Genomic Heterogeneity of Translocation Renal Cell Carcinoma · Philippe Camparo13, Xiaoping Su2,...

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Human Cancer Biology Genomic Heterogeneity of Translocation Renal Cell Carcinoma Gabriel G. Malouf 1 , Federico A. Monzon 7,8 ,J er^ ome Couturier 10 , Vincent Molini e 11 , Bernard Escudier 12 , Philippe Camparo 13 , Xiaoping Su 2 , Hui Yao 2 , Pheroze Tamboli 3 , Dolores Lopez-Terrada 9 , Maria Picken 14 , Marileila Garcia 15,16 , Asha S. Multani 4 , Sen Pathak 4 , Christopher G. Wood 5 , and Nizar M. Tannir 6 Abstract Purpose: Translocation renal cell carcinoma (tRCC) is a rare subtype of kidney cancer involving the TFEB/TFE3 genes. We aimed to investigate the genomic and epigenetic features of this entity. Experimental Design: Cytogenomic analysis was conducted with 250K single-nucleotide polymor- phism microarrays on 16 tumor specimens and four cell lines. LINE-1 methylation, a surrogate marker of DNA methylation, was conducted on 27 cases using pyrosequencing. Results: tRCC showed cytogenomic heterogeneity, with 31.2% and 18.7% of cases presenting similarities with clear-cell and papillary RCC profiles, respectively. The most common alteration was a 17q gain in seven tumors (44%), followed by a 9p loss in six cases (37%). Less frequent were losses of 3p and 17p in five cases (31%) each. Patients with 17q gain were older (P ¼ 0.0006), displayed more genetic alterations (P < 0.003), and had a worse outcome (P ¼ 0.002) than patients without it. Analysis comparing gene-expression profiling of a subset of tumors bearing 17q gain and those without suggest large-scale dosage effects and TP53 haploinsufficiency without any somatic TP53 mutation identified. Cell line–based cytogenetic studies revealed that 17q gain can be related to isochromosome 17 and/or to multiple translocations occurring around 17q breakpoints. Finally, LINE-1 methylation was lower in tRCC tumors from adults compared with tumors from young patients (71.1% vs. 76.7%; P ¼ 0.02). Conclusions: Our results reveal genomic heterogeneity of tRCC with similarities to other renal tumor subtypes and raise important questions about the role of TFEB/TFE3 translocations and other chromosomal imbalances in tRCC biology. Clin Cancer Res; 19(17); 4673–84. Ó2013 AACR. Introduction Translocation renal cell carcinoma (tRCC) is a subtype of renal cell carcinoma (RCC) that was recognized in the 2004 World Health Organization classification of renal tumors as a genetically distinct entity (1). Originally described in pediatric patients, the spectrum of the disease has recently been expanded to include adults (2). Recent studies showed that tRCC represents one third of pediatric RCCs, up to 15% of RCCs in patients younger than 45 years old, and up to 5% of all RCCs regardless of age (3–5). The hallmark of tRCC is the fusion of the TFE3 gene, located in Xp11, with various partners, including PRCC in t(X;1)(p11.2;q21), SFPQ in t (X;1)(p11.2;p34), ASPSCR1 in t(X;17)(p11.2;q25), NONO in inv(X)(p11.2q12), and CLTC in t(X;17)(p11;q23) (6). tRCC can also be related to translocations involving the TFEB gene (7). We and others have reported that the disease behaves differently in adult and pediatric patients (2, 8–10). Sim- ilarly, better outcomes in young patients were reported for alveolar soft part sarcoma, a tumor characterized by ASPSCR1-TFE3 translocation (11). Recently, we showed that patients with tRCC who had metastatic disease at presentation were older (median age 36 years) and pre- dominantly male, whereas patients who had loco-regional Authors' Afliations: Departments of 1 Leukemia, 2 Bioinformatics, 3 Pathology, 4 Genetics, 5 Urology, and 6 Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer Center; Departments of 7 Pathology & Immunology and 8 Molecular & Human Genetics, Baylor College of Medicine; 9 Department of Pathology, Texas Children's Hos- pital, Houston, Texas; 10 Department of Genetics, Institut National de la Sant e et de la Recherche Medicale (INSERM) U830, Institut Curie; 11 Department of Pathology, H^ opital Saint Joseph, Paris; 12 Department of Medical Oncology, Institut Gustave Roussy, Villejuif; 13 Department of Pathology, H^ opital Foch, Suresnes, France; 14 Department of Pathology, Loyola University Hospital, Chicago, Illinois; and Departments of 15 Med- icine and 16 Pathology, University of Colorado School of Medicine, Aurora, Colorado Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). G.G. Malouf and F.A. Monzon contributed equally to this work. Current address for G.G. Malouf: Department of Medical Oncology, Groupe Hospitalier Piti e-Salp^ etri ere, Assistance Publique Hopitaux de Paris, Fac- ulty of Medicine Pierre et Marie Curie, Institut Universitaire de Canc erologie GRC5, University Paris 6, Paris, France. Corresponding Author: Nizar M. Tannir, Department of Genitourinary Medical Oncology, Unit 1374, The University of Texas MD Anderson Cancer Center, 1155 Pressler Street, Houston, TX 77030-3721. Phone: 713-563-7265; Fax: 713-745-1625; E-mail: [email protected] doi: 10.1158/1078-0432.CCR-12-3825 Ó2013 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org 4673 on March 31, 2020. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst July 1, 2013; DOI: 10.1158/1078-0432.CCR-12-3825

Transcript of Genomic Heterogeneity of Translocation Renal Cell Carcinoma · Philippe Camparo13, Xiaoping Su2,...

Page 1: Genomic Heterogeneity of Translocation Renal Cell Carcinoma · Philippe Camparo13, Xiaoping Su2, Hui Yao2, Pheroze Tamboli3, Dolores Lopez-Terrada9, Maria Picken14, Marileila Garcia15,16,

Human Cancer Biology

Genomic Heterogeneity of Translocation Renal CellCarcinoma

Gabriel G. Malouf1, Federico A. Monzon7,8, J�erome Couturier10, Vincent Molini�e11, Bernard Escudier12,Philippe Camparo13, Xiaoping Su2, Hui Yao2, Pheroze Tamboli3, Dolores Lopez-Terrada9, Maria Picken14,Marileila Garcia15,16, Asha S. Multani4, Sen Pathak4, Christopher G. Wood5, and Nizar M. Tannir6

AbstractPurpose: Translocation renal cell carcinoma (tRCC) is a rare subtype of kidney cancer involving the

TFEB/TFE3 genes. We aimed to investigate the genomic and epigenetic features of this entity.

