Genomic Heterogeneity of Translocation Renal Cell Carcinoma

Published OnlineFirst July 1, 2013; DOI: 10.1158/1078-0432.CCR-12-3825

Clinical Cancer Human Cancer Biology Research

Gabriel G. Malouf1, Federico A. Monzon7,8,Jer ome^ Couturier10, Vincent Molinie11, 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

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 polymorphism 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; 1–12. 2013 AACR.

Introduction Translocation renal cell carcinoma (tRCC) is a subtype of Authors' Afﬁliations: Departments of 1Leukemia, 2Bioinformatics, renal cell carcinoma (RCC) that was recognized in the 2004 3Pathology, 4Genetics, 5Urology, and 6Genitourinary Medical Oncology, World Health Organization classiﬁcation of renal tumors as The University of Texas MD Anderson Cancer Center; Departments of 7Pathology & Immunology and 8Molecular & Human Genetics, Baylor a genetically distinct entity (1). Originally described in College of Medicine; 9Department of Pathology, Texas Children's Hospital, pediatric patients, the spectrum of the disease has recently 10 Houston, Texas; Department of Genetics, Institut National de la Santeet been expanded to include adults (2). Recent studies showed de la Recherche Medicale (INSERM) U830, Institut Curie; 11Department of Pathology, Hopital^ Saint Joseph, Paris; 12Department of Medical Oncol- that tRCC represents one third of pediatric RCCs, up to 15% ogy, Institut Gustave Roussy, Villejuif; 13Department of Pathology, Hopital^ of RCCs in patients younger than 45 years old, and up to 5% 14 Foch, Suresnes, France; Department of Pathology, Loyola University of all RCCs regardless of age (3–5). The hallmark of tRCC is Hospital, Chicago, Illinois; and Departments of 15Medicine and 16Pathol- ogy, University of Colorado School of Medicine, Aurora, Colorado the fusion of the TFE3 gene, located in Xp11, with various partners, including PRCC in t(X;1)(p11.2;q21), SFPQ in t Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). (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). G.G. Malouf and F.A. Monzon contributed equally to this work. tRCC can also be related to translocations involving the Current address for G.G. Malouf: Department of Medical Oncology, Groupe TFEB gene (7). Hospitalier Pitie-Salp etri^ ere, Assistance Publique Hopitaux de Paris, Fac- We and others have reported that the disease behaves ulty of Medicine Pierre et Marie Curie, Institut Universitaire de Cancerologie GRC5, University Paris 6, Paris, France. differently in adult and pediatric patients (2, 8–10). Sim- ilarly, better outcomes in young patients were reported for Corresponding Author: Nizar M. Tannir, Department of Genitourinary Medical Oncology, Unit 1374, The University of Texas MD Anderson alveolar soft part sarcoma, a tumor characterized by Cancer Center, 1155 Pressler Street, Houston, TX 77030-3721. Phone: ASPSCR1-TFE3 translocation (11). Recently, we showed 713-563-7265; Fax: 713-745-1625; E-mail: [email protected] that patients with tRCC who had metastatic disease at doi: 10.1158/1078-0432.CCR-12-3825 presentation were older (median age 36 years) and pre- 2013 American Association for Cancer Research. dominantly male, whereas patients who had loco-regional

