The Present and the Future of Cancer Immunotherapy Biomarkers: Challenges, Opportunities, and Implications for Pathologists

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1 CME The Present and the Future of Cancer Immunotherapy Biomarkers: Challenges, Opportunities, and Implications for Pathologists Course Director and Moderator David L. Rimm, MD, PhD Yale University School of Medicine New Haven, Connecticut Faculty Luis A. Diaz, MD Memorial Sloan Kettering Cancer Center New York, New York Faculty Lynette M. Sholl, MD Brigham and Women s Hospital Harvard Medical School Boston, Massachusetts What s Inside Master Class #1 Immuno-Oncology as a New Tool in the Cancer Treatment Arsenal: Understanding the Basics and Implications for Pathologists Master Class #2 Part 1 PD-L1 Testing: Evidence and Options Master Class #2 Part 2 MMR/MSI and Tumor Mutational Burden Testing: Evidence and Options Practicum Current Immunotherapy Biomarkers: Clinical Experience, Practicalities, and Recommendations Master Class #3 New and Emerging Directions in Biomarker Development for Cancer Immunotherapy: How Can We Do Better? This CME activity is jointly provided by Medical Learning Institute, Inc. and PVI, PeerView Institute for Medical Education. Participate in interactive questions, download activity slides, and obtain your instant CME credit online.

2 Activity Information Activity Description and Educational Objectives Novel cancer immunotherapies are showing remarkable clinical activity in an increasing range of solid tumors and hematologic malignancies. As the immuno-oncology (IO) landscape continues to expand, there is a growing need for biomarkers to help guide the identification of patients who are most likely to benefit from these therapies. Pathologists play an important role in cancer immunotherapy patient selection through their involvement in biomarker testing. In this unique Master Class and Practicum for pathologists, top experts examine the evolving role of IO and what this means for pathologists, exchange views on the practicalities and challenges in immunotherapy biomarker testing, and assess opportunities for further development of novel tools for predicting benefit from immunotherapies. Upon completion of this activity, participants should be better able to: Discuss the basics of cancer immunotherapy and its evolving role across the hematooncologic spectrum Describe current and novel biomarkers for immunotherapy, and the latest evidence on their predictive utility Evaluate the characteristics, benefits, and limitations of different methodologies and assays for testing PD-L1 and other current biomarkers Assess the role of evaluating PD-L1 expression and other predictive biomarkers in the selection of patients for checkpoint inhibitor therapy in different tumor types and treatment settings Outline promising emerging directions on the immunotherapy biomarker landscape and efforts to coordinate biomarker development and implementation Implement best evidence-based practices for immunotherapy biomarker testing and interpretation of results to guide selection of patients for immunotherapy in the context of effective interdisciplinary collaboration Target Audience This activity has been designed to meet the educational needs of pathologists and other professionals interested in the development of biomarkers for cancer immunotherapies and their applications in the practice of pathology and laboratory medicine. Requirements for Successful Completion In order to receive credit, participants must view the activity and complete the post-test and evaluation form. A score of 7% or higher is needed to obtain CME credit. There are no prerequisites and there is no fee to participate in this activity or to receive CME credit. Statements of Credit are awarded upon successful completion of the post-test and evaluation form. Media: Enduring Material Release and Expiration Dates: November 15, November 14, 218 Time to Complete: 9 minutes Faculty & Disclosure / Conflict of Interest Policy Before the activity, all faculty and anyone who is in a position to have control over the content of this activity and their spouse/life partner will disclose the existence of any financial interest and/or relationship(s) they might have with any commercial interest producing healthcare goods/services to be discussed during their presentation(s): honoraria, expenses, grants, consulting roles, speakers bureau membership, stock ownership, or other special relationships. Presenters will inform participants of any off-label discussions. All identified conflicts of interest are thoroughly vetted by Medical Learning Institute, Inc. for fair balance, scientific objectivity of studies mentioned in the materials or used as the basis for content, and appropriateness of patient care recommendations. The associates of Medical Learning Institute, Inc., the accredited provider for this activity, and PVI, PeerView Institute for Medical Education do not have any financial relationships or relationships to products or devices with any commercial interest related to the content of this CME activity during the past 12 months. Course Director and Moderator David L. Rimm, MD, PhD Professor of Pathology and Medicine (Medical Oncology) Director, Yale Pathology Tissue Services Yale University School of Medicine New Haven, Connecticut David L. Rimm, MD, PhD, has a financial interest/relationship or affiliation in the form of: Consultant for Ultivue, Inc. Advisory Board for Agendia; AstraZeneca; Bethyl Laboratories, Inc.; Biocept, Inc.; Bristol-Myers Squibb; Cell Signaling Technology, Inc.; Merck & Co., Inc.; OptraSCAN, Inc.; and PerkinElmer, Inc. David L. Rimm, MD, PhD, does intend to discuss either non-fda-approved or investigational use for the following products/devices: immune checkpoint inhibitors, other cancer immunotherapies or combinations, and immunotherapy biomarker testing platforms. Faculty Luis A. Diaz, MD Head, Division of Solid Tumor Oncology Memorial Sloan Kettering Cancer Center New York, New York Luis A. Diaz, MD, has a financial interest/relationship or affiliation in the form of: Consultant for Merck & Co., Inc. and Personal Genome Diagnostics, Inc. Advisory Board for Cell Design Labs, Inc. Other Financial or Material Support from Personal Genome Diagnostics, Inc. in the form of stocks and board of director member. Luis A. Diaz, MD, does intend to discuss either non-fda-approved or investigational use for the following products/devices: immune checkpoint inhibitors, other cancer immunotherapies or combinations, and immunotherapy biomarker testing platforms. Lynette M. Sholl, MD Associate Pathologist Brigham and Women s Hospital Associate Professor Harvard Medical School Boston, Massachusetts Lynette M. Sholl, MD, has no financial interests/relationships or affiliations in relation to this activity. Lynette M. Sholl, MD, does intend to discuss either non-fda-approved or investigational use for the following products/devices: immune checkpoint inhibitors, other cancer immunotherapies or combinations, and immunotherapy biomarker testing platforms. CME Reviewer Vijaya R. Bhatt, MD University of Nebraska Medical Center Omaha, Nebraska Vijaya R. Bhatt, MD, has no financial interests/relationships or affiliations in relation to this activity. Medical Director Kadrin Wilfong, MD PVI, PeerView Institute for Medical Education Kadrin Wilfong, MD, has no financial interests/relationships or affiliations in relation to this activity. Disclaimer The information provided at this CME activity is for continuing education purposes only and is not meant to substitute for the independent medical judgment of a healthcare provider relative to diagnostic and treatment options of a specific patient's medical condition. Recommendations for the use of particular therapeutic agents are based on the best available scientific evidence and current clinical guidelines. No bias towards or promotion for any agent discussed in this program should be inferred. Providership, Credit & Support This activity has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of Medical Learning Institute, Inc. and PVI, PeerView Institute for Medical Education. The Medical Learning Institute, Inc. is accredited by the ACCME to provide continuing medical education for physicians. The Medical Learning Institute, Inc. designates this enduring material for a maximum of 1.5 AMA PRA Category 1 Credits TM. Physicians should claim only the credit commensurate with the extent of their participation in the activity. Providership This CME activity is jointly provided by Medical Learning Institute, Inc. and PVI, PeerView Institute for Medical Education. Support This activity is supported through independent educational grants from AstraZeneca and Bristol-Myers Squibb. Disclosure of Unlabeled Use The faculty of this educational activity may include discussions of products or devices that are not currently labeled for use by the FDA. Faculty members have been advised to disclose to the audience any reference to an unlabeled or investigational use. No endorsement of unapproved products or uses is made or implied by coverage of these products or uses in our reports. No responsibility is taken for errors or omissions in reports. For approved prescribing information, please consult the manufacturer s product labeling. The materials presented here are used with the permission of the authors and/or other sources. These materials do not necessarily reflect the views of PeerView or any of its partners, providers, and/or supporters. 2 Go online to complete the post-test and evaluation for CME credit

3 The Present and the Future of Cancer Immunotherapy Biomarkers: Challenges, Opportunities, and Implications for Pathologists Master Class #1 Immuno-Oncology as a New Tool in the Cancer Treatment Arsenal: Understanding the Basics and Implications for Pathologists Luis A. Diaz, MD Memorial Sloan Kettering Cancer Center New York, New York Ipilimumab Augments T Cell Activation and Proliferation 1 T cell APC T cell activation TCR HLA CD28 CD8/ CD86 T cell 1. Adapted from O Day S et al. American Society of Clinical Oncology 21 Annual Meeting (ASCO 21). Plenary session presentation. Abstract 4. APC TCR HLA T cell inhibition CD28 CTLA-4 CD8/ CD86 T cell T cell remains active TCR CTLA-4 HLA CD8/ CD86 APC Ipilimumab blocks CTLA-4 Dr. Rimm: Good evening, and welcome to our educational symposium this evening, The Present and Future of Cancer Immunotherapy Biomarkers: Challenges, Opportunities, and Implications for Pathologists. The speakers today are shown here. I m Dr. David Rimm. I m Professor of Pathology at Yale University School of Medicine. Joining me is Dr. Luis Diaz, who is the Head of Solid Tumor Oncology at the Memorial Sloan Kettering Institute in New York City, and Dr. Lynette Sholl, an Associate Professor of Pathology at Brigham and Women s Hospital and the Harvard Medical School in Boston. Dr. Diaz: And the first to come into the landscape was ipilimumab, which blocks CTLA-4 and reverses that immune suppression pathway. Ipilimumab Phase 2 and 3 Data: Primary Analysis of Pooled OS Data 1 Mechanisms of Immune Suppression 1. Schadendorf D et al. J Clin Oncol. 215;33: Dr. Diaz: Thanks, so much, David. And thanks for the opportunity to present some of the data that have been emerging over the past 5 years on immunotherapy in solid tumor oncology. I m going to use a few examples of where the data are most exciting, although it s exciting in many different arenas. So, it all begins with the story of immune suppression. Tumors have developed a way to avoid the immune system. And there are a variety of different mechanisms of immune suppression, but immune checkpoints have emerged at the top as one of the most druggable and reversible ways of immune suppression. Dr. Diaz: And we saw the most exciting data, the most striking data, actually, in melanoma. This is a pooled analysis of phase 2 and phase 3 data, where we re actually seeing very longterm survivals, and potentially, even what we may call cure. How we define cure is still evolving, especially in the age of immunotherapy. In the age of chemotherapy and targeted therapy, we said it was 5 years, but we re seeing recurrences beyond 5 years in many diseases. How we define cure is a moving target, but I m hopeful that we ll have a definition in the near future. There are some medical oncologists in the audience, and I d be curious on their opinion of that definition, as well. 3

4 Role of PD-1 Pathway in Tumor Immunity 1 Recognition of tumor by T cell through MHC/antigen interaction mediates IFNγ release and PD-L1/2 up-regulation on tumor Tumor cell IFNγR MHC PD-L1 PD-L2 IFNγ T cell receptor PD-1 PD-1 Nivolumab, pembrolizumab: PD-1 receptor blocking antibodies 1. Sznol M et al. ASCO 213. Abstract CRA96. Priming and activation of T cells through MHC/antigen and CD28/B7 interactions with antigen-presenting cells PI3K NFκB Other Shp-2 T T cell Shp-2 T cell receptor CD28 PD-1 PD-1 MHC B7 PD-L1 PD-L2 Dendritic cell Atezolizumab, avelumab, durvalumab: PD-L1 blocking antibodies Dr. Diaz: But as the science evolved, on the heels of CTLA-4 blockade was the blockade of another checkpoint axis, and that was the blockade of PD-1 and its ligands, PD-L1 and PD-L2. And what this did was release the brakes. 6 Dr. Diaz: I m going to walk you through three scenarios actually, four scenarios where PD-1 blockade, the new kid on the block, is really making a lot of movement. And this is in second-line lung cancer. And when you bring a new drug into the therapeutic arena, you look for the biggest impact, but you work on areas where the treatment is refractory or the current standard of care is not working. Nivolumab vs Docetaxel in Previously Treated Advanced NSCLC: Phase 3 Trials Pts with stage IIIB/IV SQ NSCLC 1 prior platinum-based treatment ECOG PS /1 (N = 272) Pts with stage IIIB/IV non-sq NSCLC who failed 1 prior platinum-based treatment ECOG PS /1 (N = 582) CheckMate 17 1 Nivolumab 3 mg/kg IV Q2W (n = 135) Docetaxel 75 mg/m 2 IV Q3W (n = 137) CheckMate 57 2 Nivolumab 3 mg/kg IV Q2W (n = 292) Docetaxel 75 mg/m 2 IV Q3W (n = 29) Until disease progression or unacceptable toxicity Until disease progression or unacceptable toxicity Where Are We Now? 1-3 Primary endpoint (both trials): OS 1. Brahmer J et al. N Engl J Med. 215;373: Borghaei H et al. N Engl J Med. 215;373: Proportion Surviving Melanoma, PD-1 era Melanoma, IPI era Melanoma circa 21 Dr. Diaz: And so, this is an overview of CheckMate 17 and CheckMate 57. And what these studies showed was [that in] second-line lung cancer, stage IIIB and IV, that are squamous or nonsquamous treated with anti PD-1 in this case, it was nivo at 3 mg/kg versus the standard of care second-line therapy with a taxol. This is docetaxel. 1. Balch CM et al. J Clin Oncol. 21;19: Schadendorf D et al. J Clin Oncol. 215;33: Topalian SL et al. J Clin Oncol. 214;32: Survival, y Nivolumab vs Docetaxel in Advanced SQ NSCLC (CheckMate 17): OS 1 Dr. Diaz: So then, if we use melanoma as another example here s melanoma circa 21, melanoma during the ipi or CTLA-4 blockade era, and now more recently, melanoma in the PD-1 blockade era. So, we re seeing not only an evolution in our understanding of how tumors suppress the immune system, but that blocking these checkpoint inhibitors first with CTLA-4 was quite effective. And now, more recently, blocking with anti PD-1 seems to be even more effective. 1. Reckamp K et al. 16th World Conference on Lung Cancer (WCLC 215). ORAL2.1. Nivolumab (n = 135) Docetaxel (n = 137) 1 9 Median OS, mo (95% CI) ( ) ( ) 8 Events, n HR =.62 (95% CI, ; P =.4) 5 12-mo OS rate: 42% 4 18-mo OS rate: 28% mo OS rate: 24% 18-mo OS rate: 13% Time, mo No. at risk Nivolumab Docetaxel OS. % Minimum follow-up for survival: 18 mo Lung Cancer Dr. Diaz: And what was clear in both studies was that that PD-1 blockade was superior. And this is overall survival, shown as survival of 6 months versus 9.2 months in CheckMate 17. This was the squamous cell. Second-Line Setting of Metastatic NSCLC 4 Go online to complete the post-test and evaluation for CME credit

