Imaging, Diagnosis, Prognosis
Adverse Outcomes in Clear Cell Renal Cell Carcinoma with
Report by MSKCC and the KIRC TCGA Research Network
A. Ari Hakimi1, Irina Ostrovnaya2, Boris Reva3, Nikolaus Schultz3, Ying-Bei Chen4, Mithat Gonen2,
Han Liu5, Shugaku Takeda5, Martin H. Voss6, Satish K. Tickoo4, Victor E. Reuter4, Paul Russo1,
Emily H. Cheng4,5, Chris Sander3, Robert J. Motzer6, and James J. Hsieh5,6,7for The ccRCC
Cancer Genome Atlas (KIRC TCGA) Research Network investigators
Purpose: To investigate the impact of newly identified chromosome 3p21 epigenetic tumor suppressors
PBRM1, SETD2, and BAP1 on cancer-specific survival (CSS) of 609 patients with clear cell renal cell
carcinoma (ccRCC) from 2 distinct cohorts.
Experimental Design: Select sequencing on 3p tumor suppressors of 188 patients who underwent
resection of primary ccRCC at the Memorial Sloan-Kettering Cancer Center (MSKCC) was conducted to
interrogate the genotype–phenotype associations. These findings were compared with analyses of the
genomic and clinical dataset from our nonoverlapping The Cancer Genome Atlas (TCGA) cohort of 421
patients with primary ccRCC.
Results: 3p21 tumor suppressors are frequently mutated in both the MSKCC (PBRM1, 30.3%; SETD2,
7.4%; BAP1, 6.4%) and the TCGA (PBRM1, 33.5%; SETD2, 11.6%; BAP1, 9.7%) cohorts. BAP1 mutations
are associated with worse CSS in both cohorts [MSKCC, P ¼ 0.002; HR 7.71; 95% confidence interval (CI)
2.08–28.6; TCGA, P ¼ 0.002; HR 2.21; 95% CI 1.35–3.63]. SETD2 are associated with worse CSS in the
common gene mutations of ccRCC, have no impact on CSS.
BAP1 and SETD2 mutations (6%–12%) are associated with worse CSS, suggesting their roles in disease
progression. PBRM1 mutations (30%–34%) do not impact CSS, implicating its principal role in the tumor
initiation. Future efforts should focus on therapeutic interventions and further clinical, pathologic, and
molecular interrogation of this novel class of tumor suppressors. Clin Cancer Res; 19(12); 3259–67. ?2013
Renalcell carcinoma(RCC)istheeighth leading causeof
cell carcinoma (ccRCC) represents the most common and
of ccRCC have been used to successfully construct individ-
ual postoperative prognostic models that were subsequent-
survival (CSS; refs. 2–6). Increased size, advanced patho-
on multivariate analysis (2, 3). On the contrary, similar
progress is limited in the search for clinically relevant,
being made (7, 8). For example, VHL, the most commonly
mutated, silenced gene in ccRCC, has not been proven to
affect the overall clinical outlook of this disease, highlight-
ing the need for identification and validation of novel
Recent large-scale targeted and whole-exome sequencing
Atlas) Consortium, have discovered novel, prevalent geno-
mic alterations (9–11), including frequent inactivation of
Authors' Affiliations:1Urology Service, Departments of Surgery,2Epide-
ogy,5Human Oncology & Pathogenesis Program,6Genitourinary Oncol-
Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/).
A full list of principal investigators and participating institutes for the
ccRCC TCGA is listed in the Supplementary Appendix.
Program and Department of Medicine, Memorial Sloan-Kettering Cancer
Center, 415 E. 68th Street Zuckerman 801, New York, NY 10065. Phone:
646-888-3263; Fax: 646-888-3266; E-mail: email@example.com
?2013 American Association for Cancer Research.
SETD2 (3%–12%; ref. 11), and BAP1 (8%–11%; refs. 12,
13). Remarkably, all 3 genes are located on chromosome
3p21 that is in close proximity to 3p25 where VHL resides.
Emerging evidence indicates that these genes function as
tumor suppressors in diverse malignancies, for example,
PBRM1 in pancreatic cancer (14), BAP1in uveal melanoma
leukemia (17, 18). Strikingly, these mutations in ccRCC
mutations of these 4 genes would result in respective,
Our previous single-institutional sequencing effort on
185 patients with ccRCC focusing on pathologic correla-
tions, not only consolidated recently reported mutation
frequencies but also showed the association of PBRM1,
BAP1, and/or SETD2 mutations with higher tumor stage
(3 additional patients and several more months of follow-
up and events) and further show the association of BAP1
and SETD2 mutations with worse CSS (the TCGA cohort,
n ¼ 421), providing a molecular link between gene muta-
tions and cancer-specific outcomes in ccRCC.
