Emerging immune biomarkers. Athanasios Kotsakis MD, PhD Ast. Professor of Medical Oncology School of Medicine, University of Crete
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1 Emerging immune biomarkers Athanasios Kotsakis MD, PhD Ast. Professor of Medical Oncology School of Medicine, University of Crete
2 Disclosure: none
3 Cancer Immunotherapy Immunotherapy, mainly anti PD 1/PD L1 agents have been approved for the treatment of many tumor types such as melanoma, NSCLC, urothelial cancer, SCCHN and others BUT what about Toxicity and Cost?? Avoid toxic effects of the treatment to patients who will not benefit from such a therapy Spare money
4 A biomarker is defined as: European Medicines Agency: Biomarkers are tests that can be used to follow body processes and diseases in humans and animals. They can be used to predict how a patient will respond to a medicine or whether they have, or are likely to develop, a certain disease 1 European Medicines Agency. Accessed March 2, Could be: - prognostic: are biological characteristics that are objectively measured and evaluated to predict the course of a disease or - predictive: is a clinical or biologic characteristic that provides information on the likely benefit from treatment
5 Distinct types of biomarkers Physical features 2,3 Molecular variations 4 Cellular features 5 Histology Molecular Variations image adapted from: com/research education/braf/metastatic melanoma/mutations 1. OECD Policy Paper Accessed October 31, Okonkwo OC et al. Neurology. 2014;83(19): Rundle A. Cancer Epidemiol Biomarkers Prev. 2005;14(1): NCCN. Clinical Practice Guidelines in Melanoma, V Accessed February 11, NCCN. Clinical Practice Guidelines in Oncology-Breast Cancer, V Accessed February 11, Spector N et al. Breast Cancer Res. 2007;9(2): Menni C et al. Diabetes. 2013;62(12):
6 Immune-biomarkers are indicators of immune activity Immune-biomarkers are measures of activity within the tumor microenvironment, differing from established gene mutation biomarkers, such as BRAF and EGFR. As components and regulators of the immune response, immune-biomarkers include: Tumor-infiltrating immune cells Secreted peptides Cell surface proteins Immunosuppressive cells Evaluating multiple immune-biomarkers may provide a more realistic representation of the tumor microenvironment, as well as a more accurate and comprehensive assessment of clinical relevance. 15
7 Exploratory immune-biomarkers New immune-biomarkers are now being investigated across tumor types: The field of immune-biomarkers aims to characterize the ongoing g interactions between the immune system and cancer. 16
8 Tumor infiltrating lymphocytes (TILs) TILs are evaluated in 1. Hematoxylin and Eosin stained tissue sections 2. Immunostained for CD3+ (T cells), CD4+ (Th cells/cd4+ Tregs), CD8+ (CTLs/CD8+ Tregs), CD20+ (B cells) or FoxP3+ (T regulatory cell marker). Intratumoral lymphocytes (itils) were defined as intraepithelial mononuclear cells within tumor cell nests or in direct contact with tumor cells and are reported as the percentage of the tumor epithelial nests that contain infiltrating lymphocytes. y Stromal lymphocytes y (stils) are defined as the percentage of tumor stroma area that contains a lymphocytic infiltrate without direct contact to tumor cells.
9 Mandal R., et al JCI Insight. 2016;1(17):e89829
10 Mandal R., et al JCI Insight. 016;1(17):e89829 We find that both HPV+ and HPV HNSCC tumors are among the most highly immune infiltrated cancer types.