5 The Present and the Future of Cancer Immunotherapy Biomarkers: Challenges, Opportunities, and Implications for Pathologists Nivolumab vs Docetaxel in Advanced Non-SQ NSCLC (CheckMate 57): OS 1,2 OS, % yr OS rate: 39% 1-yr OS rate: 51% 18-mo OS rate: 23% Median OS, mo (95% CI) 18-mo OS rate: 39% Time, mo Minimum follow-up for 12-mo OS rate: 13.2 mo; for 18-mo OS rate: 17.1 mo 1. Horn L et al. European Society for Medical Oncology 215 Congress (ESMO 215). Abstract Borghaei H et al. N Engl J Med. 215;373: Nivolumab (n = 292) 12.2 ( ) Docetaxel (n = 29) 9.4 ( ) Events, n HR =.73 (95% CI, ; P =.2) Metastatic or locally advanced NSCLC (2L/3L), PD on prior platinum-based treatment (N = 1,225) 1. Barlesi F et al. ESMO 216. Abstract LBA44_PR. Atezolizumab vs Docetaxel in NSCLC (OAK): Phase 3 1 Stratified by PD-L1 expression, histology, prior chemotherapy regimens Primary endpoints (first 85 pts enrolled) OS in ITT population OS in pts with 1% PD-L1 expression Atezolizumab 1,2 mg IV Q3W until loss of clinical benefit No crossover allowed Docetaxel 75 mg/m 2 IV Q3W until PD Secondary endpoints ORR, PFS, DOR, safety Dr. Diaz: And in the nonsquamous cell, again, you see a survival benefit there of 9.4 months versus 12.2 months. Dr. Diaz: And this is the third agent. Pembrolizumab vs Docetaxel in Previously Treated PD-L1+ Advanced NSCLC (KEYNOTE-1) 1 Locally advanced or metastatic NSCLC with PD-L1 TPS 1, ECOG PS -1, no brain metastases (N = 1,34) Primary endpoints a : PFS, OS Secondary endpoints a : ORR, DOR, safety a In both the PD-L1 TPS 1% and 5% populations. 1. Herbst RS et al. Lancet. 215;387: Stratified by ECOG PS vs 1, region (East Asia vs not), PD-L1 TPS 5% vs 1%-49% Pembrolizumab 2 mg/kg Q3W for 24 mo (n = 345) Pembrolizumab 1 mg/kg Q3W for 24 mo (n = 346) Docetaxel 75 mg/m 2 Q3W per local guidelines (n = 343) 1. Rittmeyer A et al. Lancet. 216;389: OAK: OS 1 Landmark OS 12 mo 18 mo Atezolizumab 55% 4% Docetaxel 41% 27% HR =.73 (95% CI, ); P =.3 Time, mo Dr. Diaz: This was also shown to be true with pembrolizumab. And here, they showed two different doses of anti PD-1, at 2 mg/kg versus 1 mg/kg, versus the standard of care. KEYNOTE-1: OS for PD-L1 TPS 5% Stratum 1 1 Median HR Treatment Arm a P (95% CI), mo (95% CI) 9 Pembro 2 mg/kg 14.9 (1.4-NR).54 ( ) Pembro 1 mg/kg 17.3 (11.8-NR).5 (.36-.7) <.1 6 Docetaxel 8.2 ( ) 5 2 vs 1 mg/kg: 4 HR: 1.12 (95% CI, ) 3 Pembro 2 mg/kg 2 Pembro 1 mg/kg 1 Docetaxel Time, mo Pembro 2 mg/kg Pembro 1 mg/kg Docetaxel a Comparison of pembrolizumab vs docetaxel. 1. Herbst RS et al. Lancet. 215;387: OS, % Dr. Diaz: And the results are just as striking. You see an overall benefit for the PD-1 blockade arms superior over chemotherapy. One thing to note here is that we are also looking at patients who were exceptional expressors of PD-L1. But just to show you, these are cases where over 5% of the tumor cells there were staining positive for PD-L1. Dr. Diaz: And again, versus standard of care, a very positive result. PD-L1 as a Biomarker in Second-Line? 1-3 OS, % 1. Rittmeyer A et al. Lancet. 216;389: Barlesi F et al. ESMO 216. Abstract LBA44_PR. 3. Bass P et al. ASCO 216. Abstract 915. Nivolumab Pembrolizumab Atezolizumab In most cases, anti PD-(L)1 is best second-line option regardless of PD-L1 expression Dr. Diaz: And so, what we have here is a variety of different studies with three different PD-1 and PD-L1 blocking agents that all had similar effects in the second line. They had benefit in overall survival versus standard-of-care chemotherapy, and there was a hint that the biomarker may be beneficial as a marker to really highlight those cases that were preferentially beneficial to PD-1 blockade. But in most cases, PD-L1 or PD-1 blockade is the option of choice irrespective of PD-1/PD-L1 expression. 5

6 FDA Approvals of Anti PD-1/PD-L1 Antibodies in the Second-Line Setting of NSCLC KEYNOTE-24: Survival Outcomes 1 PFS Pembro (n = 154) Chemo (n = 151) OS Pembro (n = 154) Chemo (n = 151) Nivolumab FDA approved in Median PFS HR (95% CI).5 ( ); P <.1 1 Median OS NR NR HR (95% CI).6 ( ); P = PFS, % 6 4 OS, % First-line pembro FDA approved 216 Pembrolizumab FDA approved in 215 Atezolizumab FDA approved in Time, mo 1. Reck M et al. N Engl J Med. 216;375: Pembrolizemab Time, mo Chemotherapy 21 Dr. Diaz: And so, there were a number of breakthrough statuses and approvals with nivo, pembro, and atezolizumab in this indication so, second-line lung cancer. Dr. Diaz: We saw a profound impact in terms of progression-free survival and a profound impact in terms of overall survival, as well. And this was an FDA approval for first-line pembro in non smallcell lung cancer. First-Line Nivolumab vs CT (CheckMate-26) 1,2 Lung Cancer First-Line Setting of Metastatic NSCLC Stratified by PD-L1 expression (<5% vs 5%) and histology (squamous vs nonsquamous) Pts with stage IV/recurrent NSCLC, no previous systemic therapy, no actionable EGFR/ALK mutations, PD-L1 expression 1% (N = 541) Nivolumab 3 mg/kg IV Q2W (n = 271) Chemotherapy (histology based) for up to 6 cycles (n = 27) Until PD or unacceptable toxicity Until PD (crossover to nivolumab allowed) Primary endpoint: PFS ( 5% PD-L1 positive) Secondary endpoints: PFS ( 1% PD-L1 positive), ORR, OS 1. Socinski M et al. ESMO 216. Abstract LBA7_PR. 2. Carbone DP et al. N Engl J Med. 217;376: Dr. Diaz: In development, again, we move from second-line lung cancer, which was an area of unmet need, [and now move] into the first-line lung cancer. First-Line Pembrolizumab vs CT (KEYNOTE-24) 1 Stratified by ECOG PS ( vs 1), histology (squamous vs nonsquamous), and enrollment region Dr. Diaz: Nivo had a similar study: nivo in PD-L1 expressors that are greater than 1% so, a different cutoff than what the pembro study had versus standard of care chemotherapy. CheckMate-26: PFS by IRRC in Patients With 5% PD-L1 Expression 1,2 Pts with stage IV NSCLC and ECOG PS /1, no previous systemic therapy, no actionable EGFR/ALK mutations, and PD-L1 TPS 5% (N = 35) Pembrolizumab 2 mg IV Q3W for up to 35 cycles (n = 154) Chemotherapy (histology based) for up to 6 cycles (n = 151) Until PD or unacceptable toxicity Until PD (crossover to pembrolizumab allowed) Median PFS, mo (95% CI) Nivolumab (n = 211) 4.2 (3.-5.6) Chemotherapy (n = 212) 5.9 ( ) 1-y PFS rate, % Primary endpoint: PFS Secondary endpoints: ORR, OS, and safety 1. Reck M et al. N Engl J Med. 216;375: Socinski M et al. ESMO 216. Abstract LBA7_PR. 2. Carbone DP et al. N Engl J Med. 217;376: Dr. Diaz: Looking at PD-1 blockade versus standard of care chemotherapy in patients with non small-cell lung cancer with, again, a high PD-L1 expression. Dr. Diaz: The difference here was not significant. 6 Go online to complete the post-test and evaluation for CME credit

7 PFS Subgroup Analysis by BICR (ITT) 1,2 Lung Cancer Stage III, Locally Advanced, Unresectable NSCLC a HR and 95% CI not calculated if subgroup had <2 events. 1. Paz-Ares L et al. ESMO 217. Abstract LBA1. 2. Antonia SJ et al. N Engl J Med. 217 Sep 8 [Epub ahead of print]. Dr. Diaz: What about in earlier-stage cancers? So those two other arenas were in metastatic cancers that were nonresectable. These are in stage 3 locally advanced cancers that are traditionally treated with chemotherapy and radiation. PACIFIC Trial Study Design 1,2 Dr. Diaz: Interestingly, there appears to be no impact on PD-L1 expression, but that remains to be seen once it has overall survival data. PD-L1 as a Biomarker: First-Line Phase 3 randomized, double-blind, placebo-controlled, multicenter, international study 5% cut point 1 5% cut point 2 Stage III, locally advanced, unresectable NSCLC No progression following definitive platinum-based ccrt ( 2 cycles) 18 years of age WHO PS score or 1 Estimated life expectancy 12 wk All-comers population 1-42 days post-ccrt R Durvalumab 1 mg/kg Q2W (up to 12 mo) n = 476 2:1 randomization stratified by age, sex, and smoking history Placebo 1 mg/kg Q2W (up to 12 mo) n = 237 Endpoints Co-primary PFS by BICR a (RECIST v1.1) OS Key Secondary ORR per BICR b DOR (per BICR) Safety/tolerability PROs PD-L1 testing should be routine, and in those with >5% expression, pembrolizumab is SOC a Time from randomization to the first documented tumor progression, or death in the absence of progression. b ORR as measured from baseline scan post-crt completion Reck M et al. N Engl J Med. 216;375: Socinski M et al. ESMO 216. Abstract LBA7_PR. 1. Paz-Ares L et al. ESMO 217. Abstract LBA1. 2. Antonia SJ et al. N Engl J Med. 217 Sep 8 [Epub ahead of print]. Dr. Diaz: And so, these were locally advanced non small-cell lung cancers. This was called the PACIFIC study, and this was very creative. They took patients with locally advanced lung cancer, gave them the standard of care therapy, and then randomized them to PD-L1 blockade versus standard of care observation. Dr. Diaz: So, PD-L1 as a biomarker, in the first line, it s clear that it s an important biomarker, but a high number of tumor cells have to be expressed in the PD-L1 molecule. PFS by BICR (Primary Endpoint; ITT) 1,2 Durvalumab Placebo Median PFS (95% CI), mo 16.8 ( ) 5.6 ( ) Melanoma 12-mo PFS, % (95% CI) 55.9 ( ) 35.3 ( ) 18-mo PFS, % (95% CI) 44.2 ( ) 27. ( ) Stratified HR a =.52 (95% CI, ) Two-sided P <.1 a Time from randomization to the first documented tumor progression, or death in the absence of progression. 1. Paz-Ares L et al. ESMO 217. Abstract LBA1. 2. Antonia SJ et al. N Engl J Med. 217 Sep 8 [Epub ahead of print]. Dr. Diaz: And what they found was a dramatic difference. So, when you added PD-L1 blockade after the standard-ofcare chemoradiation, you had a dramatic difference. Here is progression-free survival, and we ll soon have readouts on overall survival. Dr. Diaz: What about melanoma? Well, when [we start] to look at responses in melanoma, because it was the earliest tumor treated, we re starting to see really how these checkpoint inhibitors are working. 7

8 Current Survival Curves 1 CheckMate 67 PFS: ITT 1 NIVO+IPI (n = 314) NIVO (n = 316) IPI (n = 315) 1 1 Median PFS, mo (95% CI) 11.5 ( ) 6.9 ( ) 2.9 ( ) OS, % N = 21 N = 17 NIVO Monotherapy (phase 3 CheckMate 66) 2 NIVO Monotherapy (phase 1 CA29-3) 3 IPI (pooled analysis) 4 Percentage of PFS PFS, % HR (95% CI) vs IPI a.43 ( ).55 ( ) HR (95% CI) vs NIVO.78 ( ) 39% N = 1, NIVO+IPI NIVO IPI 32% 1% Time, y 1. Hodi FS et al. American Association for Cancer Research Annual Meeting 216 (AACR 216). Abstract CT1. 2. Poster presentation by Dr. Victoria Atkinson at SMR 215 International Congress. 3. Current analysis. 4. Schadendorf et al. J Clin Oncol. 215;33: Time, mo Patients at risk: NIVO+IPI NIVO IPI a P <.1 Database lock: May 24, 217. Minimum follow-up of 36 months. 1. Wolchok JD et al. N Engl J Med. 217;377: Dr. Diaz: I showed you this slide earlier. These are patients treated with ipi alone. This is nivo at one dose, and this is nivo at a second dose. So, we re seeing an improvement in overall survival even in these pooled analyses when you re comparing CTLA-4 with PD-1 blockade. Dr. Diaz: This is PFS in that same population. CheckMate 67 OS by Tumor PD-L1 Expression, 1% Cutoff 1 PD-L1 Expression Level <1% PD-L1 Expression Level 1% NIVO+IPI NIVO IPI NIVO+IPI NIVO IPI Median OS, mo (95% CI) NR ( ) ( ) Median OS, mo (95% CI) NR NR 21.5 ( ) CheckMate 67 OS: ITT 1 HR (95% CI) vs NIVO.7 ( ) HR (95% CI) vs NIVO 1.2 ( ) NIVO+IPI (n = 314) NIVO (n = 316) IPI (n = 315) Median OS, mo (95% CI) NR (38.2-NR) 37.6 (29.1-NR) 19.9 ( ) HR (95% CI) vs IPI a.55 ( ).65 (.53-.8) HR (95% CI) vs NIVO.85 ( ) OS, % % 59% 45% 58% 52% 3 34% 2 1 NIVO+IPI NIVO IPI 1. Wolchok JD et al. N Engl J Med. 217;377: Time, mo No. at risk NIVO+IPI NIVO IPI a P <.1 Database lock: May 24, 217. Minimum follow-up of 36 months. 1. Wolchok JD et al. N Engl J Med. 217;377: Dr. Diaz: What about PD-L1 expression? Less important than the lung cancer. Dr. Diaz: But the next question that is the next evolution of immunotherapy is, okay, so we have CTLA-4 blockade. We have PD-1 blockade. What if we combine them? And what we re seeing is a difference in overall survival when you combine them. However, it s not significant in all settings. The problem is, we re also seeing a significant amount of toxicity. MSI-H Tumors So, improving the response rate, improving the progression-free survival, and eventually improving the overall survival will be there, but you are going to see an increase in toxicity, and some of these toxicities can be life-threatening. The majority of these are autoimmune, and oftentimes the patients don t do well on rechallenge to therapy. Dr. Diaz: So, the next area I m going to talk about is completely different: mismatch repair deficient tumors or MSI-high tumors. 8 Go online to complete the post-test and evaluation for CME credit