Experimental Design: Cytogenomic analysis was conducted with 250K single-nucleotide polymor-

phism microarrays on 16 tumor specimens and four cell lines. LINE-1 methylation, a surrogate marker of

DNA methylation, was conducted on 27 cases using pyrosequencing.

Results: tRCC showed cytogenomic heterogeneity, with 31.2%and18.7%of cases presenting similarities

with clear-cell and papillary RCCprofiles, respectively. Themost common alterationwas a 17q gain in seven

tumors (44%), followed by a 9p loss in six cases (37%). Less frequent were losses of 3p and 17p in five cases

(31%) each. Patients with 17q gain were older (P¼ 0.0006), displayedmore genetic alterations (P < 0.003),and had a worse outcome (P ¼ 0.002) than patients without it. Analysis comparing gene-expression

profiling of a subset of tumors bearing 17q gain and those without suggest large-scale dosage effects and

TP53 haploinsufficiency without any somatic TP53mutation identified. Cell line–based cytogenetic studies

revealed that 17q gain can be related to isochromosome 17 and/or to multiple translocations occurring

around17qbreakpoints. Finally, LINE-1methylationwas lower in tRCC tumors fromadults comparedwith

tumors from young patients (71.1% vs. 76.7%; P ¼ 0.02).

Conclusions: Our results reveal genomic heterogeneity of tRCC with similarities to other renal tumor

subtypes and raise important questions about the role of TFEB/TFE3 translocations andother chromosomal

imbalances in tRCC biology. Clin Cancer Res; 19(17); 4673–84. �2013 AACR.

IntroductionTranslocation renal cell carcinoma (tRCC) is a subtype of

renal cell carcinoma (RCC) that was recognized in the 2004WorldHealthOrganization classification of renal tumors asa genetically distinct entity (1). Originally described inpediatric patients, the spectrum of the disease has recentlybeen expanded to include adults (2). Recent studies showedthat tRCC represents one third of pediatric RCCs, up to 15%of RCCs in patients younger than 45 years old, and up to 5%of all RCCs regardless of age (3–5). The hallmark of tRCC isthe fusion of the TFE3 gene, located in Xp11, with variouspartners, including PRCC in t(X;1)(p11.2;q21), SFPQ in t(X;1)(p11.2;p34), ASPSCR1 in t(X;17)(p11.2;q25),NONOin inv(X)(p11.2q12), and CLTC in t(X;17)(p11;q23) (6).tRCC can also be related to translocations involving theTFEB gene (7).

We and others have reported that the disease behavesdifferently in adult and pediatric patients (2, 8–10). Sim-ilarly, better outcomes in young patients were reported foralveolar soft part sarcoma, a tumor characterized byASPSCR1-TFE3 translocation (11). Recently, we showedthat patients with tRCC who had metastatic disease atpresentation were older (median age 36 years) and pre-dominantly male, whereas patients who had loco-regional

Authors' Affiliations: Departments of 1Leukemia, 2Bioinformatics,3Pathology, 4Genetics, 5Urology, and 6Genitourinary Medical Oncology,The University of Texas MD Anderson Cancer Center; Departments of7Pathology & Immunology and 8Molecular & Human Genetics, BaylorCollege of Medicine; 9Department of Pathology, Texas Children's Hos-pital, Houston, Texas; 10Department of Genetics, Institut National de laSant�e et de la Recherche Medicale (INSERM) U830, Institut Curie;11Department of Pathology, Hopital Saint Joseph, Paris; 12Departmentof Medical Oncology, Institut Gustave Roussy, Villejuif; 13Department ofPathology, Hopital Foch, Suresnes, France; 14Department of Pathology,Loyola University Hospital, Chicago, Illinois; and Departments of 15Med-icine and 16Pathology, University of Colorado School of Medicine,Aurora, Colorado

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

G.G. Malouf and F.A. Monzon contributed equally to this work.

Current address forG.G.Malouf: Department ofMedical Oncology,GroupeHospitalier Piti�e-Salpetri�ere, Assistance Publique Hopitaux de Paris, Fac-ulty ofMedicine Pierre etMarie Curie, Institut Universitaire deCanc�erologieGRC5, University Paris 6, Paris, France.

Corresponding Author: Nizar M. Tannir, Department of GenitourinaryMedical Oncology, Unit 1374, The University of Texas MD AndersonCancer Center, 1155 Pressler Street, Houston, TX 77030-3721. Phone:713-563-7265; Fax: 713-745-1625; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-3825

�2013 American Association for Cancer Research.

ClinicalCancer

Research

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disease were younger (median age 16 years) and predom-inantly female (9). These data are in line with previousreports, suggesting a more aggressive disease in adults,especially in men (8, 12). However, the basis for thebiologic difference in tRCC between adult and pediatricpatients has not been elucidated.

Since the first description of tRCC by Argani and collea-gues, there have been few reports about a detailed cyto-genetic characterization of these tumors (13, 14). Otherrenal tumors are characterized by specific chromosomalimbalances, such as 3p loss in clear-cell RCC (ccRCC) andtrisomies 7 and 17 in papillary RCC (pRCC; refs. 15, 16).

The aims of our study were to investigate whether thereare any additional genetic/epigenetic alterations besideTFE3/TFEB translocations, and assess whether there areassociations between specific chromosomal imbalancesand classical clinicopathologic factors and overall survival(OS).

Patients and MethodsPatient selection and classification of cases included insingle-nucleotide polymorphism-array analysis

Tissue specimens from21 patients with a histopathologicdiagnosis of tRCC supported by TFE3 positivity on immu-nohistochemistry were collected after approval of the Insti-tutional Review Board of each of the participating centers.TFE3 and TFEB immunostaining were conducted at eachindividual institution as previously described (7, 17). Theflow chart of patient selection, describing the different testsconducted is depicted in Supplementary Fig. S1.

Among the total of 21 cases, 15 were confirmed to betRCC by FISH, conventional karyotyping, or reverse tran-scription PCR (RT-PCR) analysis for all known specific

fusion partners. One patient whose tumor had classicaltRCC morphology and TFEB immunohistochemical pos-itivity was also included in the final cohort (n ¼ 16). Wecollected the following clinicopathologic informationfor each patient: age, sex, ethnicity, tumor–node–metas-tasis (TNM) classification, tumor size, lymph nodeinvolvement, Fuhrman grade, and survival time (Sup-plementary Table S1). The clinicopathologic data from5 of the patients (numbers T31 through T35) werepreviously reported (9). Four patients with positive TFE3immunohistochemistry but negative by FISH analysisfor TFE3 translocations were included in the single-nucleotide polymorphism (SNP) array studies as a con-trol group.

