RCC with Sarcomatoid Dedifferentiation: New Insights. Jose A. Karam, MD, FACS Assistant Professor Department of Urology November 6, 2015

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1 RCC with Sarcomatoid Dedifferentiation: New Insights Jose A. Karam, MD, FACS Assistant Professor Department of Urology November 6, 2015

2 Farrow GM. Cancer. 1968

3 Epidemiology of srcc ~5% of all renal tumors are sarcomatoid More than 75% of patients with srcc present with metastatic disease Median overall survival of less than a year Not improved despite the advent of targeted therapies Shuch B. The Oncologist. 2012

4 Histology 2 Components Epithelial component Clear cell, papillary, chromophobe, collecting duct, MTSCC, other Sarcomatoid component Delahunt B. Am J Surg Path. 2013

5 Sarcomatoid Component Malouf G. Submitted for publication

6 Outline-Questions Can we identify srcc on preoperative imaging? Can we identify srcc on preoperative biopsy? Is srcc different from non-srcc on RNA level? DNA level? immune markers?

7 PREOPERATIVE IMAGING

8 MRI-1 Retrospective ( ) 9 patients 2 radiologists reviewed preoperative MRI in patients who later had nephrectomy 5 clear cell Irregular or infiltrative morphology and heterogeneous T2 signal intensity and enhancement Internal necrosis in all cases Non-specific Rosenkrantz AB. Clinical Imaging. 2011

9 MRI-2 Retrospective ( ) 11 patients with clear cell RCC with sarcomatoid elements Correlate preoperative MRI with pathology from nephrectomy Takeuchi M. Clinical Imaging. 2013

10 srcc showed significantly lower enhancement than the ccrcc part in each MRI phase Pseudocapsule disruption all cases Issue non-blinded, need comparator arm MRI Findings Takeuchi M. Clinical Imaging. 2013

11 Current Ongoing Research Identification of srcc using preoperative imaging PIs: Rivka Colen (Radiology) and Jose Karam (Urology), MD Anderson

12 PREOPERATIVE BIOPSY

13 FNA/Core biopsy (MD Anderson) 166 patients ( ) FNA/core biopsy of the kidney before CRN 34 patients had sarcomatoid RCC at CRN Only 4 patients (11.7%) had sarcomatoid elements detected on preoperative biopsy Abel EJ. J Urology. 2010

14 FNA/Core biopsy (MD Anderson) 405 FNA or biopsies from 378 patients ( ) 239 from mets 166 from primary tumors 76 patients had sarcomatoid RCC at time of surgery Only 7 patients (9.2%) had sarcomatoid elements detected on preoperative biopsy Abel EJ. BJU Int. 2012

FNA/Core biopsy (University of Wisconsin) 122 biopsies (ct2 or greater renal masses) 46 standard 76 multiquadrant (at least 4 separate areas) 2009 to 2014 96 were RCC

15 FNA/Core biopsy (University of Wisconsin) 122 biopsies (ct2 or greater renal masses) 46 standard 76 multiquadrant (at least 4 separate areas) 2009 to were RCC Sensitivity to detect sarcomatoid RCC Standard: 2 of 8 (25.0%) (p=0.0062) Multiquadrant: 13 of 15 (86.7%) 6 patients had some cores without sarcomatoid elements Abel EJ. J Urol. 2015

16 Current Ongoing Research Identification of molecular signature specific for srcc using needle core biopsy tissue (MD Anderson)

17 COMPARISON AT RNA LEVEL

19 Experimental Plan 8 patients with ccrcc with sarcomatoid component (FFPE) RNA-seq (N=5) RT-qPCR IHC Pal S. Mol Cancer Res. 2015