11 TILs in SCCHN: Prognostic value Increased levels of TILs (mononuclear cells) Associated with better DFS and DSS (Qiaoshi Xu et al., 2017, Translational Oncology Vol 10, no 1, pp 10-16)
12 TILs in SCCHN : Predictive value Tumor infiltrating lymphocytes predict response to definitive chemoradiotherapy in head and neck cancer High density of CD3 + and CD8 + TILs correlates with better OS, PFS, LFFS, DMFS (Balermpas P et al., 2014,BJC 110, )
13 HPV positive HNSCC had the highest levels of Treg infiltration, with HPV negative HNSCC having the second highest CD56 dim NK cell infiltration ti correlated ltdstrongly with overexpression of the KIR inhibitory receptor genes KIR2DL1 and KIR2DL3
14 higher levels of Treg infiltration were associated with superior OS Could be attributed to the ability of these cells to dampen inflammatory processes required for tumor survival/growth
15 intratumoral Treg are more immunosuppressive than circulating Treg
16 Mutational load
17 Haddad R, et al Journal of Clinical Oncology 35, no. 15_suppl (May 2017) Background: Somatic mutational load (ML) is associated with response to anti CTLA 4 and PD 1/ L1 immunotherapies in select tumors, due to formation of neoepitopes not subject to central immune tolerance. Neoepitopes specific to HPV, EBV virus infection are also present in some HNSCC. An IFNγ gene expression profile (GEP) characteristic ti of tumor inflammation is also related ltdto response to anti PD 1/ L1 therapy. This study evaluated relationships between ML and clinical outcome and independent predictive values of ML and GEP in patients with HNSCC treated with pembrolizumab. Methods: Whole exome sequencing (WES) and GEP were assessed in FFPE tumor specimens of patients with HNSCC (KEYNOTE 012; subsets of B1 [PD L1 +, n = 34] and B2 [PD L1 + /, n = 73] cohorts). ML, neoantigen load (NL), HPV/EBV status and clonality were assessed by standard WES analytical methods. GEP score is a weighted sum of normalized expression values of 18 genes. Statistical testing of ML and response, and ML and GEP relationship by HPV/EBV status was prespecified. Results: There were 73 patients identified as HPV and EBV (n = 25 in B1; n = 48 in B2). In HPV and EBV patients in B1 and B2 cohorts, respectively, associations between ML and objective response (OR) (P = and 0.055; AUROC 0.89 and 0.63), and GEP and OR (P = and 0.01; AUROC 0.82 and 0.74) were statistically significant. In combined cohorts of HPV and EBV patients, ML and GEP were significantly associated with OR (P = and 0.002; AUROC 0.70 and 0.76, respectively). ML and GEP were only weakly correlated (r = 0.173). In a joint model, ML was significantly associated with response (p = 0.020) after adjusting for GEP (also significant, p = 0.006). NL and clonality weighted ML were also significantly associated with response (P = and 0.006, respectively). In HPV + or EBV + subjects, OR association was not significant for ML, possibly due to a dominance of viral vs somatic neoepitopes; GEP was significant, ifi likely l due to tumor inflammation. Conclusions: ML and GEP are independently d predictive of response to pembrolizumab in HPV /EBV patients with HNSCC; GEP was predictive regardless of viral status. ML and GEP may have utility in characterizing responses to anti PD 1 therapies and novel cancer regimens in HNSCC. Clinical trial information: NCT
18 Haddad R, et al Journal of Clinical Oncology 35, no. 15_suppl (May 2017) Background: Somatic mutational load (ML) is associated with response to anti CTLA 4 and PD 1/ L1 immunotherapies in select tumors, due to formation of neoepitopes not subject to central immune tolerance. Neoepitopes specific to HPV, EBV virus infection are also present in some HNSCC. An IFNγ gene expression profile (GEP) characteristic ti of tumor inflammation is also related ltdto response to anti PD 1/ L1 therapy. This study evaluated relationships between ML and clinical outcome and independent predictive values of ML and GEP in patients with HNSCC treated with pembrolizumab. Methods: Whole exome sequencing (WES) and GEP were assessed in FFPE tumor specimens ML and objective of patients response with HNSCC (OR) (KEYNOTE (P = ; subsets and 0.055; of B1 [PD L1 AUROC +, n 0.89 = 34] and B2 0.63), [PD L1 and + /, n GEP = 73] cohorts). ML, and neoantigen OR (P = load (NL), and HPV/EBV 0.01; AUROC status and 0.82 clonality and were 0.74) assessed were statistically by standard WES significant. analytical methods. GEP score is a weighted