9 Background Melanoma and lung cancers Dr. Diaz: And so, the theory was that these mutations that accumulated with each cellular division, that couldn t be repaired because it was mismatch repair deficiency, that these mutations, if expressed and translated into a protein, could create a neoantigen that appeared foreign to the host immune system. Neoantigens: Further Characterization 1 Dr. Diaz: And the theory around this came about the time when we were seeing responses in melanoma and in lung cancer. And the hypothesis was that these tumors [that have a] high mutational burden, melanoma and lung cancer as compared with pediatric tumors, liquid tumors, sporadic adult tumors may be driving the immune response. Background Mismatch-repair proficient colon cancers 1. Chan DS, Mellman I. Nature. 217;541: Dr. Diaz: These mutations can take a variety of different forms and can sit in the MHC class 1 or 2 cleft and drive a T cell response. Study Design Colorectal Cancers Non-Colorectal Cancers Mismatch-repair deficient colon cancers Cohort A Deficient in Mismatch Repair (n = 28) Cohort B Proficient in Mismatch Repair (n = 25) Cohort C Deficient in Mismatch Repair (n = 3) Dr. Diaz: What would be the best test case to prove that hypothesis? And that was mismatch repair deficient tumors that didn t have a few hundred mutations per genome, but rather that had thousands of mutations per genome. And that s different from non mismatch repair deficient tumors, MS, or MSS tumors that were microsatellite stable, which had fewer mutations about 7 mutations per genome versus the MSI-high tumors, with thousands of mutations. What Is a Neoantigen? Anti PD-1 (pembrolizumab): 1 mg/kg Q2W Patients who had failed all standard of cancer therapies and had evidence of disease progression Dr. Diaz: So, in order to test the hypothesis, we designed this study, actually. This was a colorectal cancer study where we looked at mismatch repair deficient colon cancers versus those that were proficient, so MSI-high versus MSS tumors, high mutational burden versus low mutational burden and then looked a variety of other non-colon cancers that were also MSI high. These were all treatment refractory, and they were all treated with single-agent anti PD-1. A peptide that undergoes mutation in cancer leading to immune system seeing this as foreign via MHC presentation PTSMKKESL PTSMTKESL Neoantigen T cells attack Courtesy of Tim Chan. 9

10 Mismatch Repair Deficient Colorectal Cancer Dr. Diaz: There was a second study out of MD Anderson that was recently published, as well, that granted nivo approval for colorectal cancer. Single Agent Anti PD-1 Baseline Week 2 Tumor Markers Highly active in MSI-H colon cancer, with durable responses beyond 2 years Nivolumab and pembrolizumab FDA approved for treatment-refractory dmmr/msi-h metastatic colorectal cancer NCCN guidelines recommend nivolumab or pembrolizumab in secondor third-line dmmr metastatic colorectal cancer Dr. Diaz: So, this is a classic example of a patient with a large tumor here; periaortic with a great response rate. And here you can see the biomarker had a great response there. Type of Response Study Summary MMR-Deficient CRC (n = 28) MMR-Proficient CRC (n = 25) Objective response rate, % 57 Disease control rate Progression-free survival, mo NR 2.3 Overall survival, mo NR 5.98 FDA breakthrough therapy designation for pembrolizumab in advanced colorectal cancer Designation based on results in patients with mcrc with high levels of microsatellite instability Registration studies in first-line are ongoing Dr. Diaz: In terms of single-agent anti PD-1, it s clearly active in colorectal cancer, with durable responses beyond 2 years. We have several patients who are disease free, and these were previously patients who were metastatic, unresectable, nonresponsive to chemotherapy, and a handful who were already in hospice when we started treating them. Nivolumab and pembro have been FDA approved for treatment-refractory MSI-high metastatic colon cancer, and the NCCN guidelines reflect this. Registration studies in the first line are ongoing. Histology-Independent MRD Tumors Dr. Diaz: So, when you look through the study summary, the response rate was about 6% in the MSI-high tumors and % [in the] microsatellite-stable tumors. Disease control rate so, stable disease, partial response, or complete response was about 9% in the mismatch repair deficient tumors and about 16% in the proficient tumors. Progression-free survival and overall survival were not reached for the deficient tumors, but were very easily reached in the proficient tumors. So, this study was the basis for the FDA breakthrough approval for MSI-high colorectal cancer. Primary Histologies Colorectal cancer Endometrial cancer Ampullary/biliary cancer Pancreatic cancer Small bowel Gastric cancer Prostate cancer Thyroid cancer Characterization of Response (Single-Agent Nivolumab) 1 n (%) ORR 95% CI Best overall response CR PR SD PD Unable to determine Disease control for 12 wk dmmr/msi-h per Local Laboratory (N = 74) Investigator BICR 23 (31.1) 2 (27.) (2.7) 23 (31.1) 18 (24.3) 29 (39.2) 28 (37.8) 18 (24.3) 2 (27.) 4 (5.4) 6 (11.1) 51 (68.9) 46 (62.2) Dr. Diaz: But what happened after we had this breakthrough status is that we continued to collect patients, but not with just colon cancer, but also with MSI-high tumors from anywhere else in the body from the brain, lung, breast, pancreas, liver, neuroendocrine tumors, and endometrial tumors. 8/1/17: FDA granted accelerated approval to nivolumab for patients 12 yrs with dmmr or MSI-H mcrc that has progressed on fluoropyrimidine/oxaliplatin/irinotecan treatment Data from Dr. Michael J. Overman. 1. Overman MJ et al. J Clin Oncol. 217;35(suppl 4S). Abstract Go online to complete the post-test and evaluation for CME credit

11 Histology-Independent MRD Tumors (Cont d) Kaplan-Meier Estimates of DOR (RECIST v1.1, Central Review) 1 P r o g r e s s n -f r e e S u r v a l Progression-Free Survival O v e r a S u r v a l Overall Survival Percent Survival survival 5 Percent Survival survival 5 DOR, % Events, n MSI-H CRC 17 (28%) Median, mo (95% CI) NR (2.9+ to 12.5+) DOR, % MSI-H non-crc Events, n 23 (3%) Median, mo (95% CI) NR (2.4+ to 9.2+) Time, T emo Median PFS, mo NR 12-mo PFS rate 63% Time, T emo Median OS, mo NR 12-mo OS rate 8% Time, mo No. at risk No. at risk Time, mo mo PFS rate 63% 18-mo OS rate 74% 1. Diaz L et al. ASCO 217. Abstract 371. Dr. Diaz: And what we were seeing was that we had a similar response rate, a similar PFS, and a similar OS in that population of patients. So, the conversation evolved from, Let s get full approval in colon cancer because it s easy, to, Can we get approval in a tumor-agnostic fashion? So, we don t care where the tumor was born. We just care about the genetics of the tumor. Testing for MSI, for mismatch repair deficiency, had been established for years, and this, along with two other studies that were ongoing, were used as a package to present to the FDA for this case. MSI-H Tumor Types 1 Dr. Diaz: The duration of overall response is fantastic. May 23, 217 FDA granted accelerated approval to pembrolizumab for first tissue-/site-agnostic indication Dr. Diaz: And in May, earlier this year, the FDA granted accelerated approval to pembro in a tissue- and site-agnostic manner. And independently, this was the fascinating part, that it was in adults and children. So, not only it was in every tumor type for treatmentrefractory disease, but also in adults and children. 1. Diaz L et al. ASCO 217. Abstract 371. Dr. Diaz: And here, is just a slide that shows the frequency in our study of MSI-high tumors that were treated. The majority were colorectal, but we had a variety of other tumor types. Probably the most impressive in our groups were the patients with endometrial cancer, who all virtually had a complete response, and also the pancreatic cancer patients who were really in dire straits when they were enrolled in our studies. And those patients continue to do quite well. Potential Impact of Discovery 4% of all cancer diagnoses 4, total new cases (USA) 24, new stage IV cases (USA) 52, new stage IV cases (Global) Dr. Diaz: So, [what is] the impact of the discovery in this application? We believe it s about 4% of all cancers. In the United States, it s about 4, total new cases, about 24, stage IV cases, and about a half a million stage IV cases globally. 11

12 dmmr CRC dmmr non-crc pmmr CRC Baseline PD-L1 Expression and CD8 T Cell Infiltration H&E PD-L1 CD8 CD8 T T T N N N T T T N N N T T T N N N Invasive Front TIL CRC Case E Density Heat Map of PanCK+ Cells Cells/mm 2 Dr. Diaz: In terms of PD-L1 expression, it s there. It s very hot at the invasive front. It s not there in tumors that are mismatch repair proficient. The first row shows colon cancer; the last row shows endometrial cancer. Invasive-Front PD-L1 Expression and CD8 T Cell Infiltration Dr. Diaz: This is cytokeratin stain. That s very rich in the tumor, but virtually absent from the invasive front. CRC Case E Density Heat Map of CD3+ Cells Cells/mm 2 Invasive Front CD8+ T cells Invasive-Front PD-L1 Expression Tumor-Front PD-L1 Expression Dr. Diaz: These are CD3 cells that were all at the invasive front. Dr. Diaz: This is just a graph that shows that there s enrichment for CD8 cells in PD-L1 expression at the invasive front. CRC Case E Density Heat Map of CD8+ Cells CRC Case E Cells/mm 2 Red = representative tumor regions Blue = representative peri-tumor regions Dr. Diaz: CD8 cells, all at the invasive front, not within the tumor. Dr. Diaz: And this is an interesting example. So, this is a multispectral IHC. In the red we ve circled tumor, and in the blue we re circling an invasive front. 12 Go online to complete the post-test and evaluation for CME credit

13 CRC Case E Density Heat Map of CD68+ Cells CRC Case E Density Heat Map of PD-L1+/CD3+ Cells Cells/mm 2 Cells/mm 2 Dr. Diaz: CD68 cells, myeloid cells, again, at the invasive front. CRC Case E Density Heat Map of PD-L1+/PanCK+ Cells Dr. Diaz: and on CD3-positive cells at the invasive front. So, telling us a little bit about where the action is happening and where PD-L1 may be important from a functional standpoint. Mutation Burden Is Associated With Efficacy Cells/mm 2 Dr. Diaz: And PD-L1 combined with panck, absent from the tumor CRC Case E Density Heat Map of PD-L1+/CD68+ Cells Dr. Diaz: This is just a mutational burden slide showing that mutation burden is associated with efficacy. Mutation Burden vs Response to PD-1 Blockade Cells/mm 2 Objective Response Rate, % Non-responders Dr. Diaz: but present on CD68 cells in the invasive front Mutations per Genome Dr. Diaz: But I m going to end with this. If you stratify a cancer by mutational burden that s how we looked at it to begin with, that s how we picked mismatch repair deficiency to be the tumor type that we started off treating, you can look at tumors that are nonresponders, and they typically have an incredibly low mutational burden. 13

14 Mutation Burden vs Response to PD-1 Blockade (Cont d) Immune Checkpoint Inhibitor Landscape: FDA Approvals Timeline (Solid Tumors) Nivolumab (Non-SQ lung cancer) Pembrolizumab (PD-L1+ NSCLC) Ipilimumab Pembrolizumab Nivolumab Nivolumab Nivolumab + ipilimumab (MEL, first-line) Objective Response Rate, % Strong Responders (MEL) March 211 Nivolumab (RCC) Nov 215 Nivolumab (MEL) (MEL) (SQ lung cancer) Sept 214 Dec 214 March 215 Pembrolizumab Atezolizumab Pembrolizumab (MEL, first-line) (Bladder) (SCCHN) Dec 215 May 216 Aug 216 Durvalumab, avelumab, pembrolizumab (Bladder) Avelumab Pembrolizumab + chemo (NSCLC, first-line) Nivolumab Ipilimumab (MEL, adjuvant) Oct 215 Pembrolizumab (NSCLC, first-line) Nivolumab Atezolizumab (SCCHN) (NSCLC) Nov 216 Aug 216 Pembrolizumab Nivolumab (Bladder) (Merkel cell) Pembrolizumab (MSI-H cancers) (dmmr/msi-h CRC) (Gastric) (HCC) Jan 217 March 217 May 217 August 217 Sept 217 Sept 217 Mutations per Genome Dr. Diaz: You can have tumors that are strong responders, like mismatch repair deficient tumors, POLE-deficient tumors, and other DNA-repair defects, or patients with biallelic MRD both alleles of the mismatch repair deficiency machinery are not working. These are all strong responders. Mutation Burden vs Response to PD-1 Blockade (Cont d) Dr. Diaz: So, this is the last slide. This is the evolution of checkpoint inhibition and the FDA approvals across tumor types. The real excitement was on melanoma initially, with ipi, moved to PD-1 blockade. But slowly, and what made it real to us as medical oncologists, was the activity in lung cancer. I think prior to this, we thought melanoma was an immunogenic tumor. Obviously, it s going to work there. But the second it worked in lung cancer, I think it surprised everyone. Objective Response Rate, % Mixed Responders Mutations per Genome And we re then seeing an evolution of this into other tumors, like renal cell carcinoma, bladder cancer, head and neck cancers, Merkel cell cancer, and then more recently the pan-tumor in adults and children for MSI high. We saw recently the gastric approval and the HCC approval. We will continue to see approvals. But we need more biology to help define what these subgroups of response are, and I think that mutational burden will be one of those. So, I m going to stop there, and thank you very much. Dr. Diaz: And then, you have these tumors in the middle, like the ones I presented in lung or melanoma. They re the mixed responders, and they have an intermediate number of mutations. Mutation Burden vs Response to PD-1 Blockade (Cont d) Objective Response Rate, % Mixed Responders Strong Responders Non-responders Mutations per Genome Dr. Diaz: So, the question is, is, will this hold up for sporadic adult solid tumors? There are exceptions: non-hodgkin lymphoma why is that responding? Why are certain urothelial or gastric cancers responding so well, and others are not? So, is that purely defined by mutational burden, or are there other factors? And that will remain to be seen. 14 Go online to complete the post-test and evaluation for CME credit