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fusion partners. One patient whose tumor had classical Translational Relevance tRCC morphology and TFEB immunohistochemical pos- Translocation renal cell carcinoma (tRCC) is a rare itivity was also included in the final cohort (n ¼ 16). We kidney cancer subtype that mainly arises in children and collected the following clinicopathologic information young adults and often has both papillary and clear-cell for each patient: age, sex, ethnicity, tumor–node–metas- pathologic features. Little is known about whether addi- tasis (TNM) classification, tumor size, lymph node tional genetic alterations are associated with tRCC. By involvement, Fuhrman grade, and survival time (Sup- single-nucleotide polymorphism–array profiling and plementary Table S1). The clinicopathologic data from LINE-1 methylation, we found genomic heterogeneity 5 of the patients (numbers T31 through T35) were of tRCC that included alterations common with clear- previously reported (9). Four patients with positive TFE3 cell RCC (e.g., 3p loss) and papillary RCC (e.g., trisomy 7 immunohistochemistry but negative by FISH analysis and/or 17). When compared with young patients (<18 for TFE3 translocations were included in the single- years), adults with tRCC displayed distinct genomic and nucleotide polymorphism (SNP) array studies as a con- epigenetic aberrations, exemplified by lower LINE-1 trol group. methylation and frequent 17q partial gain, which were consistent with a large-scale dosage effect affecting RCC Patient selection and LINE-1 methylation analysis carcinogenesis. Our results show that besides TFE3/TFEB DNA was available for studying LINE-1 methylation in translocations, tRCC shares alterations commonly pres- 12 patients for whom SNP-array analysis was conducted ent in other RCC histologic subtypes and these are (all except the following 4 cases: LOY009, MRCC106, associated with patient outcomes. Furthermore, our MRCC107, and MRCC117). In addition, DNA was ex- study suggests that targeting tRCCs according to their tracted from 15 formalin-fixed paraffin-embedded (FFPE) genetic profile may have therapeutic relevance. tissues of patients for whom we previously reported pathologic features and outcome (9, 18). Seven of those patients had confirmed translocation by karyotyping and/or RT- PCR, and the remaining had their diagnosis confirmed by disease were younger (median age 16 years) and predom- TFE3 immunostaining, as previously described (18). Fur- inantly female (9). These data are in line with previous thermore, DNA extracted from 16 samples belonging to reports, suggesting a more aggressive disease in adults, adjacent normal kidney of patients operated on for renal especially in men (8, 12). However, the basis for the cell carcinoma was included to study LINE-1 methylation biologic difference in tRCC between adult and pediatric variability. DNA isolation was done using the Gentra Sys- patients has not been elucidated. tems Puregene DNA Purification Kit according to the Since the first description of tRCC by Argani and collea- manufacturer’s instructions. LINE-1 repetitive element, gues, there have been few reports about a detailed cyto- a surrogate of global DNA methylation, was assessed by genetic characterization of these tumors (13, 14). Other pyrosequencing as described previously (19). Assay repli- renal tumors are characterized by specific chromosomal cates for this assay were highly correlated as previously imbalances, such as 3p loss in clear-cell RCC (ccRCC) and shown (19). trisomies 7 and 17 in papillary RCC (pRCC; refs. 15, 16). The aims of our study were to investigate whether there Generation of novel cell lines from adult patients are any additional genetic/epigenetic alterations beside Two novel cell lines, HCR-59 and MDA-92, were TFE3/TFEB translocations, and assess whether there are derived from 2 adults treated at MD Anderson Cancer associations between specific chromosomal imbalances Center (MDACC; Houston, TX), as previously reported and classical clinicopathologic factors and overall survival for other RCC subtypes (20). HCR-59 was derived from a (OS). 20-year old Caucasian female, and MDA-92 was derived from a 51-year-old Hispanic male. For both of those cell Patients and Methods lines, karyotyping was conducted according to previous- Patient selection and classification of cases included in ly published methods (21). Spectral karyotyping (SKY) single-nucleotide polymorphism-array analysis was also conducted on HCR-59 by using the human Tissue specimens from 21 patients with a histopathologic chromosome HiSKY probe (Applied Spectral Imaging, diagnosis of tRCC supported by TFE3 positivity on immu- Inc.). nohistochemistry were collected after approval of the Insti- The tRCC cell lines UOK109 and UOK146, derived, tutional Review Board of each of the participating centers. respectively, from a 39-year-old male and a 42-year-old TFE3 and TFEB immunostaining were conducted at each female (22), were kindly provided by Dr. W. Marston individual institution as previously described (7, 17). The Linehan (U.S. NIH, Bethesda, MD). Both cell lines were flow chart of patient selection, describing the different tests originally reported as pRCC cell lines before tRCC became a conducted is depicted in Supplementary Fig. S1. recognized pathologic entity. The UOK109 line has been Among the total of 21 cases, 15 were confirmed to be shown to carry the NONO-TFE3 translocation (23), and the tRCC by FISH, conventional karyotyping, or reverse tran- UOK146 cell line carries the translocation t(X;1)(p11.2; scription PCR (RT-PCR) analysis for all known specific q21.2) (22).