sum of normalized expression values of 18 genes. Statistical testing of ML and response, and ML and GEP relationship by HPV/EBV status was prespecified. Results: There were 73 patients identified as HPV and EBV (n = 25 ML in B1; and n = GEP 48 in were B2). In only HPV weakly and EBV correlated patients in B1 and B2 cohorts, respectively, associations between ML and objective response (OR) (P = and 0.055; AUROC 0.89 and 0.63), and GEP ML and OR GEP (P = may have and 0.01; utility AUROC in characterizing 0.82 and 0.74) were responses statistically to anti significant. PD 1 therapies In combined and cohorts novel of HPV and EBV patients, ML and GEP were significantly cancer regimens associated in with HNSCC OR (P = and 0.002; AUROC 0.70 and 0.76, respectively). ML and GEP were only weakly correlated (r = 0.173). In a joint model, ML was significantly associated with response (p = 0.020) after adjusting for GEP (also significant, p = 0.006). NL and clonality weighted ML were also significantly associated with response (P = and 0.006, respectively). In HPV + or EBV + subjects, OR association was not significant for ML, possibly due to a dominance of viral vs somatic neoepitopes; GEP was significant, ifi likely l due to tumor inflammation. Conclusions: ML and GEP are independently d predictive of response to pembrolizumab in HPV /EBV patients with HNSCC; GEP was predictive regardless of viral status. ML and GEP may have utility in characterizing responses to anti PD 1 therapies and novel cancer regimens in HNSCC. Clinical trial information: NCT
19 Higher levels of tobacco mutational signature is associated with higher tumor mutational g g g burden, consistent with results reported in NSCLC However, tobacco mutational signature correlated inversely with the degree of T cell infiltrate, immune cell infiltrate, and IFN γ signaling Tumors with high levels of immune cell (HR = 0.66, P = 0.023), T cell (HR = 0.53, P = ) and CD8+ T cell (HR = 0.67, P = 0.029) infiltration were associated with better OS. Increasing level of tobacco mutational signature was associated with poorer survival (P = 0.005)
20 PD-L1 as a predictive biomarker
21 Nivolumab for SCCHN CheckMate 141: Overall Survival OS (%) No. at risk Median OS, mo (95% CI) HR (97.73% CI) P value Nivolumab (n = 240) 7.5 (5.5, 9.1) 0.70 Standard Therapy (n = 121) 5.1 (4.0, 6.0) (0.51, 0.96) 1-year OS rate (95% CI) 36.0% (28.5, 43.4) 16.6% 6% (8.6, Standard Therapy Nivolumab ) Months Nivolumab Standard Therapy 0.01 Adapted from Ferris et al. NEJM 2016; doi: /NEJM0a Abbreviations and references can be found in the speaker notes.
22 Nivolumab for SCCHN CheckMate 141: Overall Survival by PD-L1 Expression PD-L1 Expression 1% No. of Median OS No. of Patient mo (95% Deaths s CI) Nivolumab ( ) Standard Therapy ( ) PD-L1 Expression <1% No. of No. of Median OS Patients Deaths mo (95% CI) Nivolumab ( ) Standard Therapy ( ) Overall Surviv val (% of patien nts) No. at Risk Hazard ratio for death, 0.55 (95% CI, ) Standard Therapy Nivolumab Months Hazard ratio for death, 0.89 (95% CI, ) 30 Nivolumab Standard Therapy Months Nivolumab Standard 61 Therapy Estimates for OS HR for PD-L1 expression levels of 5% and 10% are similar to those for 1%. Adapted from Ferris et al. NEJM 2016; doi: /NEJM0a Abbreviations and references can be found in the speaker notes.
23 HNSCC Cohorts of Nonrandomized, Phase 1b KEYNOTE-012 Trial Biomarker analysis A Phase 1b Study of Pembrolizumab in Patients With HPV-Positive and HPV-Negative Head and Neck Cancer Patient population R/M HNSCC Measurable disease (RECIST v1.1) ECOG PS 0-1 PD-L1 positive (initial cohort) PD-L1 positive or PD-L1 negative (expansion cohort) Initial Cohort B N=60 Pembrolizumab 10 mg/kg q2w Expansion Cohort B2 N=132 Pembrolizumab 200 mg q3w Treat for 24 months or until progression or intolerable toxicity Pre-treatment samples collected for biomarker analyses Pre-treatment biomarker levels were correlated with efficacy outcomes (ORR, PFS, OS; central imaging vendor review) HNSCC = recurrent or metastatic squamous cell carcinoma; ORR = overall response rate; OS = overall survival; PD-L1 = programmed death ligand 1; PFS = progression-free survival; q2w = every 2 weeks; q3w = every 3 weeks; RECIST v1.1 1 = Response Evaluation Criteria In Solid Tumors version Chow LQ et al. J Clin Oncol. 2016;34(15;suppl): abstract For Internal Use Only