Patient selection and LINE-1 methylation analysisDNA was available for studying LINE-1 methylation in

12 patients for whom SNP-array analysis was conducted(all except the following 4 cases: LOY009, MRCC106,MRCC107, and MRCC117). In addition, DNA was ex-tracted from 15 formalin-fixed paraffin-embedded (FFPE)tissues of patients for whom we previously reported path-ologic features and outcome (9, 18). Seven of those patientshad confirmed translocation by karyotyping and/or RT-PCR, and the remaining had their diagnosis confirmed byTFE3 immunostaining, as previously described (18). Fur-thermore, DNA extracted from 16 samples belonging toadjacent normal kidney of patients operated on for renalcell carcinoma was included to study LINE-1 methylationvariability. DNA isolation was done using the Gentra Sys-tems Puregene DNA Purification Kit according to themanufacturer’s instructions. LINE-1 repetitive element,a surrogate of global DNA methylation, was assessed bypyrosequencing as described previously (19). Assay repli-cates for this assay were highly correlated as previouslyshown (19).

Generation of novel cell lines from adult patientsTwo novel cell lines, HCR-59 and MDA-92, were

derived from 2 adults treated at MD Anderson CancerCenter (MDACC; Houston, TX), as previously reportedfor other RCC subtypes (20). HCR-59 was derived from a20-year old Caucasian female, and MDA-92 was derivedfrom a 51-year-old Hispanic male. For both of those celllines, karyotyping was conducted according to previous-ly published methods (21). Spectral karyotyping (SKY)was also conducted on HCR-59 by using the humanchromosome HiSKY probe (Applied Spectral Imaging,Inc.).

The tRCC cell lines UOK109 and UOK146, derived,respectively, from a 39-year-old male and a 42-year-oldfemale (22), were kindly provided by Dr. W. MarstonLinehan (U.S. NIH, Bethesda, MD). Both cell lines wereoriginally reported as pRCC cell lines before tRCC became arecognized pathologic entity. The UOK109 line has beenshown to carry theNONO-TFE3 translocation (23), and theUOK146 cell line carries the translocation t(X;1)(p11.2;q21.2) (22).

Translational RelevanceTranslocation renal cell carcinoma (tRCC) is a rare

kidney cancer subtype that mainly arises in children andyoung adults and often has both papillary and clear-cellpathologic features. Little is known about whether addi-tional genetic alterations are associated with tRCC. Bysingle-nucleotide polymorphism–array profiling andLINE-1 methylation, we found genomic heterogeneityof tRCC that included alterations common with clear-cell RCC (e.g., 3p loss) andpapillary RCC (e.g., trisomy7and/or 17). When compared with young patients (<18years), adults with tRCC displayed distinct genomic andepigenetic aberrations, exemplified by lower LINE-1methylation and frequent 17q partial gain, which wereconsistent with a large-scale dosage effect affecting RCCcarcinogenesis. Our results show that besides TFE3/TFEBtranslocations, tRCC shares alterations commonly pres-ent in other RCC histologic subtypes and these areassociated with patient outcomes. Furthermore, ourstudy suggests that targeting tRCCs according to theirgenetic profile may have therapeutic relevance.

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Cytogenomic analysisDNA obtained from frozen tissues or from manually

microdissected FFPE containing at least 70% tumor cellswas quantified on a NanoDrop ND-1000 spectrophotom-eter (Thermo Fisher Scientific, Inc.). The DNA was ana-lyzed on Affy250KNsp SNP genotyping arrays (Affymetrix,Inc.) following the previously reported modified protocol(24). LOH and copy number estimates were obtained byusing the publicly available Copy Number Analyzer forAffymetrix GeneChip Arrays package (CNAG v3.0; ref. 25).The number of genetic alterations was defined as thenumber of gains and losses of segmental chromosomalregions.

RNA sequencingFor 4 translocation Xp11 cases (T31, T32, T34, T1), RNA

of good quality with RNA integrity number values between8.0 and 10.0 was available. Briefly, preparation of mRNA-seq libraries for each case was conducted according toIllumina procedures at the MDACCGenomic Core Facility,and sequencing was done on a HiSeq 2000 system (Illu-mina, Inc.). After quality control, reads were mapped andthen aligned to the UCSC hg19 version of the referencegenome.Gene expression countswere normalized using theRPKM (reads/kb/million) method (26).

Gene set enrichment analysisGene set enrichment analysis (GSEA) is a computa-

tional method that assesses whether a defined set of genesshows statistically significant differences between twoconditions (24). The average degree of change betweenthe normalized RPKM values of 2 samples with partial17q gain and 2 samples without was calculated, andgenes were ranked by order of expression in the 2 sampleswith partial 17q gain. The list was uploaded as a pre-ranked list to GSEAv2.04 (Broad Institute, Cambridge,MA; ref. 27), and GSEA was conducted using the defaultnormalization mode. Genes were classified according tothe positional gene set (C1) in the Molecular SignaturesDatabase (http://www.broadinstitute.org/gsea/msigdb/index.jsp).

Mutation of TP53 and VHL genesFor the TP53 gene, mutations occurring in exons 5 to 8

were assessed using five sets of oligonucleotide primers aspreviously described (28). VHL mutations were assessedusing four primers pairs as previously described (29). Over-all, 12 cases with available DNA were analyzed as shownin Supplementary Fig. S1.

Statistical analysisAssociations with clinicopathologic factors were ana-

lyzed with x2 and t test. OS was calculated from the dateof nephrectomy to the date of death or last visit (censored).Survival probabilities were estimated with the Kaplan–Meiermethod, and log-rank testing was applied to comparesurvival curves. Spearman correlation coefficient was usedto assess the correlation between the LINE-1 methylation

and number of genetic aberrations. All statistical tests weretwo-sided and conducted at the significance level of 0.05using Prism version 5 (GraphPad Software, Inc.).