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Cytogenomic analysis and number of genetic aberrations. All statistical tests were DNA obtained from frozen tissues or from manually two-sided and conducted at the significance level of 0.05 microdissected FFPE containing at least 70% tumor cells using Prism version 5 (GraphPad Software, Inc.). was quantified on a NanoDrop ND-1000 spectrophotom- eter (Thermo Fisher Scientific, Inc.). The DNA was ana- Results lyzed on Affy250KNsp SNP genotyping arrays (Affymetrix, Inc.) following the previously reported modified protocol Patient and tumor characteristics (24). LOH and copy number estimates were obtained by Patient and tumor characteristics are summarized in using the publicly available Copy Number Analyzer for Supplementary Table S1. With a median follow-up time of Affymetrix GeneChip Arrays package (CNAG v3.0; ref. 25). 29 months (range, 3–92 months), 5 patients (31%) were The number of genetic alterations was defined as the dead of disease, 8 patients had no evidence of disease, and 3 number of gains and losses of segmental chromosomal patients were alive with metastatic disease. regions. tRCC shows cytogenomic heterogeneity RNA sequencing Translocation carcinomas showed significant heteroge- For 4 translocation Xp11 cases (T31, T32, T34, T1), RNA neity in cytogenomic profiles (Fig. 1 and Table 1). The most of good quality with RNA integrity number values between common alteration observed was a gain of 17q, which was 8.0 and 10.0 was available. Briefly, preparation of mRNA- present in seven (44%) of the tumors. Less frequent altera- seq libraries for each case was conducted according to tions were gains of chromosomes 12 and 7, observed in 6 Illumina procedures at the MDACC Genomic Core Facility, (37%) and 5 (31%) patients, respectively. The most com- and sequencing was done on a HiSeq 2000 system (Illu- monly observed cytogenetic losses were of 9p in 6 (37%) mina, Inc.). After quality control, reads were mapped and patients and of 3p, 1p, 17p, and 18q, each of which then aligned to the UCSC hg19 version of the reference occurring in 5 (31%) patients. Three of 10 women in our genome. Gene expression counts were normalized using the cohort (30%) lost one X chromosome. RPKM (reads/kb/million) method (26). Overall, the 16 cases of tRCC with a confirmed translocation could be classified into four groups on the basis of Gene set enrichment analysis their similarities to cytogenomic profiles of other renal Gene set enrichment analysis (GSEA) is a computa- epithelial tumors (15). tional method that assesses whether a defined set of genes shows statistically significant differences between two * Group I: 5 cases had profiles similar to those of ccRCC, conditions (24). The average degree of change between owing to 3p loss and other imbalances (including the the normalized RPKM values of 2 samples with partial TFEB case). Three of those 5 cases were available for 17q gain and 2 samples without was calculated, and histologic review with 2 showing mixed clear-cell/ genes were ranked by order of expression in the 2 samples papillary features, and 1 showing only clear-cell with partial 17q gain. The list was uploaded as a pre- morphology. ranked list to GSEAv2.04 (Broad Institute, Cambridge, * Group II: 3 cases had profiles similar to those of pRCC MA; ref. 27), and GSEA was conducted using the default with gains of chromosomes 7, 12, and 17. All 3 cases normalization mode. Genes were classified according to showed mixed ccRCC and pRCC histology. the positional gene set (C1) in the Molecular Signatures * Group III: 5 cases had novel cytogenomic profiles, that is, Database (http://www.broadinstitute.org/gsea/msigdb/ not associated with those of other subsets of renal tumors. index.jsp). * Group IV: 3 cases had a balanced chromosomal complement. Mutation of TP53 and VHL genes For the TP53 gene, mutations occurring in exons 5 to 8 As Fig. 1 shows, tRCC tumors had heterogeneous chro- were assessed using five sets of oligonucleotide primers as mosome copy number profiles. 3p loss was associated with previously described (28). VHL mutations were assessed 9p loss (P < 0.04) but not associated with the loss of 1p, 17p, using four primers pairs as previously described (29). Over- or 18q (P ¼ 0.24 for each comparison). There was no all, 12 cases with available DNA were analyzed as shown significant difference in the number of genetic alterations in Supplementary Fig. S1. between patients with 3p loss and those without (11.8 vs. 6.7; P ¼ 0.23). Statistical analysis Of the 7 cases (44%) that had a gain of 17q, two tumors Associations with clinicopathologic factors were ana- had a confirmed t(X;17)(p11;q25) translocation and 1 case lyzed with x2 and t test. OS was calculated from the date had a TFEB translocation. Interestingly, in 4 of 7 cases with of nephrectomy to the date of death or last visit (censored). 17q gain, 17p loss has been shown, consistent with the Survival probabilities were estimated with the Kaplan– generation of an isochromosome 17q, or i(17q). In con- Meier method, and log-rank testing was applied to compare trast, none of 4 cases overexpressing TFE3 but negative by survival curves. Spearman correlation coefficient was used FISH had gain of 17q or loss of 17p, and 2 of them had 3p to assess the correlation between the LINE-1 methylation loss (Supplementary Table S2).

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ccRCC-like 100% Gains 80% 60% 40% 20% 0% –20% –40% –60% –80%

Percentage of samples Percentage Losses –100%

pRCC-like 100% Gains 80% 60% 40% 20% 0% –20% –40% –60% –80% Percentage of samples Percentage –100% Losses

Novel 100% Gains 80% 60% 40% 20% 0% –20% –40% –60% –80% Percentage of samples Percentage Losses –100% 1234567891011121314151618 20 22 17 19 21

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).