24 KEYNOTE-012: Overall Response by PD-L1 Status 1 PD-L1 Status Nonresponders, n Responders, n ORR % (95% CI) P TPS (tumor cells) PD-L1 positive PD-L1 negative (12 26) 26) (10 30) P values based on logistic regression one-sided testing. CI = confidence interval; CPS = combined positive score; ORR = overall response rate; PD-L1 = programmed death ligand 1; TPS = tumor proportion score. 1. Chow LQ et al. J Clin Oncol. 2016;34(15;suppl): abstract For Internal Use Only
25 KEYNOTE-012: Overall Response by PD-L1 Status 1 Incorporation of inflammatory cells improves ability to detect responders PD-L1 Status Nonresponders, n Responders, n ORR % (95% CI) P TPS (tumor cells) PD-L1 positive PD-L1 negative (12 26) 26) (10 30) CPS (either tumor and/or inflammatory cells) PD-L1 positive PD-L1 negative (15 28) (1 19) P values based on logistic regression one-sided testing. CI = confidence interval; CPS = combined positive score; ORR = overall response rate; PD-L1 = programmed death ligand 1; TPS = tumor proportion score. 1. Chow LQ et al. J Clin Oncol. 2016;34(15;suppl): abstract For Internal Use Only
26 KEYNOTE-012: Progression-free Survival by PD-L1 Status 1 og ess o ee Su a by Saus TPS (tumor cells) CPS (tumor and inflammatory cells) 1.00 P = P = Sur rvival Probab bility PD-L1+ PD-L1 Surv vival Probab bility PD-L1+ PD-L Time, days Time, days TPS< TPS CPS< CPS Median (95% CI) PD-L1 positive, 63 days (58 98) PD-L1 negative, 62 days (59 67) P values based on logistic regression one-sided testing. CI = confidence interval; CPS = combined positive score; PD-L1 = programmed death ligand 1; TPS = tumor proportion score. 1. Chow LQ et al. J Clin Oncol. 2016;34(15;suppl): abstract For Internal Use Only Median (95% CI) PD-L1 positive, 64 days (59 98) PD-L1 negative, 60 days (51 66)
27 KEYNOTE-012: Overall Survival by PD-L1 Status 1 TPS (tumor cells) CPS (tumor and inflammatory cells) 1.00 P = P = Survival Prob bability PD-L1+ PD-L1 Survival Prob bability PD-L1+ PD-L Time, days Time, days TPS< TPS CPS< CPS Median (95% CI) PD-L1 positive, 290 days ( ) PD-L1 negative, 246 days ( ) Median (95% CI) PD-L1 positive, 303 days ( ) PD-L1 negative, 151 days (84 247) P values based on logistic regression one-sided testing. CI = confidence interval; CPS = combined positive score; PD-L1 = programmed death ligand 1; TPS = tumor proportion score. 1. Chow LQ et al. J Clin Oncol. 2016;34(15;suppl): abstract For Internal Use Only
28 Challenges to use PD L1 as biomarker: Fluctuation in expression at different time points Variation within tumor tissue Lack of uniformity in cutoff points employed in different trials, in kit and antibodies used for the detection of PD L1 expression Where to look for them: TC, IC or both? Responses in PD L1 negative tumors OR No response even in highly PD L1 positive tumors While there are many doubts about how perfect PD L1 testing is, there is a belief it plays a role for enrichment!!
29 PD-L2 as a predictive biomarker
30
31 Jennifer H. Yearley et al, Clin Cancer Res; 23(12) June 15, 2017
32 KEYNOTE-012: Overall Response by PD-L2 Status 1 PD-L2 expression on tumor and inflammatory cells is predictive of response to pembrolizumab Samples from172 patients were evaluated for PD-L2 expression CPS (tumor and/or inflammatory cells) Nonresponders, Responders, ORR n n %(95% CI) PD-L (15 31) PD-L (4 20) P P values based on logistic regression one-sided testing. CI = confidence interval; CPS = combined positive score; ORR = overall response rate; PD-L2 = programmed death ligand For Internal Use Only
33 KEYNOTE-012: Correlation of PD-L1, PD-L2, and Response 1 Data suggest that PD-L2 predicts clinical outcome in pembrolizumab treated pts n=172 n = 39 PD-L1+/ PD-L2 n = 22 PD-L1 / PD-L2 n = 108 PD-L1+/ PD-L2+ PD-L1 Positive n ORR % (95% CI) PD-L1 Negative n ORR % (95% CI) n = 3 PD-L1 / PD-L2+ Significant association (P<0.001) between PD-L1 and PD-L2 expression 33 For Internal Use Only 1. Chow LQ et al. J Clin Oncol. 2016;34(15;suppl): abstract PD-L (16 32) 3 0 (0 71) PD-L (3 24) 22 9 (1 29) High response in PD-L1 positive/ PD-L2 positive tumors
34 HPV status t as a predictive biomarker
35
36 Nivolumab for SCCHN CheckMate 141: Overall Survival OS (%) No. at risk Median OS, mo (95% CI) HR (97.73% CI) P value Nivolumab (n = 240) 7.5 (5.5, 9.1) 0.70 Standard Therapy (n = 121) 5.1 (4.0, 6.0) (0.51, 0.96) 1-year OS rate (95% CI) 36.0% (28.5, 43.4) 16.6% 6% (8.6, Standard Therapy Nivolumab ) Months Nivolumab Standard Therapy 0.01 Adapted from Ferris et al. NEJM 2016; doi: /NEJM0a Abbreviations and references can be found in the speaker notes.
