with PD-L1/2 provides an immune escape for tumor cells by turning off cytotoxic T cells. Thus, blocking these interactions may subject the tumor cells to attack from cytotoxic T cells. Which PD-L1 Immunohistochemical Assay Should I Request? The enclosed insert summarizes criteria for different PD-L1 assays. It is important to note that the FDA has approved pembrolizumab [KEYTRUDA] with a companion diagnostic (22C3) to determine patient eligibility for treatment with this drug only in the case of NSCLC. While the FDA approved pembrolizumab [KEYTRUDA] for melanoma, that approval did not come with a companion diagnostic. Other indications are in clinical trials or undergoing FDA review. Watch our web site for updates. In the case of nivolumab [OPDIVO], the FDA approved this drug with a recommendation for a complementary diagnostic in the setting of NSCLC and melanoma. Nivolumab [OPDIVO] has also been approved by the FDA for treatment of renal cell carcinoma, but without the complementary diagnostic. In the case of the FDA-approved assays for NSCLC, the choice of test depends upon the drug that is being contemplated for the patient: if it is pembrolizumab [KEYTRUDA], the test is the 22C3-based assay (pharmdx by Dako), and if it is nivolumab [OPDIVO], the appropriate test is the 28-8-based assay (also a pharmdx by Dako). PD-L1 Expression in Non-Small Cell Lung Carcinoma, 22C3 clone In the case of the FDA-approved assays for melanoma, the choice of test depends upon the drug that is being contemplated for the patient: if it is nivolumab [OPDIVO], the appropriate test is the 28-8-based assay (also a pharmdx by Dako), and if it is pembrolizumab [KEYTRUDA], testing is not required. If the patient s tumor is something other than NSCLC and melanoma, PhenoPath offers a generic assay employing the E1L3N mouse monoclonal antibody (see previous Phenomena) if desired. In addition to the use of different antibodies, the PD-L1 assays for pembrolizumab [KEYTRUDA] and nivolumab [OPDIVO] employ different scoring systems. For testing with the generic E1L3N clone, there is no prescribed scoring system. At PhenoPath, we provide the percent of positive cells and the staining intensity for the E1L3N assay.

3 From the *Division of Anatomical Pathology, Vancouver General Hospital; zdivision of Anatomical Pathology, Children s and Women s Hospital of British Columbia, Vancouver, BC, Canada; and wphenopath Laboratories, Seattle, WA. Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. Correspondence: Andrew Churg, MD, Department of Pathology, Vancouver General Hospital, 910 West 10th Ave., Vancouver, BC, Canada V5Z 1M9 ( achurg@mail.ubc.ca). Copyright r 2015 Wolters Kluwer Health, Inc. All rights reserved. Copyright 2015 Wolters Kluwer Health, Inc. All rights reserved. From the *PhenoPath Laboratories, Seattle, WA; and wdepartment of Pathology, Vancouver General Hospital and University of British Columbia, Vancouver, BC, Canada. Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. Correspondence: Andrew Churg, MD, Department of Pathology, Vancouver General Hospital, 910 W 10th Ave, Vancouver, BC, Canada V5Z 1M9 ( achurg@ mail.ubc.ca). Copyright r 2014 by Lippincott Williams & Wilkins The New FDA-Approved Roche cobas EGFR Mutation Test (Version 2) Detects the EGFR T790M Mutation as Well as Other Activating EGFR Mutations PhenoPath is pleased to offer the Roche cobas EGFR Mutation Test (v2), an FDA-approved companion diagnostic for both first- and secondline therapy decisions for patients with non-small cell lung carcinoma (NSCLC). This assay is FDA approved for identifying NSCLC patients with EGFR exon 19 deletions and exon 21 (L858R) substitution mutations for whom treatment with the tyrosine kinase inhibitor (TKI) Tarceva (erlotinib) may be effective as a first-line therapy, and for identifying NSCLC patients who harbor a T790M mutation, indicating eligibility for treatment with Tagrisso (osimertinib) as a second-line TKI therapy. Per the FDA news release of November 13, 2015, Tagrisso is the only approved medicine indicated for patients with metastatic EGFR T790M mutation-positive NSCLC who have progressed on or after EGFR tyrosine kinase inhibitor therapy. Although patients with lung non-small cell adenocarcinoma may initially respond to first-line TKI therapies such as Tarceva (erlotinib), acquired resistance is common. The development of a T790M mutation accounts for over half