20 RNA-seq Pal S. Mol Cancer Res. 2015

21 qpcr Pal S. Mol Cancer Res. 2015

22 IHC Pal S. Mol Cancer Res. 2015

24 FFPE Sircar K. The Journal of Pathology: Clinical Research. 2015

25 Experiment Discovery cohort (cdna microarrays) 36 sarcomatoid RCC Epithelial component (clear cell) [E] Sarcomatoid component [S] 22 non-sarcomatoid RCC All clear cell [E*] Validation cohort (RNA-seq) 7 sarcomatoid RCC Epithelial component (clear cell) [E] Sarcomatoid component [S] 15 non-sarcomatoid RCC All clear cell [E*] Sircar K. The Journal of Pathology: Clinical Research. 2015

26 Sarcomatoid RCC vs. non-sarcomatoid RCC cdna microarray (unsupervised) RNA-seq Sircar K. The Journal of Pathology: Clinical Research. 2015

27 Sarcomatoid RCC vs. Grade 4 non-sarcomatoid RCC cdna microarray (unsupervised) RNA-seq Sircar K. The Journal of Pathology: Clinical Research. 2015

28 COMPARISON AT DNA LEVEL

29 Fox Chase Cancer Center SNP-based microarrays CNA 9 srcc (3 ccrcc, 2 prcc, 4 unclassified RCC) 71 non-srcc (39 ccrcc, 26 prcc, 6 chrrcc) Unique for srcc Loss 9q (88%), 15q (77%), 18q (66%), 22 (77%) Gain 1q (55%) and 8q (66%) Ito T. J Clin Oncol 33, 2015 (suppl 7; abstr 478)

30 7 patients with srcc with ccrcc epithelioid Whole exome sequencing MSKCC Mutations: Similar VHL gene mutations (in 6/7, in both S and E) PBRM1 mutations (in 4/7, 3 in both S and E) SETD2 mutations (in 3/7, 2 in S component only) TERT promoter mutations (in 2/7, in both S and E) No mutations were found in KDM5C, PTEN, MTOR or TP53 Chromosome changes: Chromosome 3p loss (in 6/7 E, in 3/7 S) Chromosome 14q24 loss (in 3/4, in both S and E) Chromosome 9p21 loss (in 4/5 samples, in both S and E) Chromosome 17q23-24 gain (in 3/7 S, none in E) Mano R. AUA MP47-09

31 Primary Objective MD Anderson Identify genomic alterations in srcc using a genomic profiling assay Malouf G. Submitted for publication

32 Outline of the Project Step 1: GP of 3 srcc patients using matched epithelial (ccrcc) and sarcomatoid components of srcc Step 2: GP of 26 srcc cases Step 3: Comparison with GP of 56 nonsarcomatoid cases (internal validation) Step 4: Comparison with TCGA data (external validation) Malouf G. Submitted for publication

33 FFPE Methods Targeted sequencing done at Foundation Medicine Inc. 3,230 exons of 236 cancer-related genes 37 introns from 19 genes Malouf G. Submitted for publication

34 Step 1: GP of 3 srcc patients using matched epithelial (ccrcc) and sarcomatoid components Patient Stage Histology MEDIAN EXON DEPTH A14-1 IV Epithelial/Clear cell 884 Sarcomatoid (70%) 1006 KNOWN SOMATIC SHORT-VARIANTS PTEN_c.277C>T_p.H93Y TP53_c.473G>T_p.R158L LRP1B_c.10638G>C_p.E3546D TP53_c.395A>T_p.K132M LIKELY SOMATIC SHORT-VARIANTS AMPL HOMOZYG OUS DELETIONS PTEN_c.280A>T_p.N94Y, PTEN_c G>T_p.splice none VHL PTEN_c.209+1G>C_p.splice JAK2 VHL A14-2 IV Epithelial/Clear cell 844 VHL_c.473T>C_p.L158P PBRM1_c.100A>T_p.K34* none none Sarcomatoid (60%) 892 VHL_c.473T>C_p.L158P PBRM1_c.100A>T_p.K34* none none A5 IV Epithelial/Clear cell 934 none VHL_c.513_513delG_p.K171fs*31 none none Sarcomatoid (20%) 354 none VHL_c.513_513delG_p.K171fs*31 none none Malouf G. Submitted for publication