15 Master Class #2 Part 1 PD-L1 Testing: Evidence and Options PD-L1 by IHC in NSCLC David L. Rimm, MD, PhD Yale University School of Medicine New Haven, Connecticut Master Class #2 Current Predictive Biomarkers for Cancer Immunotherapy Dr. Rimm: So, going back to 214, some of the first lung cancer patients, and all of these papers that I m showing you, and [that] Dr. Diaz just reviewed, all showed an effect of PD-L1. David L. Rimm, MD, PhD Professor of Pathology and Medicine (Medical Oncology) Director, Yale Pathology Tissue Services Yale University School of Medicine New Haven, Connecticut Lynette M. Sholl, MD Associate Pathologist Brigham and Women s Hospital Associate Professor Harvard Medical School Boston, Massachusetts PD-L1 by IHC in NSCLC (Cont d) 1 Dr. Rimm: As Dr. Diaz nicely introduced, there are kind of two pieces to the immunotherapy diagnostics. There s the diagnostic piece of PD-L1, and I ll be the one to address that. And then there s the diagnostic piece of MMR or DNA-based repair type of response to PD-[1]/L1 inhibitors and Lynette Sholl will talk about the pathologist s role in those diagnostic tests. 1. Sunshine J, Taube JM. Curr Opin Pharmacol. 215;23: Dr. Rimm: And, in fact, this review by Sunshine and Taube shows that in nearly every tumor type and you can see them in abbreviations on the bottom the PD-L1 expressors essentially always did better than the nonexpressors. PD-L1: Where we are today Companion vs Complementary PD-L1 IHC Tests: The assays PD-L1 IHC Tests: The antibodies Reading TPS vs IC vc CPS Latest addition/update PD-L1 by IHC in NSCLC (Cont d) 1 Dr. Rimm: So, let s talk about PD-L1 first. This is something that really, for the first time, arguably, since HER2, or if not, more technically, since ALK, was the first time we had a companion diagnostic test. And so, where are we today? 1. Hellmann MD et al. Lancet Oncol. 217;18: Dr. Rimm: And, in fact, even when you combined the PD-L1 inhibitor nivo[lumab] with ipilimumab, PD-L1 expression was still predictive. 15

16 Nivolumab Pembrolizumab Atezolizumab Durvalumab Avelumab Target PD-1 PD-1 PD-L1 PD-L1 PD-L1 Company BMS Merck Roche AstraZeneca First FDA approval date Cancers Diagnostics Setting Current PD-L1 Axis Drugs and Indications Pfizer/Merck KGaA NSCLC, melanoma, RCC, Hodgkin s lymphoma, SCCHN, bladder dmmr/msi-h CRC, HCC Complementary: PD-L1 IHC 28-8 pharmdx test Second-line NSCLC, melanoma, SCCHN, MMR (any histology), gastric Companion: PD-L1 IHC 22C3 pharmdx test First- and second-line NSCLC, bladder Complementary: Ventana PD-L1 (SP142) Assay Bladder, stage III NSCLC (FDA priority review status granted to sbla) Complementary: Ventana PD-L1 (SP263) Assay MCC, bladder Second-line Second-line Second-line enrollment criteria. Now, complementary diagnostics test was actually a new term to all of us just a few years ago. Mostly, this category has been used in the past when you had an all-comers trial, but it was observed that patients who were test positive were more likely to respond, but there were also responders in the test-negative category. And because of that, we saw the evolution of this new category, complementary diagnostic test. Dr. Rimm: And so, as much as the ideal or the precision medicine approach using DNA has been really stealing the day in some sense, here s a chance for pathologists to do good old IHC and actually make a contribution in picking responders to therapy. And so, what do we have and where are we really now in terms of drugs? We just got a great overview of the timing of the approvals of these drugs, but here are the key drugs that have been approved. There are first approval dates here, and then the cancers in which they re approved. PD-L1: Where we are today Companion vs Complementary PD-L1 IHC Tests: The assays PD-L1 IHC Tests: The antibodies Reading TPS vs IC vc CPS Latest addition/update PD-L1 Assay Terminology: Companion vs Complementary Companion Diagnostic Test (Cdx) Test result required for prescription of the drug Specified on the drug label Often, this category is only used when the test is among inclusion criteria for trial (gastric is the exception) Complementary Diagnostic Test Test result is predictive, but not required for prescription of the drug Nice to have, but do not need to have; no clear message on reimbursement Term invented by Liz Mansfield at the FDA Mostly, this category is used when the assay is integrated into the trial, but not used among inclusion criteria (all-comers trial) Dr. Rimm: So, what about the tests or the assays, and what about the antibodies? And I want to go over those things now. Assay Comparison Literature Antibody = Egg Assay = Cake Dr. Rimm: But I want to talk about the diagnostic tests that have improved with them, because each of them has their own diagnostic test that is an assay, and each of them has a different designation. Now, I don t have all of the sub-designations here, but just suffice it to say that you can see that there are sort of two categories. There s complementary diagnostic tests, and there s companion diagnostic tests. So, this didn t exist a few years ago, but has become a terminology now that I think we all need to be familiar with. A companion diagnostic test is a test result that s required for the prescription of the drug. So, what does it mean, require? Presumably, that means it s in the label, in the FDA label for approval of the drug, that the patients must be qualified by having a positive test. This category is usually given by the agency when the test is used to enroll patients. But as we saw just a couple weeks ago, not always. That is, sometimes they might still give a companion diagnostic designation, even if the diagnostic test wasn t an Dr. Rimm: So, there have been a number of studies that have compared the different assays, because, remember, there are five drugs with four different assays, and each assay is supplied in a different kit, or a different IVD test, and are those assays all the same? And what about LDTs? Many pathology labs do LDTs. In fact, probably most of your IHC testing is LDT based. And so, is that a possible alternative for PD-L1 testing, as well? So the first study, and perhaps the first one that came out, and the first one to be talked about a lot, is this study by Hirsch and his team, which actually represented each of the vendors of the IVD tests, as well as four of the five pharma companies that provided the material and agreement for this study. 16 Go online to complete the post-test and evaluation for CME credit

17 And then secondly, the NCCN gathered a group of academic pathologists together to produce this study, and that s why I m before you today, because I was the leader, with Ignacio Wistuba, of this comparison study. I like to use this example, because I m going to try to get this right as I speak to you. I ll try to always talk about the assay or the test, and that means the IVD itself, not the antibody. Although the antibody is the key difference, the way I see it is, the antibody s the egg. It s an ingredient. And the assay is the cake, the test. How to Read Each Lung Cancer PD-L1 Assay? The Assays for Lung Cancer Dr. Rimm: Now, how do these all compare? So, they don t look the same. These are actually images right from the Blueprint study. And the Blueprint study showed that they have different colors or different appearances, but also that SP142 seemed to be negative in the same regions or serial sections of cases that were positive for the other assays. And, in fact, we were told that the assay for SP142 was enriched for immune cells, and that s illustrated here, although it s not clear what the biology is that can have an antibody bind to an antigen in one type of cell and not another type of cell. The NCCN study found a similar thing where we found the SP142 assay seemed to be less strong than the other assays. Ab clone/epitope C3 SP142 SP263 IVD class III diagnostic partner Dako Dako Ventana Ventana Drug Nivolumab Pembrolizumab Atezolizumab Durvalumab PD-L1 scoring % tumor cells and % tumor cells % tumor cells method % immune cells % tumor cells FDA IVD status for NSCLC Complementary Companion Complementary Complementary PD-L1 thresholds 1%, 5%, or 1% 1%, 5% IC1/2/3 ( 1%, 5%, and 1%) TC1/2/3 ( 1%, 5%, and 5%) 25% Blueprint 39 cases no outcome data 3 pathologists from Dako and Ventana 4 assays FDA/IUO Not statistically powered By agreement of 6 companies (BMS, Merck, Genentech, AZ, Dako, Ventana) NCCN 9 cases no outcome data 13 pathologists from 7 academic sites 4 assays 3 FDA/IUO and 1 LDT (E1L3N on Leica Bond) Prescribed, powered, statistical protocol for ICC between pathologists, assays, and localization Led by NCCN, sponsored by BMS Dr. Rimm: So, here they are. Here are the assays for lung cancer, 28-8, which is the name of the antibody for nivolumab. And each has a different scoring system, as well. So, this was designated as a complementary diagnostic test with three cutpoints greater than 1%, greater than 5%, or greater than 1%. 22C3 matched with pembrolizumab, but in this case, [there were] only two cutpoints: greater than 1% and greater than 5%, both of these including a % cutpoint, as well. SP142 test was for atezolizumab. And here, they had a much more complicated scoring system, with three different categories or actually four, if you include % for immune cells, and then three categories, or a fourth, if you include % for tumor cells. And then, finally, SP263, the match for durvalumab, and again, still another cutpoint, at greater than 25%. So, that s what s out there. Example of PD-L1 Tumor Expression Blueprint NCCN Dr. Rimm: This is actually the summary of these two studies. Since they re the largest studies I m not mentioning the study by Sheila at this point, [which] had only 16 patients. This study, which had 39 cases with no outcome data, was read by three pathologists and had four of the five assays, or four of the FDA IUO assays, was not statistically powered, but was really a challenge, because it required all these players to all agree on it. The NCCN study, led by myself and Ignacio, was a lot easier to design in that we had a biostatistician help us, give us a power calculation which meant we need 9 cases. We had 13 pathologists from seven academic sites, and we tested three of the four IUO assays, but we also added an LDT, realizing that sometimes that s how pathologists perform their tests. This was led by NCCN. And you can see the very similar patterns that we saw. Now, you can see that the outlier here in both cases is SP142. But if you look carefully at these data, and especially if you go to the paper in JAMA Oncology, you can see there are actually subtle differences between the other markers, as well, if you look at all 13 pathologists at once. 17

18 Comparison of Immune Cell Scores Dr. Rimm: This was true not only of the tumor cells, but also of the immune cells in the NCCN study. But because the Blueprint study had fewer patients, it wasn t seen in that study. Concordance Based on PD-L1 Expression Using Matched Assay/Algorithm N = 38 cases; each using their own scoring system Cases are rank ordered from lowest to highest PD-L1 expression (above and below respective cutoffs) Assay expression prevalence decreases from left to right Cases in blue boxes (n = 24, 63.1%) indicate agreement regardless of assay/scoring method combination Cases in black box (n = 14, 36.9%) indicate discrepant cases of PD-L1 expression across the four assays Increasing PD-L1 Expression SP142 TC1/IC1 PD-L1 expression below the selected cutoff PD-L1 expression above the selected cutoff Dr. Rimm: In the summary of the Blueprint study, they tried to integrate all the scoring systems together, and what they found, if you integrate them each by their own cutoff, is that the cases in the blue box represent 63% of the population, and those are the ones where the four tests are concordant. But that means that 37% are discordant, which suggests they certainly shouldn t be used interchangeably. NCCN Distribution of Tumor Scores by Assay 22C3 1% TPS % TPS SP263 25% TPS Summary The SP142 assay selects over 5% fewer patients for both tumor cells and immune cells The other three assays are the same (more or less) Case Number Dr. Rimm: The NCCN study showed a little [bit of a] different pattern, although what we did in that study was we tried to include everybody s scoring system. And by doing that, we had a six-point scoring system, and you can see that we found that the higher percentages of scores were positive in patients who had the three tests, including the LDT test, which was E1L3N performed on a Leica Bond. The SP142 had the highest frequency of low scores, as was seen in the Blueprint. And then, this was also seen in the immune-cell scoring. This is the tumor cell scoring. This is the immune cell scoring. And again, we saw the SP142 test at a lower level. The Ratcliffe-AstraZeneca NSCLC Concordance Study Showed Strong Overall Percentage Agreement at Different Cutoffs Between SP263, 28-8, and 22C3 Then a Substudy With SP142 1 N = 493 Cutoff for assays Ventana SP263 (AZ) vs Dako 28-8 (BMS) OPA, % Lower 95% CI Dako 22C3 (Merck) vs Dako 28-8 (BMS) OPA, % Lower 95% CI Lower CI was calculated using the Clopper-Pearson method with no upper bound. 1. Ratcliffe MJ et al. AACR 216. Abstract LB-94. Ventana SP263 (AZ) vs Dako 22C3 (Merck) OPA, % Lower 95% CI 1% % % % Substudy of N = 2 Cutoff for assays OPA, % Ventana SP263 Re-stain Ventana SP263 (AZ) vs Ventana SP142 (Genentech) Lower 95% CI 1% % % % Dr. Rimm: Another study was performed and published shortly thereafter, in 217, by Dr. Ratcliffe. And [he] looked at the concordance between three different antibodies initially and showed concordance or overall percent agreement in the 9% range. And then, as a second substudy, did SP263, but saw a similar drop in overall percent agreement. PD-L1 Immunohistochemistry in Clinical Diagnostics of Lung Cancer 1 Inter-pathologist variability is higher than assay variability 1. Brunnström H et al. Mod Pathol. 217;3: Inter-assay Variation Between 5 Different Tests for PD-L1 Applying a Scoring Scale of -5 Presented as weighted kappa (95% CI) 22C3 SP263 SP A ( ).858 ( ).557 ( ).881 ( ) 22C3.753 ( ).633 ( ).99 ( ) SP ( ).819 ( ) SP ( ) Note: 28-8 pharmdx/dako, 28-8A Abcam. 18 Go online to complete the post-test and evaluation for CME credit