of such acquired resistance cases ( J Thorac Dis 2011;1:10-18). It should be noted that eligibility for Tagrisso (osimertinib), the first FDA-approved therapy for T790M positive tumors, requires identification of T790M in a post-therapy biopsy specimen. Effective November 17, 2015, the Roche cobas EGFR Mutation Test (v2) replaced the EGFR used previously. The Roche cobas assay similarly uses qualitative real-time PCR to detect clinically relevant mutations in exons 18, 19, 20, and 21 of the EGFR gene and provides improved sensitivity, as only 5% mutant allele is needed for reliable mutation detection in a background of wild-type DNA. In addition, the cobas EGFR assay covers a greater number of mutations, identifying 42 mutations in exons 18-21, as compared to the 21 mutations covered by PhenoPath s previous assay. Furthermore, this new EGFR test requires less tissue than our previous assay, which is particularly advantageous for small biopsy specimens. For more information about EGFR and PD-L1 testing at Phenopath, including pricing and ordering information, please contact our Client Services department at lab@phenopath.com or ; or visit us at For more information about the FDA s approval of the Roche cobas EGFR Mutation test or the FDA s approval of Tagrisso, see and PhenoPath Collaboration Investigates Utility of BAP1 Immunohistochemistry and P16 FISH in the Diagnosis of Malignant Mesothelioma ORIGINAL ARTICLE BAP1 Immunohistochemistry and p16 FISH to Separate Benign From Malignant Mesothelial Proliferations Brandon S. Sheffield, MD,* Harry C. Hwang, MD,w Anna F. Lee, MD,z Kim Thompson, HT, ASCP, QIHC,w Stephanie Rodriguez, HT, MB, ASCP,w Christopher H. Tse, MBBS,w Allen M. Gown, MD,w and Andrew Churg, MD* mmunohistochemistry (IHC) has been instrumental in Abstract: A variety of immunohistochemical (IHC) stains have Iallowing the pathologist to distinguish mesothelial been proposed to mark either benign or malignant mesothelial proliferations from other processes. 1 Delineation between proliferations. Loss of the p16 tumor suppressor (CDKN2A), benign reactive and malignant mesothelial proliferations through homozygous deletions of 9p21, is a good marker of continues to present a diagnostic challenge and is of mesotheliomas but lacks sensitivity. Recent reports indicate that fundamental some mesotheliomas are associated with loss of BRCA-associated protein 1 (BAP1) expression. Here we investigate BAP1 Routine morphology, and particularly stromal in- ORIGINAL importance ARTICLE to patient treatment and prognosis. and p16 as potential markers of malignancy and compare test vasion, remains the most useful way to separate benign characteristics with previously proposed markers using a wellcharacterized tissue microarray. BAP1 protein expression was diagnosis remains a challenge, numerous IHC markers from malignant mesothelial processes. 2,3 As the histologic interrogated by IHC. The locus was examined by fluorescence in situ hybridization p16 (FISH) FISH directed toward Deletion have chromosome 9p21. Loss of BAP1 was identified in 7/26 mesotheliomas in been Surface previouslyepithelial investigated for Mesothelial this purpose. Membranous staining for epithelial membrane antigen and 0/49 benign proliferations. Proliferations Loss of p16 was identified inis 14/ Predictive (EMA), p53 nuclear ofpositivity, Underlying GLUT1 and IMP3 Invasive positivity, 4,5 and lack of desmin expression have all been 27 mesotheliomas and 0/40 benign proliferations, yielding 100% specificity and positive predictive value for each marker. Together, BAP1 IHC and p16 FISH were 58% sensitive for de- Homozygous deletion of the 9p21 region has been Mesothelioma proposed as markers of malignancy; however, each of these tests lacks the specificity required for clinical use. 6 9 tecting malignancy. Various combinations Harry Hwang, of p53, MD,* EMA, IMP3, Christopher previously Tse, MBBS,* proposed as Stephanie a markerrodriguez, of malignancy. HT, 10 This ASCP,* and GLUT1 showed reasonably high specificity (96% Allen to 98%) Gown, cytogenetic MD,* and aberration Andrew Churg, results in MDw loss of the p16 but poor to extremely poor sensitivity. Combined BAP1 IHC/ (CDKN2A) tumor suppressor, among other genes, and is p16 FISH testing is a highly specific method of diagnosing detectable by fluorescence in situ hybridization (FISH). malignant mesotheliomas when the question is whether a mesothelial proliferation is benign or malignant and is particularly lignancy but only demonstrable mall pleural and in aperitoneal portion of biopsies meso-not infrequently Demonstration of p16 deletion is highly specific for ma- useful when tissue Abstract: invasion Anby atypical mesothelial mesothelial cellsproliferation cannot be along the pleural theliomas. 