Tumors of adult patients (age 18 years) showed more cluster B. Of note, 7 of 10 patients who were older than 18 genetic abnormalities than tumors from younger patients years had a 17q gain, but none of the 2 older patients in (age < 18 years; P ¼ 0.006), with no differences found in cluster A did. sex (P ¼ 0.31), TNM stage (P ¼ 0.60), and Fuhrman grade (P ¼ 0.62). Association between chromosomal imbalances and pathologic features and outcome Unsupervised clustering reveals two distinct groups of As noted earlier, a gain of 17q was the most frequent tRCC alteration (44%) we found in tRCC with a minimally To gain insights into the biology of tRCC, we con- gained region of 11.9 Mb (17q24.3-q25.3). Four of these ducted unsupervised clustering analysis using the most cases had 17p deletions, with a minimally deleted region frequent segmental alterations (losses or gains). That of22.1Mb(entireparm),includingtheTP53 locus. No analysis revealed two main clusters (Fig. 2A). The median associations were found between 17q gain and any other number of genetic alterations in cluster A was 0 (range, 0– frequently occurring genetic gain or loss. The mean 5), compared with 14 in cluster B (range, 5–22). The two number of alterations in patients with 17q gain was clusters showed no signiﬁcant differences in terms of pT 14.4 2.4 versus 3.7 2.4 in patients without 17q stage, lymph node involvement, metastasis, TNM classi- gain (P < 0.003; Fig. 2B). In addition, patients with 17q ﬁcation, gender, and Fuhrman grade. The only statistical gain were older than those without: their median age difference between clusters was age at diagnosis: the was 40.2 (range, 20.4–54.6) versus 16.7 years (range, median age of 7 patients in cluster A was 16.7 years 9.1–33.1; P ¼ 0.0006). The gain of 17q was also found (range, 9.1–33.1 years), compared with 34.2 years (range, more frequently in men than in women (71.4% vs. 16–54 years) for the 9 patients in cluster B (P ¼ 0.01). 11.1%; P ¼ 0.03). No associations were found between Overall, 5 of 7 (71%) patients in cluster A were younger 17q gain and other clinicopathologic variables, i.e., than age 18, compared with 1 of 9 (11%) patients in tumor size, ethnicity, pT stage, lymph node involvement,

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Table 1. Genetic/genomic characteristics of translocation RCC cases

Chromosomal Diagnostic Sample translocation method Gene Fusion partner Gains Losses T1 NA FISH TFE3 Unknown þ5(q33.3-q34), þ5(q34-q35.3), þ17 1(p31.1-p21.1), 1(p13.3-p11.2), 3 (q21.31-q25.3) (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), No þ12(p13.33-q24.33), þ17(p13.3- q25.3), þ20(p13-q13.33) 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 1(p36.33-p11.2), 9(p22.3-p21.1), (p15.33-q35.3), þ7(p22.3-q36.3), þ8 9(p22.1-p21.3), 9(p21.1-p21.1), (p23.3-q24.3), þ9(q12-q21.13), þ9 17(p13.3-q11.1) (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) T25 NA RT-PCR TFE3 NONO No No T29 NA IHC TFEB Presumably alpha þ1(q23.3-q44), þ3(p11.1-q29), þ4 3(p26.3-p14.1), 6(q14.1-q27), 8 (q27-q28.1), þ4(q28.3-q32.2), þ5 (p23.3-p11.1), 9(p24.3-q34.3), 13 (p15.33-p11), þ5(q23.1-q35.3), þ5 (q12.11-q34), 14(q11.2-q32.33), (q31.1-q35.3), þ7(p22.3-q36.3), þ8 17(p13.3-q11.2), 18(p11.32-q23), (q11.1-q24.22), þ12(q14.1-q24.33), 22(q11.1-q13.33) þ16(p13.3-p12.3), þ16(p12.3-q24.3), þ17(q11.2-q25.3) 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 1(p36.33-p34.3), 1(p33-p11.2), 4 (p23.3-q24.3), þ12(p13.33-q24.33), (p16.3-q35.2), 10(p15.3-q26.3), þ17(p13.3-q25.3), þ19(p13.3- 13(q11-q34), 15(q11.2-q26.3), q13.43), þ20(p13-q13.33), þ22 17(q21.31-q21.31), 18(p11.32- (q11.1-q13.33) 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 4(p16.3-q35.2), 8(p23.3-p22), 10 (p25.3-q37.3), þ6(p25.3-q27), þ7 (p15.3-q26.3), 14(q11.2-q32.33), (p22.3-q36.3), þ12(p13.33-q24.33), 17(p13.3-p11.2) þ13(q31.1-q34), þ17(q11.1-q25.3), þ20(p13-q13.33) 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- 1p, 3(p26.3-p12.3), 4, 6(q12- q34), þ14 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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P = 0.0026 AB** 25

Ward’s (minimum variance) + (nonnormalized data) 20

–1 15

0 10

5 1

Nb of genetic abnormalities 0 17q gain No 17q gain

P = 0.002 C 100

80 17q gain No 17q gain 60

20 Percentage survival Percentage

0 0204060 80 Months

Figure 2. A, unsupervised clustering analysis of most frequently gained and lost regions in patients with tRCC showing two main subtypes. B, more alterations occurred in patients with 17q gain than in patients without the gain. , P ¼ 0.0026. C, Kaplan–Meier survival estimates for patients with and without 17q gain.