ResultsPatient and tumor characteristics

Patient and tumor characteristics are summarized inSupplementary Table S1. With a median follow-up time of29 months (range, 3–92 months), 5 patients (31%) weredead of disease, 8 patients had no evidence of disease, and 3patients were alive with metastatic disease.

tRCC shows cytogenomic heterogeneityTranslocation carcinomas showed significant heteroge-

neity in cytogenomic profiles (Fig. 1 and Table 1). Themostcommon alteration observed was a gain of 17q, which waspresent in seven (44%) of the tumors. Less frequent altera-tions were gains of chromosomes 12 and 7, observed in 6(37%) and 5 (31%) patients, respectively. The most com-monly observed cytogenetic losses were of 9p in 6 (37%)patients and of 3p, 1p, 17p, and 18q, each of whichoccurring in 5 (31%) patients. Three of 10 women in ourcohort (30%) lost one X chromosome.

Overall, the 16 cases of tRCC with a confirmed translo-cation could be classified into four groups on the basis oftheir similarities to cytogenomic profiles of other renalepithelial tumors (15).

* Group I: 5 cases had profiles similar to those of ccRCC,owing to 3p loss and other imbalances (including theTFEB case). Three of those 5 cases were available forhistologic review with 2 showing mixed clear-cell/papillary features, and 1 showing only clear-cellmorphology.

* Group II: 3 cases had profiles similar to those of pRCCwith gains of chromosomes 7, 12, and 17. All 3 casesshowed mixed ccRCC and pRCC histology.

* Group III: 5 cases had novel cytogenomic profiles, that is,not associatedwith those of other subsets of renal tumors.

* Group IV: 3 cases had a balanced chromosomalcomplement.

As Fig. 1 shows, tRCC tumors had heterogeneous chro-mosome copy number profiles. 3p loss was associated with9p loss (P<0.04) but not associatedwith the loss of 1p, 17p,or 18q (P ¼ 0.24 for each comparison). There was nosignificant difference in the number of genetic alterationsbetween patients with 3p loss and those without (11.8 vs.6.7; P ¼ 0.23).

Of the 7 cases (44%) that had a gain of 17q, two tumorshad a confirmed t(X;17)(p11;q25) translocation and 1 casehad a TFEB translocation. Interestingly, in 4 of 7 cases with17q gain, 17p loss has been shown, consistent with thegeneration of an isochromosome 17q, or i(17q). In con-trast, none of 4 cases overexpressing TFE3 but negative byFISH had gain of 17q or loss of 17p, and 2 of them had 3ploss (Supplementary Table S2).

Translocation Renal Cell Carcinoma

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Tumors of adult patients (age � 18 years) showed moregenetic abnormalities than tumors from younger patients(age < 18 years; P ¼ 0.006), with no differences found insex (P ¼ 0.31), TNM stage (P ¼ 0.60), and Fuhrman grade(P ¼ 0.62).

Unsupervised clustering reveals two distinct groups oftRCC

To gain insights into the biology of tRCC, we con-ducted unsupervised clustering analysis using the mostfrequent segmental alterations (losses or gains). Thatanalysis revealed two main clusters (Fig. 2A). The mediannumber of genetic alterations in cluster A was 0 (range, 0–5), compared with 14 in cluster B (range, 5–22). The twoclusters showed no significant differences in terms of pTstage, lymph node involvement, metastasis, TNM classi-fication, gender, and Fuhrman grade. The only statisticaldifference between clusters was age at diagnosis: themedian age of 7 patients in cluster A was 16.7 years(range, 9.1–33.1 years), compared with 34.2 years (range,16–54 years) for the 9 patients in cluster B (P ¼ 0.01).Overall, 5 of 7 (71%) patients in cluster A were youngerthan age 18, compared with 1 of 9 (11%) patients in

cluster B. Of note, 7 of 10 patients who were older than 18years had a 17q gain, but none of the 2 older patients incluster A did.

Association between chromosomal imbalances andpathologic features and outcome

As noted earlier, a gain of 17q was the most frequentalteration (44%) we found in tRCC with a minimallygained region of 11.9 Mb (17q24.3-q25.3). Four of thesecases had 17p deletions, with a minimally deleted regionof 22.1 Mb (entire p arm), including the TP53 locus. Noassociations were found between 17q gain and any otherfrequently occurring genetic gain or loss. The meannumber of alterations in patients with 17q gain was14.4 � 2.4 versus 3.7 � 2.4 in patients without 17qgain (P < 0.003; Fig. 2B). In addition, patients with 17qgain were older than those without: their median agewas 40.2 (range, 20.4–54.6) versus 16.7 years (range,9.1–33.1; P ¼ 0.0006). The gain of 17q was also foundmore frequently in men than in women (71.4% vs.11.1%; P ¼ 0.03). No associations were found between17q gain and other clinicopathologic variables, i.e.,tumor size, ethnicity, pT stage, lymph node involvement,

ccRCC-like

pRCC-like

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Figure 1. Genomic subtypes of tRCC. tRCC could have different RCC profiles consistent with ccRCC (n ¼ 5), pRCC (n ¼ 3), or novel RCC karyotypes(n ¼ 5).

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Table 1. Genetic/genomic characteristics of translocation RCC cases

SampleChromosomaltranslocation

Diagnosticmethod Gene Fusion partner Gains Losses

T1 NA FISH TFE3 Unknown þ5(q33.3-q34), þ5(q34-q35.3), þ17

(q21.31-q25.3)

�1(p31.1-p21.1), �1(p13.3-p11.2), �3

(p26.3-p11.2), �9(p24.3-p21.1), �17

(p13.3-p11.2), �18(q11.2-q11.2),

�18(q12.1-q23)

T2 NA FISH TFE3 Unknown þ2(p25.3-q37.3), þ7(p22.3-q36.3),

þ12(p13.33-q24.33), þ17(p13.3-

q25.3), þ20(p13-q13.33)

No

T3 NA FISH TFE3 Presumably PRCC þ8(p23.3-q24.3), þ17(q11.1-q25.3) �1(p36.33-p11.2), �3(p26.3-q29), �5

(q15-q15), �6(p11.1-q27), �9(p24.3-

q34.3), �10(q21.1-q21.1), �10

(q21.1-q21.1), �15(q11.2-q26.3)

T24 NA FISH TFE3 Unknown þ1(p11.2-q44), þ2(p11.2-q37.3), þ5

(p15.33-q35.3), þ7(p22.3-q36.3), þ8

(p23.3-q24.3), þ9(q12-q21.13), þ9

(q21.13-q21.13), þ9(q34.11-q34.3),

þ17(q11.1-q25.3),þ17(q11.2-q11.2),

þ 17(q12-q21.2), þ17(q23.1-q23.3),

þ17(q23.3-q24.3),þ17(q25.1-q25.3),

þ17(q25.1-q25.2),þ17(q25.2-q25.3),

þ17(q25.3-q25.3)