Materials and Methods
MSKCC cohort and genomic DNA isolation
Tumor and adjacent normal kidney tissues from 189
consecutive, previously untreated patients who underwent
either radical or partial nephrectomy for sporadic, resect-
able ccRCC from December 2001 to December 2011 were
collected. All patients had previously consented to a tissue
protocol and 1 patient was excluded due to insufficient
DNA quality for mutation analysis. This study was preap-
proved by our Institutional Review Board and all patients
signed informed consent. Tumor staging was based on the
tional Union Against Cancer (AJCC/UICC) Tumor–Node–
adedicateduropathologist. Pairedfresh-frozen normaland
primary tumor tissue blocks were identified, marked, and
macrodissected for maximal tumor density. DNA was
extracted and quantified using the DNeasy Kit (Qiagen)
and a Nanodrop spectrophotometer (Invitrogen).
Integrated mutational analysis
Mutation analysis of the entire coding regions of VHL,
tumor-normal pairs. The IMPACT assay (Integrated Muta-
tion Profiling of Actionable Cancer Targets), a customized
targeted-exome capture assay of 230 cancer-associated
genes with an ultra-deep sequencing coverage (?500?)
(20), was conducted on additional 5 patients as part of a
separate study. Details of mutation analysis can be seen in
the Supplementary Methods.
Paired tumor-normal materials, genomic data, and clin-
ical information were acquired by our ccRCC TCGA Con-
sortium. This multi-institutional effort included clinical
and pathologic information on 446 retrospectively identi-
fied patients who underwent either radical or partial
nephrectomy for sporadic ccRCC from 1998 to 2010.
Whole-exome sequencing data were available on 424
cohort leaving a total of 421 patients for analysis. Full
sequencing information is detailed in the main text and
Supplementary information of the companion ccRCC
TCGA biomarker paper. Mutation data were acquired from
ical information was obtained from the file entitled
"KIRCþClinicalþDataþJul-31-2012" encompassing the
most recent follow-up information ("Max Followup"). Par-
tial mutation validation (?70%) had been conducted by
additional orthogonal platform (454 or Ion Torrent) at the
time of analysis. Vital status was determined from the field
"Composite Vital Status." Nonsilent, coding mutations
were considered for both cohorts, with truncating muta-
(within first 2 base pairs of coding region).
and Wilcoxon exact 2-tailed tests, respectively. Kaplan–
Meier method was used to estimate the survival probabil-
ities. CSS was analyzed using the competing risk method,
using the "cmprsk" R package. Cox proportional hazard
regression was used for analysis of time to recurrence (21).
Multivariate competing risk models were also fitted in the
TCGA cohort to adjust for clinical covariates (AJCC stage I
and II vs. III vs. IV and grade 1 and 2 vs. 3 and 4). No
adjustment was done for the MSKCC cohort due to shorter
follow up and thus fewer events. Tumor sizes were dichot-
omized as a categorical variable (?5 vs. <5 cm) in Table 2.
In the TCGA cohort, deceased patients were considered
dead from renal cancer-related causes if the field
Several recurrent mutations in 3p chromatin modu-
lators/modifiers (PBRM1, SETD2, and BAP1) have been
past2 years.We reportthe association ofadverse cancer-
specific outcomes with mutations of BAP1 and SETD2
in 2 large, distinct cohorts. Our results point to the need
for therapeutic interventions and further clinical, path-
Hakimi et al.
Clin Cancer Res; 19(12) June 15, 2013Clinical Cancer Research
This study was supported by grants from the Paula Moss Trust for the
research into the cure and treatment of kidney cancer and the J. Randall &
Kathleen L. MacDonald Research Fund in Honor of Louis V. Gerstner, Jr. (to
the Stephen P. Hanson FamilyFund Fellowship in Kidney Cancer (to A.A.
Hakimi), and the TCGA grant: NCI-U24CA143840 (to B. Reva, N. Schultz,
the National Cancer Institute.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
Received December 20, 2012; revised March 6, 2013; accepted April 9,
2013; published OnlineFirst April 25, 2013.
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Adverse Outcomes in ccRCC with BAP1 and SETD2 Mutations
www.aacrjournals.org Clin Cancer Res; 19(12) June 15, 2013