Pitié-Salpêtriètre Hospital Assistance-Publique Hôpitaux de Paris Molecular Genetics for the Practicing Physician: Can the Cancer Genome Atlas Impact How We Treat Patients? Gabriel G. Malouf, MD, PhD Assistant Professor Pitié-Salpêtriètre Hospital Assistance-Publique Hôpitaux de Paris Paris, France Many thanks to the organizing committee for the opportunity to present this talk, entitled “Molecular Genetics for the Practicing Physician: Can the Cancer Genome Atlas Impact How We Treat Patients?

Outline Cell of Origin of Kidney Tumors Diagnostic Markers and Therapeutic Targets Predictive Biomarkers

Renal Cell Carcinoma is NOT a Single Disease Mucinous tubular & spindle cell RCC Clear cell RCC Papillary RCC Chromophobe RCC Translocation RCC Type 1 Type 2 Collecting Duct RCC

The Cancer Genome Atlas (TCGA) Project (2006-2016) Clear-cell RCC (n=417) Papillary RCC (n=161) Chromophobe (n=66) TCGA, Nature Genetics, 2013

Practicing Physicians Pathologist Urologist Radiologist Medical Oncologist Radiation Oncologist Comorbidities Optimal therapy to improve survival and quality of life Clear-cell RCC1 Chromophobe RCC2 Papillary RCC3 Tumor biology Molecular Genetics TCGA Practicing Physicians Patients 1TCGA, Nature, 2013 2Davis C et al, Cancer Cell, 2014 3Linehan WM et al. NEJM, 2015

I. Cell of Origin of Kidney Tumors

Hierarchical Clustering of Gene Expression in Kidney Structure Proximal tubules Glomeruli Distal Convoluted tubules Thick ascending limbs of the loop of Henle Distal nephrons cTAL = cortical TAL, mTAL =medullary TAL, DCT= distal convoluted tubules, CNT= connecting tubules, OMCD = outer medullary collecting ducts Cheval L et al. Physiol. Genomics, 2010

DNA Methylation and Gene Expression Differences Between ChrRCC and ccRCC CCD = cortical collecting ducts; ccRCC = clear cell renal cell carcinoma; chRCC = chromophobe renal cell carcinoma; cTAL = cortical thick ascending limbs of the loop of Henle; DCT = distal convoluted tubules; Glom = glomeruli; mTAL =medullary thick ascending limbs of the loop of Henle; OMCD = outer medullary collecting ducts; TCGA = The Cancer Genome Atlas. Davis C et al, Cancer Cell, 2014

Survival Prediction of Clear-cell Renal Cell Carcinomas Based on Gene Expression Similarity to the Proximal Tubule of the Nephron Clustering of renal Cancer-specific survival (CSS) of clear cell renal cell carcinoma (ccRCC) tumours predicted by the S3-score Clustering of renal cell carcinoma (RCC) tumours by means of gene expression correlation between profiles of tumours and profiles of human nephron cell types Büttner et al, Eur Urol, 2015

DNA Methylation Establishes the Foundation of RCC Ontogeny Malouf GG et al, ASCO GU 2016

II. Diagnostic Markers and Therapeutic Targets

Incidence of Fusion Transcripts Detected in TCGA Clear-cell RCC TCGA KIRC dataset (n=460) No Translocation* (n=379) Translocation (n=81) (17.6%) MITF family RCC (n=7) (1.9%) Previously known gene fusions (n=12) Novel fusions (n=62) PRCC/TFE3 (n=5) KHSRP/TFE3 (n=1) KHDRBS2/TFEB (n=1) FHIT (n=2), SLC9A9 (n=2), AFF1 (n=1), MKL1 (n=1), LHFP (n=1), JAK2 (n=1), ELL (n=1), DCX (n=1), EP300 (n=1) & LNP1 (n=1) Figure 2 Malouf GG et al. Clin Cancer Res, 2014