19 Dr. Rimm: And in fact, the most recent study, which came out last week from a Scandinavian group led by Brunnström and colleagues found again the same thing for the four IVD assays that is, comparing any two, with the exception of SP142, showed a very high concordance, but lower concordance on all the comparisons with SP142. So there have now been four, arguably five studies in the literature that show this, so I think we know that the assays are certainly not interchangeable, and there seems to be relatively high similarity between three, or arguably four, including the LDT for the assays 28-8, SP263, and 22C3, and then the LDT with E1L3N. Comparison by Quantification (QIF and DAB) PD-L1: Where we are today Companion vs Complementary PD-L1 IHC Tests: The assays PD-L1 IHC Tests: The antibodies Reading TPS vs IC vc CPS Latest addition/update Dr. Rimm: But, basically, what we found quantifying by quantitative fluorescence or by DAB stains using the Aperio pixel counter quantification, is that the antibodies themselves, when not run in the IVD that is, the antibodies, with the exception of 28-8 and 22C3, which were the IVD, the rest were in LDT format are equivalent. The eggs are the same; the cakes are different. PD-L1 Expression in Melanoma: A Quantitative Immunohistochemical Antibody Comparison 1 Dr. Rimm: So now, I want to talk briefly about the antibodies, and I ll go more quickly through this. And these are the eggs themselves. Are the antibodies the same? There are actually more antibodies than we ve found. Quantitative Comparison of the Antibodies 1 % PD-L1 by 28-8 % PD-L1+ % PD-L1 by SP Sunshine JC et al. Clin Cancer Res. 217;23: H1 SP SP263 22C3 5H SP SP C Antibody PD-L1 PD-L1 PD-L1 PD-L1 PD-L1 PD-L1 Clone SP142 E1L3N SP263 E1J2J PA Isotype and Rabbit IgG Rabbit IgG Rabbit IgG Rabbit IgG Mouse Rabbit IgG Host Species Company Spring CST Ventana CST CST Abcam Bioscience C-terminus of Synthetic C-terminus of Recombinant protein specific C-terminus of Recombinant fulllength Immunogen human PD- peptide near human PD- to the amino terminus of human PD-L1 protein within L1 protein C-terminus L1 protein human PD-L1 protein protein human PD-L1 Lot 1542D 6 F2312 RM3 1 GR IHC-P, IF, WB, WB, IHC-P, IF- Applications IHC-P IHC-P WB, IHC-P IHC-P, WB, Flow IP, Flow IC Concentration 77 mcg/ml 1,1 mcg/ml 1.16 mcg/ml 5,7 mcg/ml 1 mcg/ml 967 mcg/ml Recommended 1:1 1:2 Pre-dilute 1:1 1:2 1:5 dilution 1. Gaule P et al. JAMA Oncol. 216 Aug 18 [Epub ahead of print]. Dr. Rimm: This is verified by Sunshine and colleagues in Janis Taube s lab at Johns Hopkins University, where they looked in melanoma at 5H1 this was the original antibody made by Lieping Chen and published in earlier studies and then, the four other antibodies that are in the IVD test. And Janis basically found the same thing, that the antibodies themselves, including SP142, are equivalent, although the assay in which SP142 is included is systematically lower than the other assays. Dr. Rimm: And in fact, in my own lab, we tested a wide range of antibodies actually, about ten or 12 antibodies and found that many of them did not validate. That is, they either didn t bind PD-L1, or if they bound PD-L1, they also bound something else. In fact, some of the antibodies were seen in the nucleus, which is not a place where PD-L1 has been reported. And so, this is a subset of the antibodies that we tested in the lab, and the information about each antibody. And this is all published in JAMA Oncology, so you can download it. PD-L1: Where we are today Companion vs Complementary PD-L1 IHC Tests: The assays PD-L1 IHC Tests: The antibodies Reading TPS vs IC vc CPS Latest addition/update 19

20 Dr. Rimm: So, what about the scoring system? ICC for Pathologists and Scores The ICC for each assay allows us to assess the agreement between readers for tumor cell and immune cell scores ICC for Pathologists by Each Assay in Tumor Cells 22C SP142 E1L3N Summary All, N = (.2) ICC for Pathologists by Each Assay in Immune Cells All, N = (.3) Average of all 4 assays Cutoff at >5% Cutoff at >1% Fleiss Kappa Kendall Concordance Fleiss Kappa Kendall Concordance Dr. Rimm: So, one of the things that I think is important to look at is how well the pathologist can actually score. And as part of the NCCN study, we looked at the concordance in the assays of the tumor cells and the concordance in the assays in the immune cells. And what we found is that the concordance was great when we looked at the tumor cells. These are ICCs, which any time an ICC is above.75, that s considered a highly accurate or highly concordant assay, whereas anything below.4 is considered unacceptable. And what we saw for ICC was that we were unable with 13 different pathologists to come up with concordance. Now, I should add that the time we did this was before there was more widespread training for this assay. And so, these pathologists all read these assays as, you know, board-certified pathologists without any specific training on how to read immune cells. Dr. Rimm: This concordance difference was also seen by Brunnström and colleagues in their recent paper, where they saw the discordance was higher, and you can see the discordance cases on the outside of these when they used less than 1% compared with greater than 5%, where they saw significantly higher concordance. When we use a high cutpoint, we re likely to succeed in terms of pathologist concordance. Lower cutpoints are more challenging. PD-L1: Where we are today Companion vs Complementary PD-L1 IHC Tests: The assays PD-L1 IHC Tests: The antibodies Reading TPS vs IC vc CPS Latest addition/update Dr. Rimm: So, the last thing I want to talk about happened last week, and in fact isn t even published yet, but I think it s really important for pathologists to know about, and that is that the FDA granted accelerated approval to pembrolizumab for advanced gastric cancer with a companion diagnostic test. And finally, another important observation was made from this test, and that was looking at the cutoffs. And what we found and perhaps not surprising to any pathologist in the audience when we had to choose a 5% cutoff, we had very high concordance measured by two different parameters, whereas when we were forced to choose a 1% cutoff, we saw the concordance drop pretty substantially, but [it was] still in a reasonable range. PD-L1 Immunohistochemistry in Clinical Diagnostics of Lung Cancer 1 More discordance between pathologists at cutoff >1% than at cutoff >5% Cutoff at 1% Cutoff at 5% C C C SP C SP C C SP A SP A SP SP A A A A After a median follow-up of 6 months, the investigators found an overall objective response rate of 12% with pembrolizumab alone in the pretreated patients (cohort 1). Patients who expressed PD-L1 were more likely to respond than those who did not, with objective response rates of 16% and 6%, respectively. Dr. Rimm: So, even though we had all those other complementary diagnostic tests, and it was really only pembrolizumab that required companion diagnostic tests, we now have another indication that is, in gastric cancer where a companion diagnostic test is required. It turns out this is not published yet. It was presented at ESMO. And what they found is that patients who express PD-L1 were more likely to respond than those that did not, with response rates of 16% versus 6%. 1. Brunnström H et al. Mod Pathol. 217;3: Go online to complete the post-test and evaluation for CME credit

21 Revised Scoring and Interpretation for Gastric or Gastroesophageal Cancer a No. PD-L1 staining cells (tumor cells, lymphocytes, macrophages) CPS = Total no. of viable tumor cells x 1 CPS Numerator Inclusion/Exclusion Criteria Tissue Elements Included in the Numerator Excluded From the Numerator Master Class #2 Part 2 MMR/MSI and Tumor Mutational Burden Testing: Evidence and Options Tumor cells Immune cells Convincing partial or complete linear membrane staining (at any intensity) of viable invasive gastric or GEJ adenocarcinoma tumor cells Membrane and/or cytoplasmic staining (at any intensity) of MICs within tumor nests and adjacent supporting stroma: Lymphocytes (including lymphocyte aggregates) Macrophages Only MICs directly associated with the response to the tumor are scored. Non-staining tumor cells Tumor cells with only cytoplasmic staining Adenocarcinoma, dysplasia, and carcinoma in situ Non-staining MICs MICs associated with adenoma, dysplasia, and carcinoma in situ MICs (including lymphoid aggregates) associated with ulcers, chronic gastritis, and other processes not associated with the tumor MICs associated with normal structures Neutrophils, eosinophils, and plasma cells Normal cells (including ganglion cells) Stromal cells (including fibroblasts) Necrotic cells and/or cellular debris Lynette M. Sholl, MD Brigham and Women s Hospital Harvard Medical School Boston, Massachusetts Other cells Not included a The revised label from the Dako 22C3 IVD. Dr. Rimm: And so, the last slide is, how do we score this? And the answer is, there are sessions going on, and I encourage you to go to those sessions to learn how to score it. I m not sure I m qualified to score yet. If you buy 22C3, you receive from Agilent the new scoring guide for 22C3 the updated, revised label. And that gives you this information on CPS, combined proportion score, which is the number of PD-L1-staining cells including tumor cells, lymphocytes, and macrophages divided by the total number of cells times 1. And then, the cutpoint is greater than 1% or less than 1%. And we ll talk about this a little bit more in the discussion, perhaps, but arguably it s too early. The summary of safety and effectiveness data, which is what the FDA publishes so that you can see how the assays perform, is not even published yet, so perhaps a little early, but just something to keep on your radar. And so, that was the IHC part. And now, what we re going to have next is Lynette, Dr. Lynette Sholl, is going to present the MSI/ MMR aspect of this, and then we ll have time for questions and discussion. MMR/MSI as an IO Biomarker 1 MMR deficiency results from inactivation of one of the members of the MMR pathway Lynch Syndrome = germline mutation resulting in risk of CRC, endometrial, other cancers Second (somatic) hit required for tumorigenesis Sporadic silencing of MLH1 due to promoter methylation Somatic mutation(s) (uncommon) Acceptable testing strategies: Mismatch repair immunohistochemistry MLH1/PMS2/MSH2/MSH6 Microsatellite instability testing by PCR Other testing Next generation sequencing 1. Martin SA et al. Clin Cancer Res. 21;16: Dr. Sholl: My oncology colleagues like to joke that if they don t like my PD-L1 score, they just leave and come back 1 minutes later and hope I give them a different answer. So I m glad I m talking about something that I find a little bit more concrete, the MMR/ MSI status, as well as tumor mutational burden. And so, I m sure this audience has a significant degree of familiarity with MMR and MSI testing, since this is something we ve been doing for many, many years, largely for Lynch syndrome screening. I will review it briefly and talk about the assays that we can consider using in the context of immuno-oncology. So, just to recap the biology, mismatch repair deficiency results from inactivation of one of the members of the MMR pathway. And you can see in the schematic here MLH1, PMS2, predominantly, as well as MSH2 and MSH6. The first two, MLH1 and PMS2, form a heterodimer, as do MSH2 and MSH6, and they all come together to perform the activities of mismatch repair when there s either a single base mismatch in the course of DNA replication, or when there s an error in the course of replicating repetitive stretches of DNA. So, you can see small insertion/deletion mutations that occur when there s some strand slippage as your intrinsic polymerases have trouble replicating, say, 25 A s in a row, and they maybe make 26 A s, and then the MMR proteins come in, and they clip out that extra A and return it to its baseline state. This can also repair larger insertion/deletion mutations. 21

22 So, of course, we are very familiar with the fact that when you see errors in the mismatch repair proteins that are conferred by the presence of a mutation in the germline in one of these genes, that these patients are at increased risk of hereditary cancer syndrome, named Lynch syndrome. These folks have an increased risk of colon, endometrial cancers, as well as other cancer types. Of course, these folks will walk around with a single hit in their germline in one of these genes, and a second or somatic hit is required to actually initiate tumorigenesis. The other context that we see mismatch deficiency, of course, is in patients who have sporadic silencing, particularly of MLH1 promoter. And this is actually a very common mechanism of mismatch repair deficiency in the colon and endometrium. And finally, we are also seeing somatic mutations. And I think increasingly as we re doing more sequencing of patients, we re finding somatic mutations that are actually the etiology of mismatch repair deficiency in patients who don t have an underlying Lynch syndrome. I think interestingly that there are actually different mutational phenotypes resulting from mutations in the different members of this pathway. So, for instance, patients who have mutations in PMS2 or MSH6 in the context of Lynch syndrome often have a more attenuated phenotype. They may not come to clinical attention until later in life. Dr. Sholl: So, of course, here are some requisite images of immunohistochemistry. Here is a poorly differentiated carcinoma that actually had a somatic MSH6 mutation. And you can see in this particular case, loss of staining of MSH6 with intact expression in the nuclei of surrounding stromal cells. I think, interestingly, we see a slight diminishment of the MSH2-protein expression. These two proteins, MSH2 and MSH6, form a heterodimer. MSH2 is actually the obligate member of that dimer. So, if you have a mutation in MSH2, you inevitably lose expression of MSH6, as well. But the opposite is not true. In this case, however, it s interesting that we seem to have some relative decrease in the amount of MSH2 that s actually coming into this complex. Of course, MLH1 and PMS2 retain their expression strongly. In general, we should see relatively robust staining in internal control nuclei, in particular in proliferating areas of the tissue. We also tend to see higher levels of expression in the tumor cells just because these are cells that are replicating more rapidly than the normal cells in the background. And these are the types of proteins that are going to be more highly expressed in the replicating nucleus. Microsatellite Instability (MSI) Testing 1 Gel electrophoresis 1 BAT25 BAT26 BAT4 Normal Capillary electrophoresis In addition, patients who have MSH6 mutations appear to have a somewhat different type of mutational spectrum. We tend to see an increased number of these single base mismatches, but not as many insertion/deletion mutations, whereas the insertion/ deletion mutation type of phenotype is very common in patients with, say, MLH1 or MSH2 mutations. N T N T N T D2S123 D5S346 D17S25 N T N T N T 1. Blaszyk H et al. Mod Pathol. 22;15: Tumor Mononucleotide repeat (BAT) Dinucleotide repeat So, [in terms of] acceptable testing strategies from the standpoint of hereditary colon cancer or hereditary cancer syndrome testing, we ve been using mismatch repair deficiency testing by IHC for many years. We ve been using microsatellite instability testing by PCR. But I think increasingly we re seeing entry of next-generation sequencing techniques as well. MSH2 MSH6 Dr. Sholl: We also have microsatellite instability testing, or MSI testing. This has been around for a long time. We ve recognized that these microsatellites, which essentially are repetitive elements within the genome, exist throughout the genome, and we actually have been looking at very interesting areas of the genome for a long time, and we ve been looking at these microsatellites. Many of these actually exist in very commonly studied oncogenes and tumor suppressor genes, and so you can actually infer some very interesting data about allelic imbalance or loss of heterozygosity surrounding these areas of microsatellites by studying them. MLH1 PMS2 22 Go online to complete the post-test and evaluation for CME credit

