Emerging immunotargets and primary tumor-infiltrating lymphocytes as biomarker in metastatic renal cell carcinoma

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1 Emerging immunotargets and primary tumor-infiltrating lymphocytes as biomarker in metastatic renal cell carcinoma Holger Moch Department Pathology University Hospital Zurich Boston

2 Innovation in Pathology High Throughput Scanning New Drugs Machine Learning Fusion of genomics and pathology data Systems Pathology Big Data Science New Imaging Technologies Expert/Crowd Sourcing Giesen et al. Nat Methods 2014

3 Immunotargets in RCC VHL alteration, renal cancer progression and therapy VHL protein, microvessels, TAM and TIL Quantification of microvessels and TIL

4 Clinical facts on Renal Cell Carcinoma (RCC) urologyhealth.org Incidence Mortality 2% of all cancers, 20-30% of patients are diagnosed with metastasis 30-40% die due to the disease Treatment Localized disease Advanced disease Surgery VEGF-targeted therapies or immunotherapy Source: SEER Cancer Statistics

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6 Introduction - Biological facts of RCC Renal Cell Carcinoma (RCC) subtypes RCC subtype clear cell papillary type 1 papillary type 2 chromophobe Incidence 75 % 15 % 5 % Genes for familial types VHL MET FH BHD (FLCN)

7 The VHL-Proteine Rbx1 Elongin B Cul 2 Elongin C pvhl b a HIF HIF HIF O 2 O 2 VEGF

8 Sunitinib 750 Patienten, 8/ /2005 Motzer R 2007, NEJM

9 Sunitinib Motzer R 2007, NEJM

10 Send Orders for Reprints and to Current Drug Targets, 2016, 17, Emerging Immunotargets in Metastatic Renal Cell Carcinoma John Kucharczyk a, Marc R. Matrana b, Matteo Santoni c, Francesco Massari d, Marina Scarpelli e Safety, Liang Cheng f data reflect results from patients with all, Antonio Lopez-Beltran g tumor histologies, while the efficacy, Stefano Cascinu c data focuses on renal cell carcinoma, Rodolfo Montironi e patients. RCC, renal cell carcinoma; AE, adverse event; RR, response rate; SD, stable disease; PFS, progress free survival. and Moch Holger h Table 2. a University of Queensland Ochsner Clinical School, Ochsner Medical Center, New Orleans, Louisiana, USA; b Ochsner Cancer Institute, Gayle and Tom Benson Cancer Center and University of Queensland Ochsner Clinical School, Ochsner Medical Center, New Orleans, Louisiana, USA; c Clinica di Oncologia Medica, AOU Ospedali Riuniti, Polytechnic University of the Marche Region, Ancona, Italy; d Medical Nivolumab Oncology, PD-1 Azienda Ospedaliera III Compare Universitaria OS vs everolimus in advanced Integrata, mrcc University pretreated with antiangiogenic therapy. of Verona, Piazzale L.A. Scuro 10, Verona, Italy; e Pathological Anatomy, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Ancona, Italy; f Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA; g Department of Surgery and Pathology, Cordoba University Medical School, Cordoba, Spain; h Institute of III PFS OS in patients with untreated RCC with Surgical Pathology, University Hospital Zurich, Zurich; Switzerland Abstract: Renal cell carcinoma (RCC) is one of the most immunoresponsive human cancers. High-dose IL-2 and Interferon-α were once the principle therapies for metastatic RCC, however they had harsh-tolerance profiles and limited re- II Dose-ranging study on RCC pretreated with anti-angiogenic therapy NCT sponse rates. In the last decade, targeted I therapies Biomarker have analysis supplanted of mrcc patients cytokine receiving therapy nivolumab due therapy to higher response NCT rates and more favorable Pembrolizumab toxicity profiles. PD-1 Emerging I immunotherapies Neoadjuvant therapy targeting patients the receiving PD-1 receptor RCC tumor and resections PD-L1 ligand NCT have shown promising results. Likewise, other novel targeted immunotherapies are currently under evaluation. The safety profiles and response rates of new generation immunotherapies are encouraging and justify the progression of clinical trials. However, I/II Pembrolizumab +/- pazopanib in naïve advanced RCC patients NCT longer follow-up data are needed I/II to confirm Pembrolizumab these promising + PegIFN-2b results. vs pembrolizumab In addition, + ipilimumab it is still advanced unclear if NCT an optimal sequence or combinations of new immunotherapies paired with current targeted melanoma therapies and RCC will emerge. Keywords: Immunotherapy, immunotargets, metastatic renal cell carcinoma, kidney cancer. INTRODUCTION Ongoing clinical trials on PD-1/PD-L1 blocking agents ( from clinicaltrials.gov). Drug name Target Phase Description ClinicalTrials.gov identifier Approximately 273,000 new cases of renal cell carcinoma (RCC) are annually diagnosed worldwide, representing about 2 percent of all cancers. RCC tends to occur in higher incidence in Western countries. About 45,000 patients in Europe die from kidney cancer each year. I RCC is one of the most immoresponsive human cancers I [1]. High-dose IL-2 (HDIL-2) and Interferon (IFN)-α have been approved by United States Food and Drug Administration (FDA) for patients with metastatic RCC (mrcc). However, they present harsh-tolerance profiles and limited response rates [2]. In the last decade, targeted therapies, DC, dendritic cell. such NCT II Alone vs combination with bevacizumab or ipilumab on mrcc NCT I Combination with sunitinib, pazopanib or ipilimumab NCT nivolumab/ipilimumab combination therapy vs sunitinib monotherapy NCT I/II Pembrolizumab + bevacizumab in mrcc NCT I Pembrolizumab + ziv-aflibercept in advanced solid tumors including RCC NCT optimal setting for their use? And how can we sequence or combine these agents with already approved targeted therapies? The answer to these questions NCT will constitute a major challenge for uro-oncologists in the near future and will completely revolutionize the management of mrcc patients. Pidilizumab PD-1 II Alone or with DC/RCC fusion cell vaccine NCT MPDL3280A PD-L1 II As monotherapy vs combination therapy with bevacizumab or sunitinib in untreated advanced RCC I First in human study in advanced and metastatic RCC NCT In this review, we describe the NCT recent data on the use of immunotherapies RCC in patients with mrcc, administered alone or in combination with targeted agents NCT or other immunotherapeutic RCC approaches. Combination with ipilimumab or IFN α-2b in advanced/metastatic Combination with cobimetinib in advanced solid tumors including BMS PD-L1 I Multidose study in advanced solid tumors including RCC NCT PD-1/PD-L1 EXPRESSION AS A BIOMARKER RCC, renal cell carcinoma; mrcc, metastatic renal cell carcinoma; OS, overall survival; PFS, progress free survival; PegIFN-2b, pegylated interferon α-2b; The ability to gauge tumor response by determining PD-