Incidence of Fusion Transcripts Detected in TCGA Papillary RCC TCGA KIRP dataset (n=161) No Translocation* (n=144) Translocation (n=17) (10.6%) Non MITF family RCC (n=9) MITF family RCC (n=8) 7 out of 60 Papillary Type II (11.6%) 1 out of 101 Papillary type I (1%) Figure 2 Linehan WM et al. NEJM, 2015

A Subgroup of Papillary RCC Manifests a CpG Island Methylator Phenotype (CIMP) and FH Mutations Linehan WM et al. NEJM, 2015

ChRCC with Eosinophilic Variants Resemble Oncocytoma Type II Davis C et al, Cancer Cell, 2014 Joshi et al, Cell Reports, 2015

Putative Therapeutic Targets in Clear-cell RCC TCGA, Nature, 2013

Putative Therapeutic Targets in Papillary RCC Linehan WM et al. NEJM, 2015

III. Predictive Biomarkers

TCGA Transcriptomic Classification of ccRCC Four subgroups Response to sunitinib TCGA, Nature, 2013 Beuselinck, Clin Cancer Res, 2015

Overall Survival in Two Independent Datasets of ccRCC HR= hasard ratio. UT South Western cohort TCGA cohort Kapur P et al, Lancet Oncol, 2013

While Many Biomarkers Are Associated in Univariate Analysis with Outcome in TCGA Clear-Cell RCC, only ccB Remains in Multivariate Analysis Clinical and pathological charecteristics Somatic mutations Somatic copy number expression Gene expression analysis Gulati S et al, Eur Urol, 2014

Tumor-based Biomarkers Confounded by Intratumor Heterogeneity 63-69% of all mutations not detectable across regions in same tumor Gerlinger et al. NEJM, 2012

Multi-Region Sequencing of Clear-cell Renal Cell Carcinoma How to move from studying universality to assess variability and take it in account to personalize patients therapy

Multi-Region Sequencing of RCC with IVC Thrombus

Long-non coding RNA Classification of ccRCC Reveals Four Subtypes Associated with Clinical Outcome Malouf GG et al, Molecular Oncology , 2015

CIMP Defines a Subtype of Clear-cell Renal Cell Carcinomas with Poor Outcome Cluster 1: CpG Island Methylator Phenotype (CIMP) low; Cluster 2: CIMP negative; Cluster 3: CIMP positive Malouf GG et al, Unpublished

Conclusions Understanding ontogeny of different RCC subtypes will provide insights on tumorigenesis To date, there are no approved biomarkers to predict recurrence after nephrectomy or response to therapy Frequent mutations in chromatin modifying genes in both ccRCC and pRCC imply a role for epigenetic therapy

Conclusions (continued) Inter- and intra-tumoral genetic heterogeneity might hamper the development of predictive and prognostic genetic biomarkers in RCC Mutational Convergence in chromatin modifying genes highlights the importance of epigenetic drift in the evolution of aggressive RCC clones Epigenetic biomarkers might be more stable and therefore should be pursued for precision medicine

Acknowledgements Pitié-Salpêtrière Hospital, Paris, France Pr David Khayat (Oncology) Pr Jean-Philippe Spano (Oncology) Dr Haide Boostandoost (Oncology) Pr Eva Compérat (Pathology) Pr Morgan Rouprêt (Urology) Dr Jérôme Parra (Urology) Dr Christophe Vaessen (Urology) Fondation AVEC laboratory, Paris, France Dr Roger Mouawad, PhD Frédérick Allanick, Ms Dr Souheyla Bensalma, PhD Dr Linda Denaise, MD, PhDc Dr Marion Classe, MD, PhDc Dr Ronan Flippot, MD, MS Temple University, Philadelphia, USA Jaroslav Jelinek Jean-Pierre Issa MD Anderson Cancer Center, Houston, USA Pr Nizar M. Tannir (Medical Oncology) Dr Xiaoping Su (Bionformatics) Dr Hui Yao (Bionformatics) Dr Jose A. Karam (Urology) Pr Christopher G. Wood (Urology) The patients and their families