TNM classification, or Fuhrman grade (data not shown). Cell lines recapitulate cytogenomic profiles from Finally, patients with 17q gain had a more adverse tumors prognosis, with a 19.9-fold higher risk of RCC-specific We used the established cell lines UOK109 and UOK146 death (95% confidence intervals, 3.1–129; P ¼ 0.002; and the novel cell lines HCR-59 and MDA-92. The UOK109 Fig. 2C). line, which was derived from a tumor originally reported as Patients with 3p loss had a higher pT stage than did those pRCC, had a pRCC-like cytogenomic profile, with chromo- without 3p loss (P ¼ 0.03). However, in contrast to the 17q some 7 gain (Supplementary Fig. S2A). In contrast, the gain, no association was found between 3p loss and clin- UOK146 cell line, also derived from a pRCC tumor, had icopathologic variables such as age, gender, and TNM a ccRCC-like cytogenomic profile, with loss of 1p, 3p, classification. Patients with 3p loss also had a shorter OS chromosome 4, 9p, and chromosome 18 and a gain of time than did those without 3p loss, with median 12.7 chromosomes 7 and 17q (Supplementary Fig. S2B). months versus not reached (P ¼ 0.001). To assess whether To assess whether the 17q gain is related to unbalanced 3p loss was associated with VHL mutations, as is the case for translocation or is the result of an isochromosome 17, we ccRCC, somatic mutations of VHL were analyzed in 12 conducted G-banding karyotyping of the UOK146 cells. cases, but no mutations were identified. Besides a 3p loss, an i(17q) was present. This is consistent Patients with 9p loss had more genetic alterations with a model in which a 17q gain in cancer often occurs as than did patients without 9p loss: 15.0 2.3 versus 4.3 the result of i(17q) formation and is accompanied by allelic 1.9 (P ¼ 0.003). No statistically significant associations loss at 17p (Supplementary Fig. S2C). were found between 9p loss and age, gender, TNM stage The results of FISH analysis for TFE3 (Supplementary Fig. classification, pT stage, lymph node involvement, or metas- S2D) as well as conventional karyotyping of the MDA-92 tasis; patients with 9p loss had a shorter OS time than did cell line (derived from patient T3) confirmed the TFE3 those without 9p loss, with median OS of 26 versus 66 translocation (data not shown) and showed a near-tetra- months. However, this difference did not reach statistical ploid karyotype, with 1p and 3p losses, as is the case for the significance (P ¼ 0.13). primary tumor. We also observed two interchromosomal

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1 M1 23 4 5

6 789101112M2 M3

13 14 15 16 17 18

M4 M5 19 20 21 22 XY BC

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 line derived from patient T1. C, classified karyotyping results for the novel HCR-59 cell line derived from patient T1. B and C, six different translocations enumerated as follows: t(1;3), t(5;17), t(17;22), t(18;21), t(6;10), and t(X;17). translocations involving 17q that led to 17q gains (Fig. with partial 17q gain, including patients T34 and T1, were 3A). Similarly, SKY of the HCR-59 cell line (derived from compared with 2 cases with balanced virtual karyotypes, patient T1) showed, besides 3p and 17p losses, 6 translo- including patients T31 and T32. cated chromosomes with 3 of them involving 17q (Fig. GSEA showed significant enrichment of genes located in 3B). All the genetic gains and losses reported by SNP the 17q arm as follows: 17q25 [false discovery rate (FDR) ¼ cytogenomic array analysis of the primary tumor samples 0.0001; P < 0.0001], 17q23 (FDR ¼ 0.07; P < 0.001), 17q21 [1(p31.1-p21.1), 1(p13.3-p11.2), 3(p26.3-p11.2), (FDR ¼ 0.08; P < 0.001), 17q24 (FDR ¼ 0.10; P < 0.001), þ5(q33.3-q34), þ5(q34-q35.3), 9(p24.3-p21.1), 17 and 17q22 (FDR ¼ 0.10; P < 0.001; Fig. 4A), and no (p13.3-p11.2), þ17(q21.31-q25.3), 18(q11.2-q23), and enrichment for 17p genes. Interestingly, when genes were X(p11.1-q28)] are located around the breakpoints of the classified according to the C6 oncogenic signature in the six translocations and are consistent with imbalanced Molecular Signatures Database, we found enrichment for translocations. genes downregulated by TP53 in NCI-60 panel of cell lines with mutated TP53 as compared with those classified as 17q partial gain may be related to a large-scale dosage normal (Fig. 4B). effect and p53 haploinsufficiency As gain of 17q was frequently associated with loss of Because partial 17q gain was the most common ab- 17p, which contains the p53 tumor suppressor gene, and normality after TFE3 translocations and because patients as p53 loss is commonly associated with a poor prognosis with 17q had a poorer outcome than did patients without in various malignancies, we assessed for the presence of 17q, we investigated the expression pattern on chromo- TP53 mutations. Sanger sequencing was thus conducted some 17. To identify chromosomal regions differentially on 12 tRCC including 7 cases bearing gain of 17q, but no expressed in cases with partial 17q gain and those without, mutation of TP53 was identified in any of those cases (not we conducted global gene expression analysis using RNA- shown). Interestingly, Ingenuity Pathway Analysis iden- seq in 4 Xp11 cases for which RNA was available. Two cases tified TP53 inactivation as the top transcription regulator