11 Deletions Sshow of p16 proliferations offer moreof than mesothelial a diagnostic cells that are worrisome for, as they but not alsodiagnostic, act as a biomarker, of malignancy, sometimes demonstrated. However, or peritoneal combined surface BAP1/p16 without evidence FISH testing of invasive is tumor poses a marker of malignancy, not highly sensitive, diagnostic and negative challenge. results Homozygous do not rule deletion out aof p16 (CDKN2A) conferring poorer because prognosis of cytologic in mesotheliomas, features but andmore mayoften because of mesothelioma. The bytest fluorescence characteristics in situof hybridization previously proposed (FISH) has been shown to also represent a the potential patterntarget of growth. for systemic 1,2 One therapy. most 12 difficult patterns in markers EMA, p53, be a GLUT1, good marker IMP3 of malignancy suggest that, in mesothelial even in proliferations, Recently, loss suchof specimens BRCA-associated is mesothelial protein 1 (BAP1) proliferations that are combination, thesebut markers correlations are notof useful p16 tools statusinbetween this clinical atypical surface proliferations and underlying mesothelioma have not been de- has been reported confined some to themesotheliomas. pleural or peritoneal Germline surface. These proliferations have been can appear associated awith single increased line of mesothelial cells setting. mutations in BAP1 scribed. We used p16 FISH to investigate 11 pleural and 7 Key Words: BAP1, BRCA-associated protein 1, p16, CDKN2A, risk for malignant but may mesothelioma, also grow 13 in as more well complex as uveal patterns and/or be peritoneal mesotheliomas that had both an invasive component 9p21, mesothelioma melanoma and cytologically renal cell carcinoma. very atypical; 14 Somatic however, BAP1 the current recommendation been implicated is notintononhereditary diagnose malignant meso- mesothelioma and a separate surface mesothelial proliferation. In 5/11 pleural mutations have (Am J Surg Pathol samples 2015;39: ) and 1/7 peritoneal samples, the invasive mesotheliomas theliomas. 15 BAP1 in pure IHC has surface beenproliferations, previously utilized because for even unusualappearing uveal mesothelial melanoma surface andproliferations renal cell may turn out showed homozygous deletion of p16 (all cases in excess of 90% its prognostic value of cells deleted); the surface proliferation in all 6 cases with carcinoma. 16,17 to be benign reactions. Indeed, there are no morphologic deletion in the invasive tumor was also p16 deleted. Conversely, In this study, features we examine that reliably the utility separate of BAP1 benignas from a malignant in the 12 tumors that did not show p16 deletion in the invasive marker of malignancy this setting. mesothelial 1 3 At present, proliferations, a biopsy using showing a only a surface tissue mesothelial microarray proliferation (TMA) 9 and is nondiagnostic, inves- even if compartment also did not have deletion in the surface component. We conclude that (1) surface mesothelial proliferations previously described tigate whether combining there is radiologic BAP1 IHC or clinical with p16 (thoracoscopic/laparoscopic) FISH improves the separation evidence of benign of tumor. from In malignant this situation mesothelial an additional biopsy near invasive mesotheliomas show the same pattern of p16 by FISH as the underlying tumor and may represent in situ disease proliferations. We isalso required followfor upaon tissue our previous diagnosis. data with or growth of the underlying mesothelioma along the serosal this TMA to determine Because whetherofcombining limitations multiple ofihc standard histologic surface; (2) p16 deletion in mesothelial surface proliferations is markers providesevaluation, high enougha specificity variety offor immunohistochemical clinical use. markers strongly associated with p16 deletion in underlying mesotheliomas, and biopsies consisting of pure surface mesothelial mesothelial processes, but in our opinion they are un- have been proposed for separating benign from malignant Am J Surg Pathol proliferations Volume 39, Number that are p16 7, July deleted 2015allow a diagnosis of mesothelioma without an additional biopsy if there is clinical (thorvestigators have studied the potential of p16 INK4a (also reliable (see the Discussion section). 