11 Am J Surg Pathol 37, 2013 No Predictive Biomarker for Therapy Response

12 Immunotargets in RCC VHL alteration, renal cancer progression and therapy VHL protein, microvessels, TAM and TIL Quantification of microvessels and TIL

13 Molecular Pathways and Targeted Therapies in Renal-Cell Carcinoma Brugarolas J. N Engl J Med 2007;356:

14 Tumor Blood vessel cell TKI TKI

15 PD-1/PD-L1 blocking Agents in RCC MDPL3280 McDermot DF et al. McDermott DF, et al. Immune correlates and long term follow up of a phase Ia study of MPDL3280A, an engineered PD-L1 antibody in patients with metastatic renal cell carcinoma. Annals of Oncology (2014) 25

16 PD L1 expression is regulated by HIF in clear cell RCC Ruf et al.: Int J Cancer March 2016

17 RCC attract TAM enhanced expression of PD1 association with brain mets

18 Immunotargets and TIL in RCC VHL alteration, renal cancer progression and therapy VHL protein, microvessels, TAM and TIL Quantification of microvessels and TIL

19 Morra et al.: AJP 2011

20 Evaluation of Microvessel Area by AQUA-System Automated Quantitative Analysis; McCabe et al.: JNCI 97, 2005: Mertz et al.: Epithelial/Stromal-Mask (CD10; EMA; Vimentin) Low High

21 Evaluation of Microvessel Area by AQUA-System MVA is associated with Survival P< Mertz et al.: Hum Pathol 2007

22 Vessel Segmentation, Classification and Quantification by Tissue Studio and Developer XD Tissue Studio: Vessel Extraction and Classification Developer XD: Tissue Classification in Tumor and Non Tumor Regions