�1(p36.33-p11.2), �9(p22.3-p21.1),

�9(p22.1-p21.3), �9(p21.1-p21.1),

�17(p13.3-q11.1)

T25 NA RT-PCR TFE3 NONO No No

T29 NA IHC TFEB Presumably alpha þ1(q23.3-q44), þ3(p11.1-q29), þ4

(q27-q28.1), þ4(q28.3-q32.2), þ5

(p15.33-p11), þ5(q23.1-q35.3), þ5

(q31.1-q35.3), þ7(p22.3-q36.3), þ8

(q11.1-q24.22), þ12(q14.1-q24.33),

þ16(p13.3-p12.3),þ16(p12.3-q24.3),

þ17(q11.2-q25.3)

�3(p26.3-p14.1), �6(q14.1-q27), �8

(p23.3-p11.1), �9(p24.3-q34.3), �13

(q12.11-q34), �14(q11.2-q32.33),

�17(p13.3-q11.2), �18(p11.32-q23),

�22(q11.1-q13.33)

T31 t(X;1)(p11.2;q21) RT-PCR TFE3 PRCC No No

T32 t(X;1)(p11.2;q21) RT-PCR TFE3 PRCC No �X

T33 t(X;1)(p11.2;q21) RT-PCR TFE3 PRCC No No

T34 t(X;17)(p11;q25) RT-PCR TFE3 ASPSCR1 þ1(p11.2-q44), þ7(p22.3-q36.3), þ8

(p23.3-q24.3), þ12(p13.33-q24.33),

þ17(p13.3-q25.3), þ19(p13.3-

q13.43), þ20(p13-q13.33), þ22

(q11.1-q13.33)

�1(p36.33-p34.3), �1(p33-p11.2), �4

(p16.3-q35.2), �10(p15.3-q26.3),

�13(q11-q34), �15(q11.2-q26.3),

�17(q21.31-q21.31), �18(p11.32-

q23)

T35 t(X;1)(p11.2;q21) RT-PCR TFE3 PRCC �6(q12-q25.3), �22(q11.1-q13.33) �6(q12-q25.3), �22(q11.1-q13.33)

T44 NA FISH TFE3 Unkown þ1(p11.2-q44), þ1(p11.2-q44), þ2

(p25.3-q37.3), þ6(p25.3-q27), þ7

(p22.3-q36.3), þ12(p13.33-q24.33),

þ13(q31.1-q34), þ17(q11.1-q25.3),

þ20(p13-q13.33)

�4(p16.3-q35.2), �8(p23.3-p22), �10

(p15.3-q26.3), �14(q11.2-q32.33),

�17(p13.3-p11.2)

T009 NA FISH TFE3 Unknown þ12(p13.33-q24.33), 2(p25.3-q37.3) �1(p36.33-p34.3), �6(p25.3-q27), �9

(p24.3-q34.3), �13(q11-q34), �14

(q11.2-q32.33), �18(p11.32-q23),

�19(p13.3-q13.43), �X(p11.4-q28)/

�4(p16.3-q35.2), �7(p22.3-q36.3),

�11(p15.5-q25), �22(q11.1-q13.33)

T106 t(X;1)(p?;q?) Karyotype TFE3 Unknown þ2(q21.2-q37.1), þ12, þ13(q14.3-

q34), þ14

�1p, �3(p26.3-p12.3), �4, �6(q12-

q27), �9p, �11(q14.1-q25), �18

(q12.1-q23)

T107 t(X;1)(p11.2;p34) Karyotype TFE3 Presumably PSF No No

T117 NA FISH TFE3 Unknown 7 �3, �15, �17(p13.3-p11.2), �20p

Abbreviations: NA, not available; IHC, immunohistochemistry; NED, no evidence of disease.

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TNM classification, or Fuhrman grade (data not shown).Finally, patients with 17q gain had a more adverseprognosis, with a 19.9-fold higher risk of RCC-specificdeath (95% confidence intervals, 3.1–129; P ¼ 0.002;Fig. 2C).

Patients with 3p loss had a higher pT stage than did thosewithout 3p loss (P¼ 0.03). However, in contrast to the 17qgain, no association was found between 3p loss and clin-icopathologic variables such as age, gender, and TNMclassification. Patients with 3p loss also had a shorter OStime than did those without 3p loss, with median 12.7months versus not reached (P ¼ 0.001). To assess whether3p loss was associatedwith VHLmutations, as is the case forccRCC, somatic mutations of VHL were analyzed in 12cases, but no mutations were identified.

Patients with 9p loss had more genetic alterationsthan did patients without 9p loss: 15.0 � 2.3 versus 4.3� 1.9 (P ¼ 0.003). No statistically significant associationswere found between 9p loss and age, gender, TNM stageclassification, pT stage, lymph node involvement, or metas-tasis; patients with 9p loss had a shorter OS time than didthose without 9p loss, with median OS of 26 versus 66months. However, this difference did not reach statisticalsignificance (P ¼ 0.13).

Cell lines recapitulate cytogenomic profiles fromtumors

We used the established cell lines UOK109 and UOK146and the novel cell lines HCR-59 andMDA-92. TheUOK109line, which was derived from a tumor originally reported aspRCC, had a pRCC-like cytogenomic profile, with chromo-some 7 gain (Supplementary Fig. S2A). In contrast, theUOK146 cell line, also derived from a pRCC tumor, hada ccRCC-like cytogenomic profile, with loss of 1p, 3p,chromosome 4, 9p, and chromosome 18 and a gain ofchromosomes 7 and 17q (Supplementary Fig. S2B).

To assess whether the 17q gain is related to unbalancedtranslocation or is the result of an isochromosome 17, weconducted G-banding karyotyping of the UOK146 cells.Besides a 3p loss, an i(17q) was present. This is consistentwith a model in which a 17q gain in cancer often occurs asthe result of i(17q) formation and is accompanied by allelicloss at 17p (Supplementary Fig. S2C).

The results of FISH analysis for TFE3 (Supplementary Fig.S2D) as well as conventional karyotyping of the MDA-92cell line (derived from patient T3) confirmed the TFE3translocation (data not shown) and showed a near-tetra-ploid karyotype, with 1p and 3p losses, as is the case for theprimary tumor. We also observed two interchromosomal

A B

17q gain

Ward’s (minimum variance) + (nonnormalized data)

0 20 40

Months

Perc

en

tag

e s

urv

ival

60 80

Nb

of

gen

eti

c a

bn

orm

aliti

es

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17q gain

P = 0.002

–1

0

1

P = 0.0026

No 17q gain

25

20

15

10

5

0

100

80

60

40

20

0

**

C

Figure 2. A, unsupervised clustering analysis of most frequently gained and lost regions in patients with tRCC showing twomain subtypes. B,more alterationsoccurred in patients with 17q gain than in patients without the gain. ��,P¼ 0.0026. C, Kaplan–Meier survival estimates for patients with and without 17q gain.

