RCC with Sarcomatoid Dedifferentiation: New … 6, 2015 RCC with Sarcomatoid Dedifferentiation: New Insights . Farrow GM. Cancer. 1968. ... FNA/Core biopsy (University of Wisconsin)

Jose A. Karam, MD, FACSAssistant Professor

Department of Urology

November 6, 2015

RCC with Sarcomatoid Dedifferentiation: New Insights

Farrow GM. Cancer. 1968

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

Histology 2 Components

• Epithelial component

– Clear cell, papillary, chromophobe, collecting duct, MTSCC, other

• Sarcomatoid component

Delahunt B. Am J Surg Path. 2013

Sarcomatoid Component

Malouf G. Submitted for publication

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?

PREOPERATIVE IMAGING

MRI-1

• Retrospective (2003-2009)• 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

MRI-2

• Retrospective (2004-2012)

• 11 patients with clear cell RCC with sarcomatoid elements

• Correlate preoperative MRI with pathology from nephrectomy

Takeuchi M. Clinical Imaging. 2013

MRI Findings

• sRCC showed significantly lower enhancement than the ccRCC part in each MRI phase

• Pseudocapsule disruption all cases

• Issue non-blinded, need comparator arm

Takeuchi M. Clinical Imaging. 2013

Current Ongoing Research

• Identification of sRCC using preoperative imaging

– PIs: Rivka Colen (Radiology) and Jose Karam (Urology), MD Anderson

PREOPERATIVE BIOPSY

FNA/Core biopsy (MD Anderson)

• 166 patients (1991-2007)

• 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

FNA/Core biopsy (MD Anderson)

• 405 FNA or biopsies from 378 patients (1991-2007)– 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• 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

Current Ongoing Research

• Identification of molecular signature specific for sRCC using needle core biopsy tissue (MD Anderson)

COMPARISON AT RNA LEVEL

Experimental Plan

• 8 patients with ccRCC with sarcomatoid component (FFPE)

– RNA-seq (N=5)

– RT-qPCR

– IHC

Pal S. Mol Cancer Res. 2015

RNA-seq


qPCR


IHC


FFPE

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

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*]


Sarcomatoid RCC vs. non-sarcomatoid RCC

cDNA microarray (unsupervised) RNA-seq


Sarcomatoid RCC vs. Grade 4 non-sarcomatoid RCC

cDNA microarray (unsupervised) RNA-seq


COMPARISON AT DNA LEVEL

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)

MSKCC

• 7 patients with sRCC with ccRCC epithelioid• Whole exome sequencing

• 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 2015. MP47-09

MD Anderson

• Primary Objective

– Identify genomic alterations in sRCC using a genomic profiling assay


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 non-

sarcomatoid cases (internal validation)• Step 4: Comparison with TCGA data (external

validation)


Methods

• FFPE

• Targeted sequencing done at Foundation Medicine Inc.

– 3,230 exons of 236 cancer-related genes

– 37 introns from 19 genes


Step 1: GP of 3 sRCC patients using matched epithelial (ccRCC) and sarcomatoid components

Patient Stage Histology MEDIAN

EXON

DEPTH

KNOWN SOMATIC

SHORT-VARIANTS

LIKELY SOMATIC SHORT-VARIANTS AMPL HOMOZYG

OUS

DELETIONS

A14-1 IV Epithelial/Clear cell 884PTEN_c.277C>T_p.H93Y

TP53_c.473G>T_p.R158L

PTEN_c.280A>T_p.N94Y,

PTEN_c.1027-1G>T_p.splicenone VHL

Sarcomatoid (70%) 1006LRP1B_c.10638G>C_p.E3546D

TP53_c.395A>T_p.K132MPTEN_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




EXON

DEPTH

KNOWN SOMATIC

SHORT-VARIANTS


OUS

DELETIONS


TP53_c.473G>T_p.R158L












EXON

DEPTH

KNOWN SOMATIC

SHORT-VARIANTS


OUS

DELETIONS


TP53_c.473G>T_p.R158L










Step 2: GP of 26 sRCC cases

• 7 females and 19 males

• Stage

– III = 7

– IV = 18

– n/a = 1



• 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)






Step 3: Comparison with GP of 56 non-sarcomatoid 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


Step 3: Comparison with GP of 56 non-sarcomatoid 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


Step 4: Comparison with TCGA data (external validation)

TP53 (%) NF2 (%)

05

1015

20

25

30

354045

50

TCGA. Nature. 2013TCGA. Cancer Cell. 2014Durinck S. Nat Genet. 2015TCGA. NEJM. 2015

05

1015

20

25

30

354045

50

PD-1 AND PD-L1 IN SARCOMATOID RCC

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 561273 – ≥1 PD-1+ TIL/mm2

• PD-L1: – 2 Abs (R&D systems, Clone 130021 and Spring Bioscience, Clone SP142)– ≥5% staining – Score of 2+ or 3+

Joseph RW. Cancer Immunol Res. 2015

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

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 176611– Similar to PD-L1

Shin SJ. Ann Surg Onc. 2015

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

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

Control Group: ccRCC

• All clear cell RCC

– No sarcomatoid components

• 92 patients

– Grade 2 = 14

– Grade 3 = 58

– Grade 4 = 20


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


Future Directions

• Identifying sRCC on imaging• Identifying sRCC using molecular signature on

biopsy• Validation of DNA sequencing findings• Mechanistic work• Animal models• Novel therapeutics

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

THANK YOUJose A. Karam

[email protected]

Documents

RCC with Sarcomatoid Dedifferentiation: New … 6, 2015 RCC with Sarcomatoid Dedifferentiation: New Insights . Farrow GM. Cancer. 1968. ... FNA/Core biopsy (University of Wisconsin)