35 Step 1: GP of 3 srcc patients using matched epithelial (ccrcc) and sarcomatoid components Patient Stage Histology MEDIAN EXON DEPTH A14-1 IV Epithelial/Clear cell 884 Sarcomatoid (70%) 1006 KNOWN SOMATIC SHORT-VARIANTS PTEN_c.277C>T_p.H93Y TP53_c.473G>T_p.R158L LRP1B_c.10638G>C_p.E3546D TP53_c.395A>T_p.K132M LIKELY SOMATIC SHORT-VARIANTS AMPL HOMOZYG OUS DELETIONS PTEN_c.280A>T_p.N94Y, PTEN_c G>T_p.splice none VHL PTEN_c.209+1G>C_p.splice JAK2 VHL A14-2 IV Epithelial/Clear cell 844 VHL_c.473T>C_p.L158P PBRM1_c.100A>T_p.K34* none none Sarcomatoid (60%) 892 VHL_c.473T>C_p.L158P PBRM1_c.100A>T_p.K34* none none A5 IV Epithelial/Clear cell 934 none VHL_c.513_513delG_p.K171fs*31 none none Sarcomatoid (20%) 354 none VHL_c.513_513delG_p.K171fs*31 none none Malouf G. Submitted for publication

36 Step 1: GP of 3 srcc patients using matched epithelial (ccrcc) and sarcomatoid components Patient Stage Histology MEDIAN EXON DEPTH A14-1 IV Epithelial/Clear cell 884 Sarcomatoid (70%) 1006 KNOWN SOMATIC SHORT-VARIANTS PTEN_c.277C>T_p.H93Y TP53_c.473G>T_p.R158L LRP1B_c.10638G>C_p.E3546D TP53_c.395A>T_p.K132M LIKELY SOMATIC SHORT-VARIANTS AMPL HOMOZYG OUS DELETIONS PTEN_c.280A>T_p.N94Y, PTEN_c G>T_p.splice none VHL PTEN_c.209+1G>C_p.splice JAK2 VHL A14-2 IV Epithelial/Clear cell 844 VHL_c.473T>C_p.L158P PBRM1_c.100A>T_p.K34* none none Sarcomatoid (60%) 892 VHL_c.473T>C_p.L158P PBRM1_c.100A>T_p.K34* none none A5 IV Epithelial/Clear cell 934 none VHL_c.513_513delG_p.K171fs*31 none none Sarcomatoid (20%) 354 none VHL_c.513_513delG_p.K171fs*31 none none Malouf G. Submitted for publication

37 Step 2: GP of 26 srcc cases 7 females and 19 males Stage III = 7 IV = 18 n/a = 1 Malouf G. Submitted for publication

38 Step 2: GP of 26 srcc cases Epithelial histology Clear cell = 12 Unclassified = 9 Collecting duct = 2 Papillary = 1 MTSCC = 1 n/a = 1 Tumor assayed Primary renal tumor in 23 cases Metastatic site in 3 cases (lymph node, liver metastasis, and peritoneal nodule) Malouf G. Submitted for publication

39 Step 2: GP of 26 srcc cases Malouf G. Submitted for publication

40 Step 2: GP of 26 srcc cases Malouf G. Submitted for publication

41 Step 3: Comparison with GP of 56 nonsarcomatoid cases (internal validation) Compared our 26 srcc cases with 56 advanced stage clear cell RCC cases Evaluated by the same CGP Grade at diagnosis Grade 2 = 21 Grade 3 = 19 Grade 4 (non-sarcomatoid) = 16 Stage at diagnosis Stage I = 1 Stage II = 4 Stage III = 12 Stage IV = 39 Malouf G. Submitted for publication

42 Step 3: Comparison with GP of 56 nonsarcomatoid cases (internal validation) Mutations in these 56 clear cell RCC (non-sarcomatoid) VHL = 73% PBRM1 = 47% SETD2 = 31% BAP1 = 13% TP53 only 9% NF2 only 2% KRAS, NRAS or HRAS None Malouf G. Submitted for publication

40 35 30 25 20 15 10 5 0 TCGA. Nature. 2013 TCGA.