977 More recently, inacosopic/laparoscopic) or radiologic evidence of diffuse pleural called CNKN2A) as another marker in this diagnostic or peritoneal tumor; (3) however, the absence of p16 deletion in area. p16 INK4a is a cyclin-dependent kinase inhibitor and surface proliferations does not rule out underlying invasive a member of the INK4 (INhibitor of CDK4) family of mesothelioma. cell cycle regulatory proteins that bind and generally inhibit D-type cyclin-dependent kinases. Homozygous de- Key Words: malignant mesothelioma, atypical mesothelial proliferation, p16 FISH letion of the p16 gene locus has been described in a number of human tumors including carcinomas primary (Am J Surg Pathol 2014;38: ) to the bladder, breast, and prostate. Recent studies have shown that some proportion of mesotheliomas demonstrate homozygous deletion of p16 when assayed by fluorescence in situ hybridization (FISH), either using cytology preparations or tissue biopsies. 4 9 In this study we have used p16 FISH testing on a series of pleural and peritoneal mesotheliomas that, on biopsy, have a surface mesothelial proliferation as well as an invasive component to determine whether surface proliferations ever show p16 deletion and, if so, whether that finding correlates with p16 deletion in the underlying mesothelioma. Am J Surg Pathol Volume 38, Number 5, May The evaluation of mesothelioma versus atypical reactive mesothelial proliferations remains a challenging area in diagnostic surgical pathology. Recently alterations of the BRCA1-associated protein-1 (BAP1) and p16ink4a genes have been described in mesothelioma. In particular, identification of BAP1 mutations in familial mesothelioma has spurred studies to examine the use of BAP1 IHC to diagnose sporadic mesothelioma in surgical pathology specimens. Homozygous loss of the cell cycle regulatory protein P16INK4a detected by FISH is also an active area of study for this differential diagnosis. In a series of studies in the American Journal of Surgical Pathology (AJSP), Drs. Allen Gown and Harry Hwang with PhenoPath technical staff, in collaboration with Drs. Andrew Churg and Brandon Sheffield (UBC Department of Pathology), evaluated the combined use of BAP1 IHC and p16 FISH in diagnosing mesothelioma versus atypical mesothelial proliferations. In the May 2014 issue of AJSP, we showed that P16 deletion detected by FISH can be predictive of detecting an underlying invasive mesothelioma in a subsequent biopsy specimen (ref 1). In the July 2015 issue of AJSP, using tissue microarrays, we showed that BAP1 IHC and P16 FISH in combination is a specific marker of mesothelioma, though with somewhat limited sensitivity (ref 2). In the January 2016 issue of AJSP, we further showed that in matched cytology and tissue biopsy specimens, that the combined use of BAP1 IHC and P16 FISH is both sensitive and specific in detecting mesothelioma (ref 3). Overall these studies highlight PhenoPath s continuing commitment and interest in refining the use of both immunohistochemical and molecular tools in diagnostic surgical pathology through collaborations with academic colleagues. References: 1. Hwang HC, et al. P16 FISH Deletion in Surface Epithelial Mesothelial Proliferations Is Predictive of Underlying Invasive Mesothelioma. AJSP 38(5):681-8, Sheffield BS, et al. BAP1 Immunohistochemistry and P16 FISH to Separate Benign from Malignant Mesothelial Proliferations. AJSP 39(7):977-82, Hwang HC, et al. Utility of BAP1 Immunohistochemistry and p16 (CDKN2A) FISH in the Diagnosis of Malignant Mesothelioma in Effusion Cytology Specimens. AJSP 40(1):120-6, 2016 Meet Our Pathologists at the Upcoming ASCP Course in Chicago, May 11-12! Allen M. Gown, MD and Regan Fulton, MD, PhD of PhenoPath, and Dr. Jason L. Hornick, MD, PhD, FASCP of Brigham and Women s Hospital present the ASCP course, Diagnostic Immunohistochemistry: Basics, Clinical Applications, and Challenges, on Wednesday, May 11 - Thursday, May 12, 2016 in Chicago, IL. For the complete program, please visit Please check for detailed information about this and other upcoming events at which PhenoPath pathologists are speaking.