23 The old-school way of looking at microsatellites was by doing gel electrophoresis, and performing PCR around an area where a microsatellite exists, performing gel electrophoresis. Of course, if you just turn that 9 degrees and you put a fluorescent tag on it, you can look at it on a capillary electrophoresis and get a much more kind of clean and precise look at the microsatellite. And in this particular example, we re looking at two different patterns of microsatellites. One is a mononucleotide repeat. This is a BAT B- A-T, which stands for bit A tract and we ll see what that looks like in a sequencing context in a couple of slides. Another type of microsatellite that is commonly tested are dinucleotide repeats, so AT-AT-AT-AT. And you can see that even in the normal tissue, we tend to see what is really a Gaussian distribution around these areas of microsatellites. And that is because even in a normal tissue, the polymerases that are replicating these areas of the DNA are going to have some slippage. So, in the test tube, you re going to see a little bit of variation around that microsatellite. So potentially, the true length of the microsatellite is 25 nucleotides. But within the test tube, you re going to get a population anywhere from, say, 2 to 3 nucleotides, and as a result, you get this distribution. However, if the tumor has a defect in repairing these repetitive regions, you end up getting this very stretched-out population, and you get what s called a little bit of shouldering. You can get very strange morphologies, rather than this sort of nice, normal distribution. And the same thing can be seen both in mononucleotide repeats as well as dinucleotide repeats. We know that MSI-high status correlates with Lynch syndrome and also correlates with sporadic loss of mismatch repair function, whereas MSI-low and microsatellite-stable cases appear to be largely clinical, pathologically similar, and we really don t know exactly what this MSI-low category means. In particular in relevance to immuno-oncology response, it does not appear to be associated with Lynch syndrome. A couple things I want to point out that are kind of in the fine text here in the Bethesda guidelines are that when you don t have paired normal tissue to test to compare your tumor with, you are suggested to use what are called quasi-monomorphic mononucleotide repeats. So, essentially these are areas of the genome that are quite stable in the population and tend to have the same conformation on both alleles in the genome. So, they have a very predictable appearance when you perform PCR. By choosing your mononucleotide repeats or by choosing your microsatellites carefully, you can potentially run an assay without having paired normal tissue, and I think that that can be very helpful in today s laboratory when you may be asked to run MSI PCR on a greater number of cases. Normal Big A Tract (BAT26) Revised Bethesda Guidelines for MSI PCR 1 If 5 microsatellites tested and are unstable: MSS 1 is unstable: MSI-Low 2 are unstable: MSI-High MSI-H status correlates with Lynch Syndrome MSI-L and MSS cases are clinicopathologically similar MSI-L is not associated with Lynch; relevance to IO response unknown Recommendations for the Evaluation of MSI-H and MSI-L The original NCI microsatellite panel included BAT25, BAT26, D2S123, D5S346, and D17S25; however, the following caveats may apply: 1. If only dinucleotide repeats are mutates, test a secondary panel of microsatellite markers with mononucleotides (eg, BAT4 and/or MYCL) to exclude MSI-L 2. Dinucleotide repeats are less sensitive than mononucleotide repeats for MSI-H; however, they provide an internal control for the prevention of sample mix-up 3. A pentaplex panel of 5 quasimonomorphic mononucleotide repeats may be more sensitive for MSI-H tumors than other microsatellite markers and may obviate the need for normal tissue for comparison; this approach requires 3 or more mutant alleles to indicate MSI-H MSI-H in tumors refers to changes in 2 or more of the 5 NCI-recommended panels of microsatellity markers in tumors. MSI-L in tumors refers to changes in only 1 of the 5 NCI-recommended panels of microsatellity markers in tumors. 1. Umar A et al. J Natl Cancer Inst. 24;96: Dr. Sholl: So, there have been many, many years of testing use for microsatellite instability. The revised Bethesda guidelines have been around for a long time. In fact, this particular set of guidelines was published in 24, and anybody who s been working in a molecular lab is probably quite familiar with these. Most labs are testing five microsatellites throughout the genome. If none of these are unstable, you consider the tumor to be microsatellite stable. If one is unstable, it s considered to be MSI low. And two or more is considered MSI high. MSI-H colon cancer Dr. Sholl: So, this is what these microsatellites look like, if you actually look at the genome directly. So, here is a big A tract, and it s actually aptly name BAT26, because there are 26 A s here. This one s actually located just next to the spliced region of MSH2, and will very commonly be altered in a patient who has mismatch repair deficiency. You can see that even in a normal specimen that there is some slippage around these BATs which we are able to see in those gel electrophoresis and capillary electrophoresis images. However, in a mismatch repair deficient tumor, you can see that there s a significant amount of error around this tract in terms of inability to repair correctly. 23

24 Universal MMR/MSI Testing? Why Does MMR-D Predict Response to IO? Many institutions offer universal MMR IHC screening in colon cancer and/or endometrial cancer for Lynch Syndrome Low rates of MSI reported in other tumor types; can screening of ALL tumor types for selection of patients for immunotherapy be rationalized or operationalized? Important to understand the patterns of MMR across tumors Identify high-yield tumor types (gastric) or clinical scenarios (progressive disease) Pan-cancer MSI analysis 1 Your average MMR-proficient colon cancer: KRAS c.35g>t (p.g12v) APC c.637c>t (p.r213*) TP53 c.742c>t (p.r248w) Your average MMR-deficient colon cancer: KRAS c.38g>a (p.g13d), exon 2 - in 35% of 521 reads FUS c.454c>a (p.p152t), exon 5 - in 38% of 486 reads*** MSH6 c.3254delc (p.f188sfs*2), exon 5 - in 47% of 594 reads* GATA2 c.223g>a (p.a75t), exon 2 - in 4% of 236 reads*** MSH6 c.2942delt (p.p982lfs*15), exon 4 - in 31% of 513 reads* GLI1 c.53c>a (p.p168h), exon 5 - in 4% of 48 reads*** c.166c>t (p.r554*), exon 14 - in 3% of 172 reads** GLI2 c.2815c>t (p.r939c), exon 13 - in 17% of 96 reads*** APC c.1738dela (p.k581rfs*9), exon 14 - in 35% of 196 reads** GNAS c.2431g>a (p.e811k), exon 6 - in 32% of 449 reads*** FBXW7 c.832c>t (p.r278*), exon 5 - in 36% of 379 reads** KAT6A c.416c>g (p.t1369s), exon 17 - in 33% of 524 reads*** RNF43 c.1976delg (p.g659vfs*41), exon 9 - in 31% of 745 reads** KAT6B c.4264g>a (p.e1422k), exon 18 - in 35% of 325 reads*** SOX9 c.133delc (p.p346rfs*37), exon 3 - in 5% of 242 reads** KDR c.2337g>a (p.w779*), exon 16 - in 33% of 249 reads*** TP53 c.844c>t (p.r282w), exon 8 - in 35% of 432 reads** KIF1B c t>c () - in 5% of 361 reads*** ALK c.11c>g (p.p367r), exon 4 - in 42% of 228 reads*** KIF1B c c>t () - in 11% of 395 reads*** APC c.547a>g (p.n1824d), exon 16 - in 32% of 43 reads*** KIF1B c.3278a>g (p.q193r), exon 29 - in 34% of 422 reads*** ARID1A c.2397delg (p.g81vfs*32), exon 7 - in 27% of 523 reads*** KIT c.1847c>t (p.a616v), exon 12 - in 37% of 4 reads*** ARID1A c.38c>t (p.s13l), exon 1 - in 38% of 65 reads*** KLLN c.362a>c (p.k121t), exon 1 - in 35% of 631 reads*** ARID1A c.5495g>t (p.g1832v), exon 2 - in 4% of 577 reads*** MAP2K4 c.219-1g>c () - in 44% of 295 reads*** ARID1B c g>a () - in 36% of 211 reads*** MAP3K1 c.3914a>c (p.n135t), exon 16 - in 33% of 227 reads*** ARID1B c.5146g>t (p.d1716y), exon 2 - in 38% of 398 reads*** MED12 c g>t () - in 76% of 247 reads*** ARID1B c.5226g>c (p.k1742n), exon 2 - in 38% of 393 reads*** MGA c.325c>t (p.r19c), exon 2 - in 36% of 497 reads*** ARID1B c.5584g>a (p.v1862i), exon 2 - in 33% of 488 reads*** MGA c.3737g>a (p.r1246q), exon 11 - in 32% of 415 reads*** ATM c.2941c>t (p.r981c), exon 2 - in 37% of 347 reads*** MLH3 c.1499c>t (p.p5l), exon 2 - in 37% of 675 reads*** ATM c.6679c>t (p.r2227c), exon 46 - in 37% of 359 reads*** MTA1 c c>t () - in 36% of 364 reads*** ATM c delt () - in 32% of 349 reads*** MTOR c.518g>a (p.r173h), exon 5 - in 32% of 25 reads*** AXIN2 c.863g>a (p.g288d), exon 3 - in 39% of 44 reads*** MUTYH c.1432t>g (p.f478v), exon 14 - in 37% of 521 reads*** B2M c.343_344insg (p.d116gfs*12), exon 2 - in 25% of 552 reads*** NF1 c.374g>a (p.r125h), exon 4 - in 37% of 532 reads*** BAP1 c.182c>t (p.t67m), exon 14 - in 34% of 558 reads*** NF1 c.7815g>t (p.l265f), exon 53 - in 36% of 53 reads*** BARD1 c.113g>a (p.r38h), exon 1 - in 34% of 394 reads*** NF1 c.8434c>t (p.r2812*), exon 58 - in 34% of 425 reads*** BCL2 c.67g>a (p.g23s), exon 2 - in 3% of 336 reads*** NFE2L2 c.886a>g (p.s296g), exon 5 - in 37% of 64 reads*** BCORL1 c.2593g>a (p.v865i), exon 4 - in 73% of 299 reads*** NOTCH1 c.667g>a (p.a223t), exon 32 - in 4% of 556 reads*** BCORL1 c.3248g>a (p.r183q), exon 4 - in 3% of 225 reads*** NOTCH3 c.551g>a (p.r1837h), exon 3 - in 36% of 48 reads*** BRIP1 c.986a>g (p.q329r), exon 8 - in 16% of 51 reads*** NOTCH3 c g>a () - in 23% of 173 reads*** C1orf86 c t>c () - in 34% of 147 reads*** NRG1 c.1139c>t (p.s38f), exon 12 - in 35% of 266 reads*** CBLB c.482t>c (p.f161s), exon 4 - in 5% of 453 reads*** PBRM1 c.1463c>t (p.a488v), exon 13 - in 39% of 423 reads*** CDH1 c.268c>t (p.r9w), exon 3 - in 34% of 449 reads*** PIK3CA c.544t>c (p.y182h), exon 3 - in 35% of 22 reads*** CDH4 c.899c>t (p.t3m), exon 7 - in 36% of 343 reads*** PIK3R1 c.1123delg (p.g376efs*5), exon 1 - in 9% of 494 reads*** CHEK1 c.668dela (p.t226hfs*14), exon 7 - in 32% of 37 reads*** PML c.1745c>t (p.s582f), exon 8 - in 36% of 343 reads*** CIC c.12delc (p.w343gfs*7), exon 2 - in 35% of 751 reads*** PML c.2263c>t (p.r755c), exon 9 - in 35% of 48 reads*** CIC c.4145g>a (p.r1382q), exon 17 - in 37% of 546 reads*** POLD1 c.2677g>a (p.d893n), exon 21 - in 46% of 416 reads*** CIITA c.315-5g>a () - in 35% of 372 reads*** POLE c.2747c>t (p.p916l), exon 24 - in 39% of 477 reads*** CIITA c.2323g>a (p.g775r), exon 11 - in 38% of 469 reads*** POLQ c.6289g>a (p.a297t), exon 2 - in 5% of 451 reads*** COL7A1 c.681delc (p.p229lfs*177), exon 73 - in 5% of 293 reads*** PTCH1 c.366delc (p.s123afs*52), exon 22 - in 29% of 46 reads*** COL7A1 c.5895g>t (p.e1965d), exon 72 - in 35% of 394 reads*** RAD21 c.59c>t (p.a2v), exon 2 - in 36% of 499 reads*** 1. Cortes-Ciriano I et al. Nat Commun. 217;8:1518. Dr. Sholl: So, I think the question that probably many of us have asked ourselves and our oncology colleagues is, what do we do now that there is essentially a universal indication for testing MSI in any solid tumor type for adults or for children? Many of us already offer universal testing for colon and potentially also for endometrial cancers for the indication of Lynch syndrome. The challenge, however, is that you can see MSI in many different contexts, as Dr. Diaz has already pointed out. And how do you make a decision as a lab in terms of what kind of additional testing you are going to offer on a routine basis for your different indications? This is a very nice analysis that was published using TCGA data that essentially did a pan-cancer evaluation of MSI status. I would point out that it seems like it s fairly obvious that we should indeed be doing universal testing in endometrial cancer, because there is nearly a 3% rate of MSI-high status in this context. Colon cancer, as well, not surprisingly, has more than a 15% rate of MSI high. And here s gastric cancer, 22%. So, you might argue that we should be doing routine gastric cancer MSI or MMR testing. If you start looking at the bottom of the list, however, you see there are certain tumor types where MSI is very rare, and we do know from Dr. Diaz s data that there are thyroid cancers out there that are MSI high, but apparently none of them made it into the pan-cancer or TCGA analysis, possibly because these have somewhat different morphologies than our traditional thyroid cancers, and were not actually included in these types of analyses. Also, if you look at some other very common tumor types, such as lung adenocarcinoma, where only one out of 482 tumors that were sequenced seem to have MSI, it becomes very difficult to rationalize, say, doing universal MSI or MMR testing in that tumor type. Rather, you re probably looking at scenarios where there s potentially a higher yield from the clinical standpoint to do this type of testing. PD-L1 testing might be the better biomarker in this scenario, as well, but potentially both of these things may be informative in some cases. Dr. Sholl: So, I think that Dr. Diaz has already covered this very clearly. I just think having access to sequencing data is also very informative to really see what is going on underneath the hood in these tumors. Here is a very characteristic MMR-proficient colon cancer compared with the genome of a mismatch repair deficient colon cancer. You can see two very striking features, a very low number of mutations in our MMR-proficient, with a striking degree of genomic instability, so lots of genomic gains and losses, as compared with our MMR-deficient colon cancers, which have huge numbers of mutations and a virtually silent copy number profile, so a very different mechanism of tumorigenesis going on in these two contexts. Image courtesy of Cathy Wu, DFCI. Dr. Sholl: And we ve already talked about neoantigen presentation. So, mismatch repair looks like a pretty good biomarker. Are there other kinds of mutation-based biomarkers that we should be thinking about in predicting response to immuno-oncology? 24 Go online to complete the post-test and evaluation for CME credit

25 Neoantigen Burden Predicts Response to IO Across Tumor Types 1 Different mechanisms of (hyper)mutation across human tumors Dr. Sholl: If we look at these tumors that have high mutational signatures more closely, we can see very discrete patterns arising here, and potentially these patterns of mutations within these tumors may be informative, as well. These are publicly available data that you can get from the COSMIC database, the Sanger COSMIC database. And you can actually look at these different signatures and see that these will correlate specifically with certain types of mutagenesis. 1. Desrichard A et al. Clin Cancer Res. 216;22: Dr. Sholl: Neoantigen burden. So, we ve talked about the fact that MMR deficiency leads to an increased number of neoantigens. I think that in some of these studies, the numbers are still too small to know for sure how neoantigen burden in and of itself is predictive of response, but there s certainly a trend towards predicting as much. Tumor Mutation Burden Varies Widely Across and Within Cancer Subtypes Dr. Sholl: So, for instance, Signature 7, which is characterized by a large number of C-to-T transitions, is characteristic of UV mutagenesis. Signature 4, which is characterized by a large number of G-to-T transversions, is characteristic of smoking. And these are very obvious signatures that you can see within sequencing data, and may be very informative in understanding which tumor will and will not respond to treatment. 1. Alexandrov LB et al. Nature. 213;5: In Lung Cancers, Smoking High Tumor Mutational Burden Neoantigen Formation Adaptive Immunity 1 Dr. Sholl: Let s talk a little bit about tumor mutation burden, because MMR is certainly not the whole story across all tumor types. It s certainly a mechanism to drive very high levels of mutation and potentially neoantigen presentation. But again, I think as Dr. Diaz pointed out, there s huge variability across tumor types in terms of the number of mutations that are present, and that s probably largely driven by the types of mutational processes that are associated with tumorigenesis in these different tumor types. In lung adenocarcinoma, high neoantigen burden associated with: Longer overall survival Upregulated immune related genes including PD-L1 and IL-6 High PD-1 expression in neoantigen-reactive T cells 1. McGranahan et al. Science PD-L1 CD8 Dr. Sholl: This is an interesting study that was published earlier this year, led by McGranahan, showing that lung cancer [patients] who are smokers, tend to have a higher tumor mutational burden, which I think is no surprise. These tend to have higher levels of neoantigen formation, and ultimately have a phenotype of increased adaptive immunity. This is a type of phenotype that we would predict would be responsive to immunotherapeutics. This I think is really kind of getting into the weeds, but when you actually dissect out the different neoantigens that actually exist within these lung cancers, understanding the patterns of neoantigen formation actually seems to predict response, as well