43 Step 4: Comparison with TCGA data (external validation) TP53 (%) NF2 (%) TCGA. Nature TCGA. Cancer Cell Durinck S. Nat Genet TCGA. NEJM. 2015

44 PD-1 AND PD-L1 IN SARCOMATOID RCC

45 PD-1 and PD-L1 (Mayo Clinic, AZ and Caris) Sarcomatoid (40 26)?cc ccrcc non-sarcomatoid (91 29) Only 6 of 91 (7%) were Grade 4 Whole sections PD-1: BD Pharmingen, Clone PD-1+ TIL/mm 2 PD-L1: 2 Abs (R&D systems, Clone and Spring Bioscience, Clone SP142) 5% staining Score of 2+ or 3+ Joseph RW. Cancer Immunol Res. 2015

46 PD-1 and PD-L1 (Mayo Clinic, AZ and Caris) PD-1+ TILs Sarcomatoid 25 (96%) ccrcc non-sarc 18 (62%) P=0.003 PD-L1 Sarcomatoid 14 (54%) ccrcc non-sarc 5 (17%) P=0.006 Joseph RW. Cancer Immunol Res. 2015

47 PD-L1 and PD-L2 (Asan Medical Center) Sarcomatoid RCC 54 Clear cell non-sarc RCC 150 TMA PD-L1: Cell Signaling Technology, Clone E1L3N moderate expression in 5 % of tumor cells strong expression in 5 % of tumor cells PD-L2: R&D systems, Clone Similar to PD-L1 Shin SJ. Ann Surg Onc. 2015

48 PD-L1 and PD-L2 (Asan Medical Center) PD-L1 Sarcomatoid 16 (29.6%) ccrcc non-sarc 11 (7.3%) P<0.001 PD-L2 Sarcomatoid 24 (44.4%) ccrcc non-sarc 46 (30.7%) P=0.067 Shin SJ. Ann Surg Onc. 2015

49 Sarcomatoid RCC (MD Anderson) 118 patients (94 clear cell) FFPE Re-reviewed by GU pathology Epithelial and sarcomatoid components Whole slides and TMA Kawakami F. Submitted

50 All clear cell RCC Control Group: ccrcc No sarcomatoid components 92 patients Grade 2 = 14 Grade 3 = 58 Grade 4 = 20 Kawakami F. Submitted

51 PD-1 and PD-L1 PD-1 Staining manually evaluated Positive: PD-1 cell numbers 1/HPF PD-L1 Digital analysis Semiquantitative method (H-score=0-300) Results will be presented at ASCO GU 2016 Kawakami F. Submitted

52 Future Directions Identifying srcc on imaging Identifying srcc using molecular signature on biopsy Validation of DNA sequencing findings Mechanistic work Animal models Novel therapeutics

53 Acknowledgements Urology Christopher Wood Surena Matin Mehrad Adibi GU Medical Oncology Eric Jonasch Nizar Tannir Interventional Radiology Kamran Ahrar Sabir Sharjeel Translational Molecular Pathology Fumi Kawakami Ignacio Wistuba Jaime Rodriguez-Canales Pathology Pheroze Tamboli Kanishka Sircar Urology Fellows/Residents Megan Merrill Patrick Kenney Kara Babaian Arun Thomas Zachary Compton Radiology Rivka Colen Statistics Rebecca Slack Bioinformatics and Computational Biology Xiaoping Su Foundation Medicine Siraj Ali Kai Wang City of Hope Sumanta Pal Pitie-Salpetriere Gabriel Malouf

54 THANK YOU Jose A. Karam

Fifteenth International Kidney Cancer Symposium

Fifteenth International Kidney Cancer Symposium The following presentation should not be regarded as an endorsement of a particular product/drug/technique by the speaker. The presentation topics were assigned to the speakers by the scientific committee