4 PhenoPath Diagnoses you can count on 551 North 34th Street, Suite100 Seattle, Washington, P (206) F (206) FEATURED At the PhenoPath Conference Martin Mac Cheever, MD Fred Hutchinson Cancer Research Center, Seattle, WA PhenoPath, Thursday, March 31, 2016, 6:00PM (light dinner), 7:00PM (talk) Martin Mac Cheever, MD will present Cancer Immunotherapy During the First Half of 2016 at the PhenoPath Conference at 7:00pm on Thursday, March 31, Dr. Cheever will give a related lecture at noon the same day. Dr. Cheever is the Director of the NCI-funded Cancer Immunotherapy Trials Network (CITN), a Member of Fred Hutchinson Cancer Research Center (FHCRC) and a Professor of Medicine/Oncology at the University of Washington. The CITN has brought together leading immunotherapists from 32 foremost universities and cancer centers to design and conduct innovative early phase cancer immunotherapy trials. The CITN is currently conducting trials of a checkpoint inhibitor (anti-pd1), an antigen presenting dendritic cell growth factor (Flt3-Ligand), an antigen presenting dendritic cell activator (anti-cd40), the T cell growth factors (IL-7 and IL-15), and a small molecule inhibitor of the inhibitory enzyme, IDO. Dr. Cheever has extensive experience with cancer immune therapy clinical trials, as well as cancer antigen discovery, vaccine development and principles of T cell therapy. He received his MD from the University of Michigan. He completed his internship and residency at the University of Washington, training and working as a faculty member in Dr. E. Donnall Thomas s bone marrow transplant program. In 1994 he co-founded a biotech company, Corixa Corporation, in order to develop cancer vaccines as therapeutic products. He served as Corixa s Vice President of Clinical Research and Medical Affairs from 1997 to From 2007 to 2013 he returned to the Fred Hutch to serve as Director of Solid Tumor research for the Fred Hutch/University of Washington Cancer Consortium. Currently he is focused full time on the CITN.

5 Anti-PD-L1 antibodies 22C3, 28-8, and E1L3N Which PD-L1 test should I order? Is it NSCLC? for pembrolizumab (KEYTRUDA ) Companion 22C3 pharmdx Only on Dako Link 48 for nivolumab (OPDIVO ) EIL3N (or 22C3 or 28-8 upon request) Complementary 28-8 pharmdx Only on Dako Link 48 Is it melanoma? for nivolumab (OPDIVO ) Complementary 28-8 pharmdx Only on Dako Link 48 for pembrolizumab (KEYTRUDA ) No PD-L1 Testing Required EIL3N (or 22C3 or 28-8 upon request) PhenoPath Diagnoses you can count on Questions? Contacts: Allen M. Gown, MD or Regan Fulton, MD, PhD

6 Non-Small Cell Lung Carcinoma (NSCLC) Testing EGFR Mutation Test (PCR) & PD-L1 IHC 22C3 May be eligible for Pembrolizumab (if progression on chemo or TKI) Erlotinib ALK FISH ROS FISH OR PD-L1 IHC 28-8 May be ineligible for Pembrolizumab May be eligible for Nivolumab (if progression on chemo or TKI) May be ineligible for Nivolumab Progression on or after EGFR TKI EGFR T790M Mutation Analysis on re-biopsy Crizotinib Chemo Emerging Targeted Therapies RET FISH MET FISH BRAFV600 (PCR) Cabozantinib Crizotinib Vemurafenib Dabrafenib Reference: NCCN NSCLC Guidelines v Chemo PhenoPath Diagnoses you can count on Osimertinib Chemo Questions? Contacts: Harry Hwang, MD or Sandra Bohling, MD