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translocations involving 17q that led to 17q gains (Fig.3A). Similarly, SKY of the HCR-59 cell line (derived frompatient T1) showed, besides 3p and 17p losses, 6 translo-cated chromosomes with 3 of them involving 17q (Fig.3B). All the genetic gains and losses reported by SNPcytogenomic array analysis of the primary tumor samples[�1(p31.1-p21.1), �1(p13.3-p11.2), �3(p26.3-p11.2),þ5(q33.3-q34), þ5(q34-q35.3), �9(p24.3-p21.1), �17(p13.3-p11.2), þ17(q21.31-q25.3), �18(q11.2-q23), and�X(p11.1-q28)] are located around the breakpoints of thesix translocations and are consistent with imbalancedtranslocations.

17q partial gain may be related to a large-scale dosageeffect and p53 haploinsufficiencyBecause partial 17q gain was the most common ab-

normality after TFE3 translocations and because patientswith 17q had a poorer outcome than did patients without17q, we investigated the expression pattern on chromo-some 17. To identify chromosomal regions differentiallyexpressed in cases with partial 17q gain and those without,we conducted global gene expression analysis using RNA-seq in 4 Xp11 cases for which RNAwas available. Two cases

with partial 17q gain, including patients T34 and T1, werecompared with 2 cases with balanced virtual karyotypes,including patients T31 and T32.

GSEA showed significant enrichment of genes located inthe 17q arm as follows: 17q25 [false discovery rate (FDR)¼0.0001; P < 0.0001], 17q23 (FDR¼ 0.07; P < 0.001), 17q21(FDR ¼ 0.08; P < 0.001), 17q24 (FDR ¼ 0.10; P < 0.001),and 17q22 (FDR ¼ 0.10; P < 0.001; Fig. 4A), and noenrichment for 17p genes. Interestingly, when genes wereclassified according to the C6 oncogenic signature in theMolecular Signatures Database, we found enrichment forgenes downregulated by TP53 in NCI-60 panel of cell lineswith mutated TP53 as compared with those classified asnormal (Fig. 4B).

As gain of 17q was frequently associated with loss of17p, which contains the p53 tumor suppressor gene, andas p53 loss is commonly associated with a poor prognosisin various malignancies, we assessed for the presence ofTP53 mutations. Sanger sequencing was thus conductedon 12 tRCC including 7 cases bearing gain of 17q, but nomutation of TP53was identified in any of those cases (notshown). Interestingly, Ingenuity Pathway Analysis iden-tified TP53 inactivation as the top transcription regulator

A

1

6

13

19 20 21 22 X Y

14 15 16 17 18

7 8 9 10 11 12

2 3 4 5M1

M2

M4 M5

M3

B C

Figure 3. A, karyotype for the novel MDA-92 cell line, derived from patient T3, shows the 3p loss and five different translocations enumerated as follows:M1 ¼ t(1;3), M2 ¼ t(9;17), M3 ¼ t(6;10), M4 ¼ t(18;21), and M5 ¼ different translocations involving t(X;17). B, SKY results for the novel HCR-59 cell linederived from patient T1. C, classified karyotyping results for the novel HCR-59 cell line derived from patient T1. B and C, six different translocationsenumerated as follows: t(1;3), t(5;17), t(17;22), t(18;21), t(6;10), and t(X;17).

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factor predicted to be inactivated in the 2 cases (RCC-T34and RCC-T1) with concomitant partial 17q gain as com-pared with the 2 cases (RCC-T31 and RCC-T32) withbalanced karyotype (Supplementary Table S3). Overall,there were 51 of 89 genes whose expression direction wasconsistent with inhibition of TP53 (z-score ¼ �3.84; P ¼5.37 � 10�11; Fig. 4C).

Also consistent with TP53 inactivation, GSEA showedenrichment for multiple pathways related to mitosis asfollows: the M-phase of the mitotic cycle (FDR ¼ 0.10;P < 0.001), mitosis (FDR ¼ 0.10; P < 0.001), and the M-phase (FDR ¼ 0.10; P < 0.001), in keeping with the aggres-sive phenotype of cases with partial 17q gain (Supplemen-tary Fig. S3A).

KEGG (Kyoto Encyclopedia of Genes and Genomes) andBioCarta (BioCarta LLC) pathways (C2 gene set) usingGSEA and Ingenuity Pathway Analysis (Ingenuity Systems,Inc.) showed consistent results with T-cell activation in thesubgroup of patients with partial 17q gain. In particular,

BioCarta pathways showed enrichment of the CTLA-4 path-way (FDR < 0.001; P < 0.001), and the KEGG pathwayshowed enrichment of the T-cell receptor signaling pathway(FDR ¼ 0.01; P < 0.001). Those results were also obtainedusing Ingenuity Pathway Analysis, which showed activationof inducible costimulator–inducible costimulator ligandsignaling in T-helper cells (P ¼ 2.4E�4) and activation ofthe CTLA-4 signaling pathway (P¼ 1.6E�3). Of note, 17 of95 genes (17.9%) related to the CTLA-4 pathway wereupregulated in patients with 17q gain (Supplementary Fig.S3B). Immunohistochemistry for CD3 and CD5 T-cellmarkers showed increase of T lymphocytes in the 2 caseswith 17q gain (10%–20%) as compared with less than 1%to 2% in the 2 cases without 17q gain (not shown).

LINE-1 methylation is lower in adults than in youngpatients (<18 years old)

We conducted LINE-1 methylation analyses in a total of27 tRCC cases (12 of which had SNP array results). LINE-1

Figure 4. Partial 17q gain and gene expression. GSEA of genes expressed differently between selected caseswith partial 17q gain andwithout was conductedbyRNA-seq. A, significant correlationwas observed between chromosomal 17q gain and expression of genes located in the 17q arm (e.g., 17q21 and 17q25).B, C6 oncogenic signature in the Molecular Signatures Database showing enrichment for genes downregulated by TP53 in NCI-60 panel of cell lineswith mutated TP53 as compared with those classified as normal. C, Ingenuity Pathway Analysis showing the network of genes consistent with TP53inactivation. Overall, there were 51 of 89 genes that have expression direction consistent with inhibition of TP53.