26 Clonal Neoantigen Burden (ie, Derived From Smoking) Predicts Favorable Response to IO 1 Subclonal neoantigens (such as from chemo?) do not predict benefit. analyses, if you take into account the patient s tumor mutation burden plus the PD-L1 status, you actually see a 75% response rate to this drug in the first line, as compared with 25% with chemo alone. If, however, you look at a high PD-L1, greater than 5%, with a low tumor mutation burden, you see no difference relative to chemotherapy. So, there s some signal surrounding tumor mutation burden in this context. And indeed, if you look at tumor mutation burden in and of itself, irrespective of PD-L1 status, you see an improved response rate and progression-free survival. 1. McGranahan et al. Science Computational Modeling Supports Use of 3+ Gene Panel for Accurate Tumor Mutational Burden Determination 1 Dr. Sholl: And so, what this essentially is telling us is that if you have a tumor that has a clonal neoantigen burden so every single tumor cell in the population has neoantigens presented on its surface, and you can see that in some cases you have 5 or 6 neoantigens per tumor, that these are much more likely to respond to immunotherapy. Also, interestingly, these tumors that have a clonal neoantigen burden tend to have a very striking tobacco mutational signature. In contrast, those tumors that have a subclonal neoantigen ie, neoantigens that are only present in a subset of the tumor cells do not seem to respond as well to immunotherapies, and the authors here speculated that potentially the evolution of subclonal neoantigens may be a result of prior chemotherapies. NSCLC CheckMate-26: Nivolumab Fails to Outperform Chemotherapy in the First-Line Setting, Irrespective of PD-L1 Status 1 BUT high TMB + PD-L1 expression DOES correlate with response to this drug High TMB RR (47% vs 28%) and PFS (median, 9.7 vs 5.8 mo; HR:.62; 95% CI,.38-1.) with nivolumab vs chemotherapy 1. Garofalo A et al. Genome Med. 216;8:79 Dr. Sholl: Do you need whole-exome sequencing data? No. You can actually derive high tumor mutational burden from targeted panels, as well, down to about 3 genes, as far as we can tell. If you drop far below that, that correlation with the tumor burden from whole-exome sequencing begins to drop off. P.S. You Can Also Use Targeted Gene Panels to Detect MMR (and Other Mutational Signatures) 1 1. Carbone DP et al. N Engl J Med. 217;376: Dr. Sholl: Then, just finally, I think that practically speaking, thinking about what we might need to consider in a clinical laboratory, I think most of us are not going to be running neoantigen analysis on our patients tumors. You generally need whole-exome or whole-genome sequencing to do that. Many times I can t figure out if my patient has a KRAS mutation, let alone whether they ve got a neoantigen that s going to predict response. But is there kind of a happy medium? Is there something that we can use as a potential biomarker that s practical? 1. Nowak JA et al. J Mol Diagn. 217;19: Dr. Sholl: And then finally, you can also use more targeted gene panels, such as a 3- or 4-gene panel, to actually detect mismatch repair deficiency. So, if you re looking for a universal way to do mutational profiling as well as MMR testing, targeted nextgen sequencing can actually provide that information. And with that, we ll pass it over to Dr. Rimm. Tumor mutation burden in and of itself may actually be a viable biomarker. This is a little bit more analysis of the study that Dr. Diaz presented earlier from CheckMate 26 that showed that nivolumab did not perform better than chemo in the first line in patients with non small-cell lung cancer. But in the retrospective 26 Go online to complete the post-test and evaluation for CME credit

27 Practicum Current Immunotherapy Biomarkers: Clinical Experience, Practicalities, and Recommendations Featured Presentations David L. Rimm, MD, PhD Luis A. Diaz, MD Dear Dr. Sholl, You recently signed out a PD-L1 stain for my patient, Mrs. Smith. She is 55 and a smoker, with a large suprahilar adenocarcinoma with metastases to lymph nodes and soft tissue. You said her BAL fluid was 9% positive. However, your colleague also reviewed a lymph node sample from this patient and said THAT specimen was 4% positive. She is in the infusion center awaiting her first-line chemotherapy. But should I switch to pembrolizumab? Please help!!! Sincerely, Your confused oncologist Lynette M. Sholl, MD Practicum Current Immunotherapy Biomarkers Clinical Experience, Practicalities, and Recommendations Dr. Sholl: I open my . Dr. Sholl, you recently signed out a PD-L1 stain for my patient, Mrs. Smith. She is 55 and a smoker, with a large suprahilar adenocarcinoma with mets to lymph nodes and soft tissue. You said her BAL fluid was 9% positive. However, your colleague also reviewed a lymph node sample from this patient and said that specimen was 4% positive. She is in the infusion center awaiting her first-line chemotherapy, but should I switch to pembrolizumab? Please help. Bronchoalveolar Lavage: PD-L1 IHC Reported at 9% Tumor Cells Staining Dr. Rimm: Dr. Sholl will present a case that really happened at her institution, and that will lead off the discussion. Biological Pitfalls: Intratumoral Heterogeneity Field #1: <1% tumor cells staining Field #2: 5% tumor cells staining Field #3: >9% tumor cells staining Dr. Sholl: Here s the BAL. PD-L1 IHC was reported at 9% tumor cell staining. You can see the very nice, crisp membranous staining there in the majority of the tumor cells in the field. Obviously, a little bit paucicellular, but as you scan around, you can get probably 2 or 3 cells in the field. Dr. Rimm: So, what we want to do now is talk a little bit about heterogeneity. These are actually all IHC images that Dr. Sholl has provided in the context of this case. So, Dr. Sholl? Simultaneous Scalene Lymph Node Biopsy: PD-L1 IHC Reported at 4% Tumor Cells Staining PD-L1 27

28 Dr. Sholl: Here is a scalene lymph node biopsy, which was reported at 4% tumor cell staining. And there is a low-power view. Simultaneous Scalene Lymph Node Biopsy: PD-L1 IHC Reported at 4% Tumor Cells Staining (Cont d) Dr. Sholl: Well, I actually recommended chemotherapy to the clinician based on the fact that the label strictly has not examined cytology specimens in particular, and having reviewed both of them side by side and realizing that there was quite a bit of discrepancy between the percentage that was showing in the lymph node versus in the cytology. Dr. Rimm: So, there is no right answer here? Dr. Sholl: Yes, there s no right answer. PD-L1 Dr. Sholl: There s a higher-power view, and there s a little bit of variability in terms of the intensity. Sort of stare it long enough, and you can maybe hit about 4% between weak to strong staining of the tumor cells. So, how would you advise the confused oncologist? Dr. Rimm: This is just state of the art. This is where we are today. And we re fortunate to have an oncologist with us. So, let s see what Dr. Diaz thinks. Dr. Diaz: So, I would probably treat with immunotherapy. Chemotherapy does have limitations, and you can always go back and treat with chemotherapy afterwards. Faculty Q&A What guidance would you provide to the confused oncologist? Let s Discuss! Dr. Rimm: The vast majority of the people recommended giving immunotherapy, and I assume that that was on the basis of cytology. So, I want to take that, because, remember, the first specimen is a cytology specimen, and actually, cytology specimens aren t in the label for this test. But you all kind of converted it and said, Well, it s a cytology. It s probably going to work. It s a cell block, and in fact move forward. And in fact, what I didn t get to present is, a number of cytology comparison studies have been done between cytology, IHC on cell blocks, not on fresh specimens, but on cell blocks and tumors, and those show generally very high concordance, in the 9-plus percent range. Now, you could arguably recommend giving chemotherapy, because in the lymph node specimen, there was less than 5% positivity, so thereby it wouldn t be appropriate for immunotherapy in the first line. But I ll let Dr. Sholl speak on this topic. Dr. Rimm: The first question is about MLH1 and PMS1. It says, MLH1 and PMS1 are expressed in all tumor cells of a given mass. When one has heterogeneity, how is the treatment option considered, or how are treatment options considered? Do you want to field that one, Dr. Sholl? Dr. Sholl: Well, I guess the first thing is, heterogeneity is fairly unusual. I think that when we initiated endometrial cancer screening, which we did about 2 years ago, we started seeing more heterogeneity there than we were seeing in the colon cancer space. And we are interpreting the heterogeneity that we re seeing there as acquisition of somatic mutations in one of the mismatch repair genes in the course of tumorigenesis. We have not looked at it comprehensively to know if those tumors actually are POLE mutated, because we know these tumors have exceptionally high rates of mutation, and you can certainly see multiple mutational signatures within a single tumor. You can have a POLE mutation initiating the process, acquiring mutations within your mismatch repair genes, and going on to develop mismatch repair deficiency in subclones of the tumor. 28 Go online to complete the post-test and evaluation for CME credit

29 You could probably argue, if that s the scenario, the patient s likely to be responsive to immunotherapy. If it s a different kind of just sporadic alteration that s a subclonal alteration, I think it s harder to know how the patient would respond overall. Dr. Rimm: Okay, very good. The next question that has come up was a question, why do we even do PD-L1 on cytology if it is not labeled use? And so, I guess we can each opine on that. I would say clearly cytology specimens come from the same source of the tumor, and so arguably you could say In fact, in some labs, it s a very fine line. What is a cytology specimen, and what is a small biopsy? And a small biopsy is actually what is on the label, often endoscopic, but could be also confused for cytology. So what is a cytology by definition as a cytopathologist is that it s often not formalin fixed. And I think that s the variable that people are concerned about. But the minimum number of cells that is required for the on-label test is 1 cells. And we all know that many times our cytology specimens have many more than 1 cells, and many times our biopsy specimens have many fewer, or some fewer than 1 cells, [which] are completely inadequate. So, I think if you use the 1-cell guideline, I m comfortable with returning the data. In fact, in sign-out, I return cytology specimen data to the clinicians with a disclaimer on the bottom that says, Technically, this is not in the label since it is a cytology specimen. Lynette? Dr. Sholl: We re not even that sophisticated. We take what we can get, I think. Of course, the challenge is, you know, that s all you have on a patient, so you don t have the luxury of saying, Oh, go back and get me the biopsy specimen. It s either, you test this, or you test nothing at all. So, we report it out just as we would a surgical specimen. Dr. Diaz: From a medical oncologist s perspective, I think reimbursement and getting the drug can be an issue, and if the payers, or the hospital, or the pharmacy and the hospital, the P&T committee, has strict guidelines, that can really make things difficult. So, it s hospital to hospital, payer to payer, and institution to institution. Okay, here s a question for the oncologist. What is the rationale for combining ipi[limumab] and nivo[lumab] instead of sequential treatment, considering the expression of CTLA-4 early and PD-1 late? So, the hope was that since they re working by different mechanisms of action, that you would potentiate the response. We were seeing mixed responses in melanoma and in lung cancer, and even renal cell, with response rates of 2% to 4%, varying by first and second line. And the hope was that we could achieve a higher response rate, and therefore then a higher durable overall response rate and better PFS and OS. There are studies looking at these sequentially so, for instance, in ipi[limumab] failures, treating with PD-1, or in PD-1 failures, adding ipi[limumab]. So, the real answer is that we don t know, and we ll know better probably in 2 to 3 years. But the initial rationale was to potentiate the response. Dr. Rimm: Thank you. Back to a pathology question. Does companion diagnostic apply to the antibody cloned only, or both the clone and the platform? It seems that the 22C3 clone is now available to be validated and run on other platforms, like the Ventana platform. So, I think that it s very important to realize that the FDA approval is for the whole platform that is, the stainer, the antibody, all the reagents that go with it, and the scoring system. So as soon as you take things apart and don t do exactly what s in the entire platform, then it s no longer an FDA-approved [assay]. Now, is it correct to say that using 22C3 is the FDA-approved antibody? Well, it s part of an in vitro diagnostic test that is FDA approved, but it s probably not correct to say And in fact, it is an LDT if you use the 22C3 antibody not in the context of the Dako Link 48 test, in fact, using the exact criteria that are stipulated in the testing manual. Any comment? Do you want to comment on that, or Dr. Sholl: No further comment. Dr. Rimm: Okay, no further comment. Okay. And another question is more for oncologists. Has resistance ever been reported or identified post immunotherapy? Dr. Diaz: So, that s a great question. It s still evolving, but there s primary resistance and secondary resistance. And there are data that suggest that mutations in the machinery responsible for class I presentation, either in β2 microglobulin or other genes related to the signaling or expression of class I, may be responsible for acquired resistance. I don t think that there are convincing data yet in that area, certainly not convincing with mismatch repair deficiency tumors. Most of the data are in melanoma. So, the simple answer s no, we don t have a good feel for the mechanism of acquired resistance. There is clinical resistance, but we don t know why. 29