37 Nivolumab for SCCHN CheckMate 141: Overall Survival by p16 Status t p16-positive p16-negative No. of No. of Median OS No. of No. of Median OS Patients Deaths mo (95% CI) Patients Deaths mo (95% CI) Nivolumab ( ) Nivolumab (3.0 NA) Standard Therapy ( ) Standard Therapy ( ) Hazard ratio for death, 0.56 (95% CI, 0.32 Hazard ratio for death, 0.73 (95% CI, ) ) patients) Overall Survival (% of Standard Therapy Nivolumab No. at Risk Months Nivolumab Standard Therapy Nivolumab Standard Therapy Months mos appeared longer for patients treated with nivolumab than standard therapy regardless of p16 status Adapted from Ferris et al. NEJM 2016; doi: /NEJM0a Abbreviations and references can be found in the speaker notes.
38 IFN γ signature
39 FOR INTERNAL USE ONLY; NOT TO BE SHARED OR DISTRIBUTED TO ANYONE OUTSIDE THE COMPANY
40 T-Cell Inflamed Phenotype Gene Expression Signatures Predict Benefit from Pembro across Multiple Tumor Types Patients enrolled FFPE tumor in clinical study tissue NanoString Gene expression data RNA Evaluate genes and signatures associated with anti-pd-1 response Collected at baseline prior to receiving anti-pd-1 therapy Transcripts for genes of interest are counted 680 genes on platform immune focused (custom design) six IFN-γ regulated genes with a gene signature panel IFN-γ (6 gene) significantly associated with ORR, PFS (IFN CXCL9, CXCL10, IDO1, HLA-DRA, STAT1 - Nanostring) Consistent data across CRC, Esophageal, Biliary, Anal and Ovarian 40 For Internal Use Only
41 Inflamed-phenotype gene expression signatures to predict benefit from the anti-pd-1 antibody pembrolizumab in PD-L1+ head and neck cancer patients Tanguy Y. Seiwert et al abstr 6017 They investigated 4 multi-gene expression signatures previously described in melanoma and gastric patients (pts). FFPE-extracted RNA was analyzed on the NanoString ncounter system which is being developed as a companion diagnostic Interferon γ γ signature correlated strongly with the previously independently discovered inflamed/mesenchymal HNSCC intrinsic subtype
42 IMS subtype is characterized among others by CD8+ T-cell infiltration
43 Seiwert TY et al, Lancet Oncol Jul;17(7): The gene expression composite score provided a positive predictive value of 40% and a negative predictive value of 95% th ti f ti t ith ll i th bi k l t d the proportion of patients with an overall response in the biomarker selected population was nearly double that seen in the trial overall
44 KEYNOTE-012: Progression-free Survival and Overall Survival by IFN- 6-gene esg Signature auescoe Score 1 Progression-free Survival Overall Survival 1.00 IFN- Score < Q1 IFN- Score Q IFN- Score < Q1 IFN- Score Q1 Su urvival Proba ability Su urvival Proba ability Time, days IFN- Score < Q IFN- Score Q Time, days IFN- Score < Q IFN- Score Q Signature score was associated with improved PFS and OS Nonresponder Q1 defined as IFN = interferon; OS = overall survival; PFS = progression-free survival. 1. Chow LQ et al. J Clin Oncol. 2016;34(15;suppl): abstract For Internal Use Only
45
46 Slide 20 Presented By Drew Pardoll at 2015 ASCO Annual Meeting
47 T cell inflamed phenotype (TCIP) defined by a 12 gene chemokine signature (CCL2, CLL3, CLL4, CCL5, CCL8, CCL18, CCL19, CCL21, CXCL9, CXCL10, CXCL11 and CXCL13) was evaluated in a cohort of 134 HNSCC from the University of Chicago and 424 HNSCC samples from TCGA The presence of the TCIP was associated with infiltration of CD8+ cells in a subset of HNSCCs; 21% of HPV negative tumors were TCIP high and 51% of HPV positive tumors were TCIP high. The TCIP high phenotype was associated with mesenchymal subtype and higher prevalence of PD L1 expression, suggesting that this phenotype could represent the sensitivity to anti PD1/PD L1 therapies.
48 Inflamed phenotype correlates with Immune escape mechanisms Presented by T. Seiwert ESMO 2016
49
50 Conclusion Immunotherapy is active in SCCHN Identification of pts who will more likely benefit from ICI Most research is focused in TME Intensive research in this field; PD L1, PD L2, IFN γ gene signature and others have been proposed p The role of peripheral blood?? Lot of suppressive Treg, MDSC subpopulations have been suggested as potential biomarkers for response
51 Thank you
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