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methylation levels correlated inversely with the number ofgenetic abnormalities in cases with both SNP-array andLINE-1methylationdata (Spearmanr¼�0.6;P¼0.04; Fig.5A). Consistent with the greater number of genetic abnor-malities in adult patients, in the 27-patient cohort, adultsshowed lower LINE-1methylation, comparedwith youngerpatients (71.1% vs. 76.7%; P ¼ 0.02; Fig. 5B). Methylationlevels of normal kidney tissue showed little variability withan averagemethylation of 73.07% and SDof�0.50, and noassociation with age or gender.

DiscussionTo our knowledge, this is the first study to describe

cytogenomic aberrations and DNA methylation status ina cohort of pediatric, adolescent, and adult patients withtranslocation RCC. Our study shows cytogenomic andepigenetic heterogeneity of tRCC and unexpectedly revealsthat these tumors share genomic alterations that are com-mon to ccRCCs and pRCCs. Our results show that tRCCtumors from young patients (<18 years) display fewergenetic alterations and higher levels of global DNA meth-ylation, compared with tumors from adult patients (�18years). Moreover, our data show that a partial 17q gain isfrequent in tumors from adult patients, particularly inmen,and this genetic lesion is associated with poor outcome.Although the identification of significant correlations

between cytogenetic alterations and histopathologic sub-types of RCC is well established for common renal cancer

subtypes, such as ccRCC (e.g., 3p loss) and pRCC (e.g.,trisomy 7 and/or 17; refs. 15, 30), little is known aboutchromosome copy number alterations in tRCC. Here, weshow that a large proportion of tRCCs present with chro-mosome copy number changes similar to those observed inccRCC or pRCC, but almost 30% of them display cytoge-nomic profiles that cannot be related to these or character-ized RCC subtypes.

Morphologic diagnosis of tRCC is challenging, especiallyin adult patients. Sukov and colleagues showed that tRCCtumors have significant histologic variability (31), andKlatte and colleagues showed that only 2 of 75 cases withmicroscopic features suggestive of Xp11.2 translocationRCC or occurring in patients 40 years or younger haveidentifiable translocations with FISH and/or RT-PCR(32). Historically, the translocation involving TFE3 (orTFEB) is considered as the defining feature for this disease.Mechanistic studies have shown the transforming effect ofthe TFE3 and TFEB translocations in cell lines (33, 34).Mathur and Samuels conducted knockdown of PSF-TFE3 inUOK-145 cell line and showed that it led to impairedgrowth, proliferation, invasion potential, and long-termsurvival (33). The same authors also showed that theexpression of PSF-TFE3 in normal proximal tubular cellsleads to initiation and maintenance of an oncogenic phe-notype (33). Moreover, in the UOK109 cell line (NONO-TFE3 translocation), TFE3-directed short hairpin RNA(shRNA) prevented all colony growth (35). The identifica-tion of "ccRCC-like" and "pRCC-like" cytogenomic profilesin tRCC cases raises the question of whether tumors har-boring such profiles are in reality ccRCC and pRCCs thathave acquired a TFE3 translocation during tumor progres-sion or whether tRCC tumors (initiated by a TFE3 translo-cation) can then develop different types of chromosomalinstability leading to different cytogenomic profiles. Are 3ploss and gain of 7/17 important for the initiation of tRCCtumors or are these changes acquired during tumorprogression? Are tumors in these tRCC subgroups, carcino-mas that initiated as ccRCC and pRCC, and then acquiredTFE3/TFEB translocations as they progressed? Or are thesetumors initiated by a TFE3/TFEB translocation, whichacquired patterns of chromosomal imbalances similar tothose observed in ccRCC and pRCC? The 3p loss is arequired event in ccRCC, as it leads to loss of the wild-typeVHL allele and abrogation of VHL hypoxia-regulating activ-ity. VHL mutations do not seem to play a role in tRCC, asVHLmutations were not identified in our cohort. However,other tumor suppressor genes with roles in RCC biologyhave been recently identified in 3p (PBRM1, SETD2, andBAP1), and thus 3p loss in tRCC tumorsmight be associatedwith inactivation of one or more of these genes (36–38).Alternatively, tumor initiation by the TFE3/TFEB transloca-tion might trigger chromosomal instability pathways thatresemble those in ccRCCandpRCC subtypes. Future studiesare needed to clarify whether the presence of these translo-cations is enough to determine a tRCC diagnosis and iftumor behavior (and response to therapy) depends on theoverall genomic profile. Interestingly, patients with tRCC

A 85

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e-1

meth

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tio

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%)

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15

P = 0.04

Spearman R = –0.6

20 25

*

Figure 5. Genomic aberrations and LINE-1 methylation. A, Spearmancorrelation between the number of chromosomal armswith gain or loss intRCC samples and LINE-1 methylation (n ¼ 12). B, LINE-1 methylationlevel is lower in tumors of adult as compared with young patients. ns, notsignificant. �, P ¼ 0.02.

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with 3p loss (ccRCC-like profile) had worse outcomescompared with those without 3p loss. In contrast, 9p lossin our studywasnot associatedwith apoor prognosis, as it isin early-stage ccRCC (32).

Another interesting finding of our study is the greaternumber of genetic abnormalities and the lower level ofglobal methylation found in tRCC tumors from adultpatients, comparedwith tumors fromyoung patients. Thesedata are concordant with the significant inverse correlationbetween the degree of genomic alterations and the LINE-1methylation levels reported in other tumor types (39).Although the difference inmethylation levels between adultand young patients is small, it achieved statistical signifi-cance due to the robustness of the assay used. This is shownby the fact that methylation levels of normal kidneysshowed little variability with an average methylation of73.07% (�0.50%). Using this assay, no significant associa-tions were previously found between LINE-1 methylationand age, gender, or diet in colonic tissues (19); thus, webelieve that the observed differences are related to intrinsiccharacteristics of tRCC in adult and young patients, ratherthan to age. Additional studies with a larger group ofpatients are necessary to confirm these findings.