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Figure 4. Partial 17q gain and gene expression. GSEA of genes expressed differently between selected cases with partial 17q gain and without was conducted by RNA-seq. A, signiﬁcant correlation was 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 lines with mutated TP53 as compared with those classiﬁed as normal. C, Ingenuity Pathway Analysis showing the network of genes consistent with TP53 inactivation. Overall, there were 51 of 89 genes that have expression direction consistent with inhibition of TP53.

factorpredictedtobeinactivated in the 2 cases (RCC-T34 BioCarta pathways showed enrichment of the CTLA-4 path- and RCC-T1) with concomitant partial 17q gain as com- way (FDR < 0.001; P < 0.001), and the KEGG pathway pared with the 2 cases (RCC-T31 and RCC-T32) with showed enrichment of the T-cell receptor signaling pathway balanced karyotype (Supplementary Table S3). Overall, (FDR ¼ 0.01; P < 0.001). Those results were also obtained there were 51 of 89 genes whose expression direction was using Ingenuity Pathway Analysis, which showed activation consistent with inhibition of TP53 (z-score ¼3.84; P ¼ of inducible costimulator–inducible costimulator ligand 5.37 10 11;Fig.4C). signaling in T-helper cells (P ¼ 2.4E4) and activation of Also consistent with TP53 inactivation, GSEA showed the CTLA-4 signaling pathway (P ¼ 1.6E3). Of note, 17 of enrichment for multiple pathways related to mitosis as 95 genes (17.9%) related to the CTLA-4 pathway were follows: the M-phase of the mitotic cycle (FDR ¼ 0.10; upregulated in patients with 17q gain (Supplementary Fig. P < 0.001), mitosis (FDR ¼ 0.10; P < 0.001), and the M- S3B). Immunohistochemistry for CD3 and CD5 T-cell phase (FDR ¼ 0.10; P < 0.001), in keeping with the aggres- markers showed increase of T lymphocytes in the 2 cases sive phenotype of cases with partial 17q gain (Supplemen- with 17q gain (10%–20%) as compared with less than 1% tary Fig. S3A). to 2% in the 2 cases without 17q gain (not shown). KEGG (Kyoto Encyclopedia of Genes and Genomes) and BioCarta (BioCarta LLC) pathways (C2 gene set) using LINE-1 methylation is lower in adults than in young GSEA and Ingenuity Pathway Analysis (Ingenuity Systems, patients (<18 years old) Inc.) showed consistent results with T-cell activation in the We conducted LINE-1 methylation analyses in a total of subgroup of patients with partial 17q gain. In particular, 27 tRCC cases (12 of which had SNP array results). LINE-1

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subtypes, such as ccRCC (e.g., 3p loss) and pRCC (e.g.,

A 85 Spearman R = –0.6 trisomy 7 and/or 17; refs. 15, 30), little is known about P = 0.04 chromosome copy number alterations in tRCC. Here, we 80 show that a large proportion of tRCCs present with chromosome copy number changes similar to those observed in 75 ccRCC or pRCC, but almost 30% of them display cytoge- 70 nomic profiles that cannot be related to these or characterized RCC subtypes. 65 Morphologic diagnosis of tRCC is challenging, especially Line-1 methylation (%) Line-1 methylation 60 in adult patients. Sukov and colleagues showed that tRCC 051015 20 25 tumors have significant histologic variability (31), and Number of gain/loss Klatte and colleagues showed that only 2 of 75 cases with microscopic features suggestive of Xp11.2 translocation B 100 ns * RCC or occurring in patients 40 years or younger have 80 identifiable translocations with FISH and/or RT-PCR (32). Historically, the translocation involving TFE3 (or 60 TFEB) is considered as the defining feature for this disease. Mechanistic studies have shown the transforming effect of 40 the TFE3 and TFEB translocations in cell lines (33, 34).