30 Dr. Rimm: So, we do have a few examples where there have been for example, the MHC-II gene mutation, but the acquired resistance is certainly going to be an area of, I think, a lot of interesting studies in the not-too-distant future. The more and more patients that you get that show resistance, the more substrate, if you will, for future assessment of what the cause of that acquired resistance is. Dr. Diaz: Just one point on that. The question, is it tumor cell intrinsic or extrinsic? And I think, at least with targeted therapy, it s always been intrinsic. We don t know, is it microenvironment or tumor cell? Master Class #3 New and Emerging Directions in Biomarker Development for Cancer Immunotherapy: How Can We Do Better? David L. Rimm, MD, PhD Yale University School of Medicine New Haven, Connecticut Dr. Rimm: One of the pathologists in the audience, I assume, asked, Since the lymph node met is the aggressive clone, shouldn t PD-L1 score be based more on the score from the lymph nodes than from the primary? What do you think? Dr. Sholl: Well, I think there s such extraordinary heterogeneity at all of the sites that it s very difficult to know what the best site is to go after. I think in theory, yes, we d like to know what s happening at the metastasis in particular, but it s rare that we actually see the whole thing. So, we re looking at a very small subset of a subset of the tumor, and we re extrapolating from there. So I think, you know, it s really a very inexact science at best. Dr. Diaz: Could I ask a question based on that? So what about focusing on the invasive front, it seems like that s where all the action is at least in the example I showed, the tumor was quite cold for PD-L1 expression. Dr. Rimm: So actually, I m going to talk a little bit about doing something similar to what you did, which is looking at multiple lymphocyte parameters, as opposed to tumor cell parameters. In the last 1 minutes or so here, I ll talk about some tests on the horizon. But I would say in answer to that, that in some tumors that s clearly the case, and colon and melanoma are two tumor types where that s clearly the case. In lung cancer, we can t always tell what s the invasive front, and in fact, in breast cancer, where it s more of a spiculated tumor, would the ends of those spicules be the invasive front or not? And so, people have started talking about the tumor stromal margin or the tumor stromal border as a way to sort of say, Okay, we know there s something going on here, but is that really the leading edge? RNA-based methods - Expression profiling Protein-based methods - Activated T cells - Capacity to express PD-L1 Dr. Rimm: So, new and emerging directions in biomarker development for cancer immunotherapy, how can we do better than what we re doing? And I ll start this by saying that we started with IHC, and you can see that there s a lot of interest in MMR as a diagnostic test, and there s a lot of interest that didn t come out so much here, but in other sessions will probably come out related to total mutational burden, or TMB. And so, I think that in some ways, those are on the horizon, as well, but we kind of already covered that. What I want to talk about is RNA expression based methods that is, expression profiling. And then, some protein-based methods, a way to assess activated T cells or T cells not too dissimilar from what Dr. Diaz showed. And then, finally, the potential to determine the capacity to express PD- L1, which is active in a few different labs, including my own. Dr. Diaz: Right. Dr. Rimm: And so, in some tumors, I think it s likely to be more possible to even do, but it s tricky, because we never know, especially if we get a tiny biopsy, we don t really know if it s at the invasive front. 3 Go online to complete the post-test and evaluation for CME credit

31 IFNγ-Related mrna Profile Predicts Clinical Response to PD-1 Blockade 1 FFPE tumor tissue collected at baseline before receiving pembrolizumab RNA NanoString platform 1. Ayers M et al. J Clin Invest. 217;127: Gene expression data Discover genes and signatures associated with anti PD-1 response 4-68 genes on custom platform Immune Most focused samples yield >2 ng of usable RNA per slide 5 ng of RNA requires for 1 assay Melanoma discovery set 19 patients IFNγ Score 1-gene Preliminary IFNγ signature developed to correlate with clinical response Nonresponder Responder Best Overall Response, RECIST v1.1 Dr. Rimm: So, this is some work from Ayers and colleagues, where they looked at tumors, and then, took the FFPE and put them into a NanoString platform to actually discover a series of genes and they went through a few different series of genes that would predict response that is, would be correlated with response to therapy and not to nonresponse. IFNγ-Related mrna Profile Predicts Clinical Response to PD-1 Blockade (Cont d) 1 Dr. Rimm: So, I think this represents sort of a potential future direction. You can see how they sort of pick the genes that they picked. And the genes that they picked the RNA from all play a role in either tumor cell T cell interactions or the actual PD-L1 itself: PD-L2, which is a related family member, some other immune inhibitors and other less characterized genes, including antigenpresenting signature cells and T cell or NK signature cells. So, this has now been published, and there are a number of other similar signatures that are now in process. Most significantly, we ll see a few of them at the World Lung Cancer Congress in Tokyo in a few weeks, but I can t present those to you now. But I think it s a space that you should keep your eye on, since it s a way that pathologists can still do in their lab. RNA-based methods - Expression profiling Protein-based methods - Activated T cells - Capacity to express PD-L1 Dr. Rimm: The other platform was alluded to by Dr. Diaz in his talk, and has been work that we ve been doing in our lab. 1. Ayers M et al. J Clin Invest. 217;127: Dr. Rimm: And what they found is that they could do this by doing a heat map. As you can see here, they could define a series of genes where the responders, are clustered in this group here, and not so much down here. And so, these 18 genes were put into a signature, and then that signature was tested. And this was just published a few weeks ago, actually, and you can see that when they tested that signature, they looked at the area under the curve. And the area under the curve for the signature, the GEP score, is.75 compared with PD-L1 IHC, which was.65 in the same cohort. Four Areas of Immune Biology Are Represented in the Tissue Inflammation Signature 1 IFNγ Biology CCL5 CXCL9 CD27 CXCR6 IDO1 STAT1 T Cell Exhaustion TIGIT CD8A LAG3 PD-L1 PD-L2 CD276 Association Between TILs and Response to PD-1 Blockade 1 1. Tumeh PC et al. Nature. 214;515: Dr. Rimm: And it was in fact first described in some of the earliest papers, where Dr. Tumeh and his colleagues showed that patients with high CD8 were more likely to respond than patients with low levels of CD8. And this makes sense. These are sort of the tumorinfiltrating lymphocytes we ve all seen. TIS has been clinically verified in SCCHN, gastric, TNBC, urothelial, anal, biliary, colorectal, esophageal, and ovarian cancer T Cell/ NK Signature HLA-E NKG7 CMKLR1 Antigen Presenting Cell Signature PSMB1 HLA-DQA1 HLA-DRB1 The TIS for use on the ncounter Dx Analysis System is for investigational use only. Limited by United States law to investigational use. 1. Piha-Paul SA et al. ASCO 216. Abstract

32 Converting the Lung Tumor Subclasses to T Cell Activation Subclasses 1 Testing the T Cell Activation in Treated Patients 1 PD-L1-/TIL- PD-L1+/TIL+ PD-L1-/TIL+ PD-L1+/TIL- 45% Type I Type 1= Low CD3 Type 1 = Low CD3 17% 26% Type II Type III Type 2 = High CD3/low GzmB Type 2=High CD3/low GZB & Ki-67 and Ki-67 12% Type IV Type 3 = High CD3/high GzmB Type 3=High CD3/high GZB or Ki-67 or Ki-67 CD3 Gzmb in CD3 Ki-67 in CD3 GzmB or Ki-67 in CD3 Score (AU) DAPI/CK/CD3/GZB/Ki-67 DAPI/CK/CD3/GzmB/Ki-67 DAPI/CK/CD3/GzmB/Ki-67 DAPI/CK/CD3/GZB/Ki-67 DAPI/CK/CD3/GzmB/Ki-67 DAPI/CK/CD3/GZB/Ki-67 CD3 GzmB Ki Nikita Mani and Kurt Schalper. 1. Nikita Mani and Kurt Schalper. Dr. Rimm: These are the categories of tumor types identified by Lieping Chen in some early work on melanoma where he sort of put them in four categories that is, no TILs and no PD-L1, high PD-L1 and high TILs, no PD-L1 but high TILs, and then high PD-L1 and low TILs. And you can see the rough percentages here. What we ve been doing is trying to put these into categories, so that we can actually look at activation of these T cells. That is, instead of just asking, can we look at how many T cells are present, can we ask, if the T cells are present, are they activated? And the way we did this is, if they re not present that is, they re TIL-negative well, then, they re going to be in this category we just call CD3 low or TIL low. Dr. Rimm: And so, with those three types, we could actually ask the question, can we assess T cell activation as a future method to predict response to therapy? And you can see that in this cohort, a relatively small cohort of lung cancer patients in a retrospective study, there s a broad range of expression of these different markers, and neither granzyme or Ki-67 by themselves predict clinical benefit, although CD3 does in fact predict clinical benefit by itself. Outcome in PD-1 Axis Therapy Treated NSCLC Patients 1,2 Converting the Lung Tumor Subclasses to T Cell Activation Subclasses (Cont d) 1 PD-L1-/TIL- PD-L1+/TIL+ PD-L1-/TIL+ PD-L1+/TIL- 45% Type I 17% Type II I 26% Type III 12% Type IV Type 2 = High CD3/low GzmB Type 3 = High CD3/high GzmB Type 1= Low CD3 Type 2=High CD3/low GZB & Ki-67 Type 3=High CD3/high GZB or Ki-67 Type 1 = Low CD3 and Ki-67 or Ki-67 DAPI/CK/CD3/GZB/Ki-67 DAPI/CK/CD3/GzmB/Ki-67 DAPI/CK/CD3/GzmB/Ki-67 DAPI/CK/CD3/GZB/Ki-67 DAPI/CK/CD3/GzmB/Ki-67 DAPI/CK/CD3/GZB/Ki Nikita Mani and Kurt Schalper. 2. Gettinger et al, in revision. 1. Nikita Mani and Kurt Schalper. Type 1 - Absent Type 2 - Dormant? Type 3 - Active Dr. Rimm: If they re present, then there are two possibilities. They could either be high granzyme and high-ki-67, which would be activated, or they could be neither high granzyme nor [high] Ki-67, and those would be dormant T cells. Dr. Rimm: But when you combine them for progression-free survival, and especially overall survival, you can see that it s the type 2s, it s the dormant ones that benefit most, which actually, at first, we didn t think would be true, but then on further consideration, we thought, Wait a sec, those are the ones that are checked. So, if you inhibit the checkpoint, then those are the ones that are most likely to be capable of actually responding to this therapy, and you can see that for progression-free and overall survival. This was a pilot study presented last year at the World Lung Conference by Dr. Kurt Schalper and his team, and Scott Gettinger and colleagues. Scott is a first author as an oncologist, and this work is now under revision, not published yet. 32 Go online to complete the post-test and evaluation for CME credit

33 C D 3 D A P I C R / P R / S D P D C D 3 C a s e s C R / P R / S D P D + C a s e s / t o t a l G Z M B C D 3 C R / P R / S D P D G Z M B C a s e s C R / P R / S D P D + C a s e s / C D K i 6 7 K i 6 7 C D 3 C R / P R / S D P D C a s e s C R / P R / S D P D + / C D 3 C a s e s + The Present and the Future of Cancer Immunotherapy Biomarkers: Challenges, Opportunities, and Implications for Pathologists Each Marker Split by Response in Melanoma (CR/PR/SD vs PD) 1 QIF/measure (AQUA) Expression of PD-L1 Is Heterogeneous and Varies With Antibodies Used 1 H&E E1L3N SP142 P =.2 A Q U A s c o r e A Q U A s c o r e A Q U A s c o r e Negative QIF/count (inform) C e l l s ( % ) P =.39 C e l l s ( % ) P =.59 C e l l s ( % ) 1 mm Positive Immunofluorescence shows stroma and epithelial staining are often concordant and adjacent Green = Cytokeratin Blue = Nuclei Red = PD-L1 (SP142) 1. Pok Fai Wong, Kim Blenman, and Harriet Kluger. 1. Joe McLaughlin and Roy Herbst. Dr. Rimm: But we were very interested in this, so we looked at it in melanoma and here s the same markers in melanoma to see if something crosses tumor types, perhaps it s more convincing. And you can see high CD3 certainly by two different quantitative software measurements seems to be predictive to response, but not so much for the others. The T Cell Activation Panel in Melanoma 1 Dr. Rimm: This was the issue that was addressed that in the same piece of tumor, you can have both PD-L1-negative and PD-L1- positive regions of the tumor. And in fact, given that, maybe we should look at how PD-L1 is expressed or how it s controlled. IRF-1 Drives PD-L1 Transcription in Response to Interferon Gamma Signaling Lymphocyte infiltration PD-L1 expression 1. Pok Fai Wong, Kim Blenman, and Harriet Kluger. Dr. Rimm: But when you combine them, in fact, you can see and this is early studies using both cell counts and quantitative software, that again, it s group 2 that does the best, whereas group 1 and group 3 do less well in this context. Dr. Rimm: And in fact, there s a number of different transcription factors NF-κΒ and IRF-1, and even some micrornas that control the expression of PD-1 mrna. And so, the thought was that if we can look at how it s expressed, perhaps that could be a biomarker. IRF-1 Is Higher in PR-CR and Associated With Improved PFS 1 RNA-based methods - Expression profiling Protein-based methods - Activated T cells - Capacity to express PD-L1 IRF-1 AQUA Score Percent Survival P e r c e n t s u r v i v a l 1 5 IIRF-1 R F - 1 H High I G H ( n (n = = 3 131) ) IIRF-1 R F - 1 L OLow W ( n (n = = 1 616) ) p P = T i m e f r o m s t a r t o f t h e r a p y ( D a y s ) Time From Start of Therapy, d 1. Smithy J et al. J Immunother Cancer. 217;5:25. Dr. Rimm: And then, the last thing that I want to discuss in the last couple minutes is the capacity to express PD-L1 what assays this will be done by remains to be seen. 33

34 Dr. Rimm: And in fact, that was found to be the case, that patients that had high IRF-1 this is a small melanoma cohort done by Smithy and colleagues showed that there is actually a relationship between response and nonresponse, or IRF-1, shown here with overall survival, compared with PD-L1 in this small melanoma cohort, and in fact validated in a somewhat larger cohort. However, that effect was not a prognostic marker. That is, if you just looked at IRF-1 retrospectively, that doesn t actually predict good outcome in melanoma. You only see that effect when the patients have been treated with an immune checkpoint inhibitor. Tumor PD-L1 NFκB Relationship to PD-L1 (in Lung) YTMA79 Tumor NFκB 9% NFκB+ PD-L1- y =.35x R 2 =.97 Tumor PD-L1 YTMA25 Tumor NFκB y =.2334x R 2 = % NFκB+ PD-L1- Validated IRF-1 Antibody Shows IRF-1 Is Not Prognostic Dr. Rimm: This is an example of NF-κΒ, and other people are looking at NFAP and other markers, as well. IRF-1 AQUA Score IRF-1 Positive IRF-1 Negative Percent Survival We Envision a Transcription Factor/Modulator Signature For example: Predictor = x(irf-1) + y(nf-κb) + z(nfat) a(stat1) b(t-bet) Melanoma cases Dr. Rimm: In fact, this is a test that has the potential to be converted to a DAB test. This is a DAB staining of IRF-1 and PD-L1 in the same area. Relationship Between PD-L1 and IRF-1 Expression 1 Dr. Rimm: So, this is sort of another sort of test that you might look out for. You might imagine someday a transcription factor or modulator signature, in the same way that we saw the signatures constructed on the NanoString platform. And so with that, thank you very much for your attendance, and I hope to see you all again, and hope we answered some of your questions tonight. Thank you. 1. Smithy J et al. J Immunother Cancer. 217;5:25. Dr. Rimm: You can see that there may be cases that are high in IRF-1 that suggest the tumor has the capacity to express PD-L1, but hasn t done so yet. 34 Go online to complete the post-test and evaluation for CME credit