The cytogenomic differences between tumors from adultand young patients were independent of classical clinico-pathologic prognostic factors such as TNM stage and Fuhr-man grade. Although those results might be related to asmall number of cases, cytogenomic array analysis in 3 ofour 4 young patients with American Joint Committee onCancer (AJCC) stage III–IV disease found no genetic abnor-malities. Another age-related difference is that gains in 17qwere identified in tumors from 70% (n ¼ 7/10) of all ouradult patients, whereas this abnormality was not found inyounger patients. Patients with 17q gain were more fre-quently men, displayed more genetic alterations, and hadworse outcomes. Five of those patients had a partial gain of17q, whereas 2 had trisomy 17. Of interest, 17q gains arerare in ccRCC and pRCC, in contrast to trisomy 7 and 17,which are very frequent in pRCC (15). On the basis of ourstudy of patient-derived cell lines, 17q gains seem to arisefrom i(17q) and/or unbalanced translocations and com-plex rearrangements. Together with G-banding and SNPresults available for three cell lines (HCR-59, MDA-92, andUOK146), our data showed that 2 cases had multipletranslocations involving 17q, leading to 17q gain concom-itantwith i(17q) formation in 1 case, and 1 case had only ani(17q) without other associated translocations. Two caseshad breakpoints in 17p11.2 and were thus consideredisodicentric chromosomes. Of note, 17p11.2 has beenreported to contain a cluster region characterized by largepalindromic and low copy-repeat sequences in chronicmyelogenous leukemia (CML), medulloblastoma withidic(17)(p11), and high hyperdiploid childhood acutelymphoblastic leukemia (40–43). This complex architec-ture suggests that i(17q) does not occur randomly, butrather because of susceptibility in the genomic structure.However, the reasonwhy 17q is associatedwith age remainsunclear; and whether adults with tRCC without a 17q gain

have a different prognosis than adults with the 17q gainremains unknown. A larger cohort is needed to answer thisquestion. Nonetheless, our results strongly suggest that 17qgain is more frequent in adult tRCCs and our preliminaryGSEA analysis suggest a dosage effect of genes on 17q andp53 haploinsufficiency. It is important to note that CMLthat bear an i(17q) have a poor prognosis (44), andmedul-loblastomas with an i(17q) or a 17q gain also have a pooroutcome (43). In our study, GSEA analysis of a subset ofXp11 cases with and without partial 17q gain found enrich-ment of pathways involved in mitosis and the cell cycle inpatients with 17q gain, suggesting a proliferative advantagein tumors harboring this chromosomal abnormality. It isnotable that those cases also had gene expression patternsconsistent with activation of T-helper cells and the CTLA-4signaling pathway. Further studies are required to confirmthese findings.

Abnormalities involving 17q have been previouslydescribed in tRCC but to date 17q gain had not beenrecognized as a recurrent abnormality in these tumors, norits possible association with outcomes. Among our 14previously reported cases (9), we found 1 case, that of a2-year-old girl,whohada t(5;17)(q23;q25), in addition to at(X;17)(p11.2,17q25) leading to theASPSCR1-TFE3 fusion,consistent with multiple translocations occurring on the17q arm. This patient had lymph node involvement andlung metastasis at the time of her diagnosis, which is rarein pediatric cases of tRCC (9). A recent case report of thet(X;17)(p11;q25) translocation, in a 24-year-old Japanesewoman (45), also showed the presence of a second trans-location involving 17q: t(13;17)(p11;q11). Moreover,in the original report by Argani and colleagues that des-cribed tRCC (13), they identified a complex nonreciprocalt(X;17)(p11.2;q25), involving not only the translocationof Xp11 to 17q25 but also of 17q25 to another chromo-some. Our results and these reports in the literaturesuggest an important role of 17q in the biology of a subsetof tRCC tumors.

Although we did not study whether there is intratu-moral heterogeneity with respect to the presence of 17qand other chromosomal abnormalities identified in thisstudy, we have previously shown that intratumoral het-erogeneity at the level of chromosomal abnormalities isseen in only up to 20% of RCC cases (46), in contrast withthe level of mutational heterogeneity reported by Gerlin-ger and colleagues (47). However, intratumoral hetero-geneity studies might be helpful to understand whether17q is an initiating event or acquired during tumorprogression, as cytogenomic heterogeneity in our priorstudies is restricted to abnormalities acquired duringtumor progression (46).

In conclusion, our data show that tRCC is geneticallyheterogeneous and share cytogenomic profiles with otherrenal cell tumors, raising important questions about therole for TFE3/TFEB translocations in tumor initiation and/or progression. Furthermore, we have shown that cytoge-nomic and methylation differences exist between tRCCtumors arising in adult and pediatric/adolescent patients.

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Thesedatamaybeused todevelopprognostic biomarkers intRCC patients.

Disclosure of Potential Conflicts of InterestF.A. Monzon has honoraria from Speakers Bureau of Affymetrix. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: G.G. Malouf, F.A. Monzon, B. Escudier, C.G.Wood, N.M. TannirDevelopment ofmethodology:G.G.Malouf, F.A. Monzon, B. Escudier, M.Garcia, N.M. TannirAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): G.G. Malouf, F.A. Monzon, B. Escudier, P.Camparo, P. Tamboli, D. Lopez-Terrada, M. Picken, M. Garcia, C.G. Wood,N.M. TannirAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis):G.G.Malouf, F.A.Monzon, V.Molini�e, P.Camparo, X. Su, H. Yao, M. Garcia, N.M. TannirWriting, review, and/or revision of the manuscript: G.G. Malouf, F.A.Monzon, J. Couturier, V. Molini�e, P. Tamboli, M. Garcia, A.S. Multani, S.Pathak, C.G. Wood, N.M. Tannir

Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): G.G. Malouf, M. Picken, N.M.TannirStudy supervision:G.G.Malouf, F.A. Monzon, B. Escudier, M. Picken, C.G.Wood, N.M. Tannir

AcknowledgmentsThe authors thank Dr. W. Marston Linehan, the UOB Tumor Cell Line

Repository, National Cancer Institute (Bethesda, MD), for providing theUOK146 andUOK109 cell lines. The authors also thankMs. Karla Alvarez ofThe Methodist Hospital Research Institute for generating the SNP arraycytogenomic profiles.

Grant SupportThis study was supported in part by the NIH through MD Anderson’s

Cancer Center Support Grant, 5 P30 CA016672, and in part by the Nelia andAmadeo Barletta Foundation (to G.G. Malouf).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 17, 2012; revised June 10, 2013; accepted June 12,2013; published OnlineFirst July 1, 2013.

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2013;19:4673-4684. Published OnlineFirst July 1, 2013.Clin Cancer Res   Gabriel G. Malouf, Federico A. Monzon, Jérôme Couturier, et al.   Genomic Heterogeneity of Translocation Renal Cell Carcinoma

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