Methylation (%) Methylation 20 Mathur and Samuels conducted knockdown of PSF-TFE3 in UOK-145 cell line and showed that it led to impaired 0 Normal Tumor (<18 y) Tumor (≥18 y) growth, proliferation, invasion potential, and long-term survival (33). The same authors also showed that the expression of PSF-TFE3 in normal proximal tubular cells Figure 5. Genomic aberrations and LINE-1 methylation. A, Spearman leads to initiation and maintenance of an oncogenic phe- correlation between the number of chromosomal arms with gain or loss in notype (33). Moreover, in the UOK109 cell line (NONO- tRCC samples and LINE-1 methylation (n ¼ 12). B, LINE-1 methylation TFE3 translocation), TFE3-directed short hairpin RNA level is lower in tumors of adult as compared with young patients. ns, not significant. , P ¼ 0.02. (shRNA) prevented all colony growth (35). The identification of "ccRCC-like" and "pRCC-like" cytogenomic profiles methylation levels correlated inversely with the number of in tRCC cases raises the question of whether tumors har- genetic abnormalities in cases with both SNP-array and boring such profiles are in reality ccRCC and pRCCs that TFE3 LINE-1 methylation data (Spearman r ¼0.6; P ¼ 0.04; Fig. have acquired a translocation during tumor progres- TFE3 5A). Consistent with the greater number of genetic abnor- sion or whether tRCC tumors (initiated by a translo- malities in adult patients, in the 27-patient cohort, adults cation) can then develop different types of chromosomal showed lower LINE-1 methylation, compared with younger instability leading to different cytogenomic profiles. Are 3p patients (71.1% vs. 76.7%; P ¼ 0.02; Fig. 5B). Methylation loss and gain of 7/17 important for the initiation of tRCC levels of normal kidney tissue showed little variability with tumors or are these changes acquired during tumor an average methylation of 73.07% and SD of 0.50, and no progression? Are tumors in these tRCC subgroups, carcino- association with age or gender. mas that initiated as ccRCC and pRCC, and then acquired TFE3/TFEB translocations as they progressed? Or are these tumors initiated by a TFE3/TFEB translocation, which Discussion acquired patterns of chromosomal imbalances similar to To our knowledge, this is the first study to describe those observed in ccRCC and pRCC? The 3p loss is a cytogenomic aberrations and DNA methylation status in required event in ccRCC, as it leads to loss of the wild-type a cohort of pediatric, adolescent, and adult patients with VHL allele and abrogation of VHL hypoxia-regulating activ- translocation RCC. Our study shows cytogenomic and ity. VHL mutations do not seem to play a role in tRCC, as epigenetic heterogeneity of tRCC and unexpectedly reveals VHL mutations were not identified in our cohort. However, that these tumors share genomic alterations that are com- other tumor suppressor genes with roles in RCC biology mon to ccRCCs and pRCCs. Our results show that tRCC have been recently identified in 3p (PBRM1, SETD2, and tumors from young patients (<18 years) display fewer BAP1), and thus 3p loss in tRCC tumors might be associated genetic alterations and higher levels of global DNA meth- with inactivation of one or more of these genes (36–38). ylation, compared with tumors from adult patients (18 Alternatively, tumor initiation by the TFE3/TFEB transloca- years). Moreover, our data show that a partial 17q gain is tion might trigger chromosomal instability pathways that frequent in tumors from adult patients, particularly in men, resemble those in ccRCC and pRCC subtypes. Future studies and this genetic lesion is associated with poor outcome. are needed to clarify whether the presence of these translo- Although the identification of significant correlations cations is enough to determine a tRCC diagnosis and if between cytogenetic alterations and histopathologic sub- tumor behavior (and response to therapy) depends on the types of RCC is well established for common renal cancer overall genomic profile. Interestingly, patients with tRCC