23 TissueStudio

24 Overall survival (%, x100) Blood vessel density (CD34) p= N=27 N=23 low high mean Time

25 Peritumoral lymph vessels D2-40

26 D2-40

27 D2-40

28 Intra- and peritumoral lymph vessels! Lyve1

29 Lyve1

30 Lyve1

31 D2-40

32 D2-40

33 D2-40

34 Overall survival (%, x100) Overall survival (%, x100) Intratumoral lymphatic vessel density (LYVE1) p= N=12 N=35 low high mean p= N=22 N=25 low high median Time Time Hemato-Lymphangiogenic Switch

35 Results high MVD of blood vessels associated with better prognosis Peritumoral lymph vessels (PTL) not associated with prognosis Intratumoral lymph vessels (ITL) associated with poor outcome ITL associated with high TIL count ITL or PTL associated with tertiary lymphatic structures?

36 Future Projects Quantification of TIL Quantification of TAM Quantification of peritumoral TLS Lymphatic aggregates w/wo germinal centers Tertiary lymphatic structures Correlation of TIL, TAM and TLS with prognosis and therapy response Co-registration of different biomarkers in RCC (CD70-positive RCC and CD27-positive TIL)

37 Quantitative pathology approach for quantification of TILs 1. Multi-colour IF staining (Opal TSA kit) Ventana Discovery Raw multispectral image 2. Multispectral imaging of slides Vectra Un-mixing of fluorescent signals InForm software 4. Training of cell phenotype algorithm InForm software 5. Quantification of different cell phenotypes InForm software 6. Statistical analysis MS Exel, SPSS Un-mixed composite image CD3 CD20 Lyve1 DAPI Cell phenotype map CD3 CD20 Lyve1 Other

38 Patient ID: Image ID: TLS_[42861,9265] Tumour IF: CD3 CD20 Lyve1 DAPI

39 Patient ID: Image ID: TLS_[42861,9265] Cell phenotyping by Inform software: CD3 CD20 Lyve1 Other

40 Patient ID: Image ID: TLS_[45214,11661] TLS in tumour periphery IF: CD3 CD20 Lyve1 DAPI

41 Overall survival (%, x100) Tertiary Lymphatic Structures TLS (mm 2 ) N=38 N=11 absent present mean.2 0 p=ns Time

42 CD70 is frequently expressed in primary ccrcc and metastases Clinical Trials: anti-cd70 agents (antibody-drug conjugates): MDX-1203 (NCT ) MDX-1411 (NCT ) SGN-75 (NCT ) Ruf et al.: Clin Cancer Res 2015

43 Co-Registration: CD27 + TIL preferentially infiltrate CD70-expressing RCC

44 PBMCs trigger the release of scd27 in a CD70- dependent manner CD70 expression is regulated by HIF Increased serum levels of CD27 suggest existence of CD70- expressing ccrcc scd27 as potential serum marker Ruf et al.: Clin Cancer Res 2015

45 TIL: Lymphocyte Segmentation, Classification and Quantification by Developer XD First Step: applying Definiens Image Analysis solution for Nuclei Object Detection (developed by Dr. Nicolas Brieu). Nuclei Objects are the different types of lymphocytes: CD3, CD8 and CD20 Second Step: embedding lymphocytes into tumor and non-tumor regions

46 Tissue Phenomics Applications Virtual multiplexing Co-registration of images from different biomarkers Analysis of relations between specific biomarkers and corresponding objects Statistics of mutual relations: Tumor heterogeneity and distribution of lymphatic vessels (ITL, PTL) and bloodvessels (MVD) Mutual relation of the local lymphatic vessel distribution to the local blood vessel distribution Relative position of blood vessels and lymphatic vessels to TIL, TAM and peritumoral tertiary lymphatic structures (TLS)

47 CD3

48 CD8

49 CD34

50 d2-40

51 lyve1

52 CD3 CD8 CD34 d2-40 lyve1

53

54 Developer XD

55 Developer XD

56 Conclusion need of predictive biomarker for immunotherapy in RCC Potential vessel biomarker: MVD, ITL, PTL Potential lymphocyte biomarker: TIL, TAM and peritumoral TLS Existence of intratumoral lymphatics (ITL) is associated with poor prognosis Tissue Phenomics allows quantification of complex morphological structures Tissue phenomics as a tool to identify mutual relations between different biomarkers

57 Maria Athelogou Ralf Huss Günter Schmidt Gerd Binnig