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with 3p loss (ccRCC-like profile) had worse outcomes have a different prognosis than adults with the 17q gain compared with those without 3p loss. In contrast, 9p loss remains unknown. A larger cohort is needed to answer this in our study was not associated with a poor prognosis, as it is question. Nonetheless, our results strongly suggest that 17q in early-stage ccRCC (32). gain is more frequent in adult tRCCs and our preliminary Another interesting finding of our study is the greater GSEA analysis suggest a dosage effect of genes on 17q and number of genetic abnormalities and the lower level of p53 haploinsufficiency. It is important to note that CML global methylation found in tRCC tumors from adult that bear an i(17q) have a poor prognosis (44), and medul- patients, compared with tumors from young patients. These loblastomas with an i(17q) or a 17q gain also have a poor data are concordant with the significant inverse correlation outcome (43). In our study, GSEA analysis of a subset of between the degree of genomic alterations and the LINE-1 Xp11 cases with and without partial 17q gain found enrich- methylation levels reported in other tumor types (39). ment of pathways involved in mitosis and the cell cycle in Although the difference in methylation levels between adult patients with 17q gain, suggesting a proliferative advantage and young patients is small, it achieved statistical signifi- in tumors harboring this chromosomal abnormality. It is cance due to the robustness of the assay used. This is shown notable that those cases also had gene expression patterns by the fact that methylation levels of normal kidneys consistent with activation of T-helper cells and the CTLA-4 showed little variability with an average methylation of signaling pathway. Further studies are required to confirm 73.07% (0.50%). Using this assay, no significant associa- these findings. tions were previously found between LINE-1 methylation Abnormalities involving 17q have been previously and age, gender, or diet in colonic tissues (19); thus, we described in tRCC but to date 17q gain had not been believe that the observed differences are related to intrinsic recognized as a recurrent abnormality in these tumors, nor characteristics of tRCC in adult and young patients, rather its possible association with outcomes. Among our 14 than to age. Additional studies with a larger group of previously reported cases (9), we found 1 case, that of a patients are necessary to confirm these findings. 2-year-old girl, who had a t(5;17)(q23;q25), in addition to a The cytogenomic differences between tumors from adult t(X;17)(p11.2,17q25) leading to the ASPSCR1-TFE3 fusion, and young patients were independent of classical clinico- consistent with multiple translocations occurring on the pathologic prognostic factors such as TNM stage and Fuhr- 17q arm. This patient had lymph node involvement and man grade. Although those results might be related to a lung metastasis at the time of her diagnosis, which is rare small number of cases, cytogenomic array analysis in 3 of in pediatric cases of tRCC (9). A recent case report of the our 4 young patients with American Joint Committee on t(X;17)(p11;q25) translocation, in a 24-year-old Japanese Cancer (AJCC) stage III–IV disease found no genetic abnor- woman (45), also showed the presence of a second trans- malities. Another age-related difference is that gains in 17q location involving 17q: t(13;17)(p11;q11). Moreover, were identified in tumors from 70% (n ¼ 7/10) of all our in the original report by Argani and colleagues that des- adult patients, whereas this abnormality was not found in cribed tRCC (13), they identified a complex nonreciprocal younger patients. Patients with 17q gain were more fre- t(X;17)(p11.2;q25), involving not only the translocation quently men, displayed more genetic alterations, and had of Xp11 to 17q25 but also of 17q25 to another chromo- worse outcomes. Five of those patients had a partial gain of some. Our results and these reports in the literature 17q, whereas 2 had trisomy 17. Of interest, 17q gains are suggest an important role of 17q in the biology of a subset rare in ccRCC and pRCC, in contrast to trisomy 7 and 17, of tRCC tumors. which are very frequent in pRCC (15). On the basis of our Although we did not study whether there is intratu- study of patient-derived cell lines, 17q gains seem to arise moral heterogeneity with respect to the presence of 17q from i(17q) and/or unbalanced translocations and com- and other chromosomal abnormalities identified in this plex rearrangements. Together with G-banding and SNP study, we have previously shown that intratumoral het- results available for three cell lines (HCR-59, MDA-92, and erogeneity at the level of chromosomal abnormalities is UOK146), our data showed that 2 cases had multiple seen in only up to 20% of RCC cases (46), in contrast with translocations involving 17q, leading to 17q gain concom- the level of mutational heterogeneity reported by Gerlin- itant with i(17q) formation in 1 case, and 1 case had only an ger and colleagues (47). However, intratumoral hetero- i(17q) without other associated translocations. Two cases geneity studies might be helpful to understand whether had breakpoints in 17p11.2 and were thus considered 17q is an initiating event or acquired during tumor isodicentric chromosomes. Of note, 17p11.2 has been progression, as cytogenomic heterogeneity in our prior reported to contain a cluster region characterized by large studies is restricted to abnormalities acquired during palindromic and low copy-repeat sequences in chronic tumor progression (46). myelogenous leukemia (CML), medulloblastoma with In conclusion, our data show that tRCC is genetically idic(17)(p11), and high hyperdiploid childhood acute heterogeneous and share cytogenomic profiles with other lymphoblastic leukemia (40–43). This complex architec- renal cell tumors, raising important questions about the ture suggests that i(17q) does not occur randomly, but role for TFE3/TFEB translocations in tumor initiation and/ rather because of susceptibility in the genomic structure. or progression. Furthermore, we have shown that cytoge- However, the reason why 17q is associated with age remains nomic and methylation differences exist between tRCC unclear; and whether adults with tRCC without a 17q gain tumors arising in adult and pediatric/adolescent patients.

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These data may be used to develop prognostic biomarkers in Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): G.G. Malouf, M. Picken, N.M. tRCC patients. Tannir Study supervision: G.G. Malouf, F.A. Monzon, B. Escudier, M. Picken, C.G. Wood, N.M. Tannir Disclosure of Potential Conflicts of Interest F.A. Monzon has honoraria from Speakers Bureau of Affymetrix. No Acknowledgments potential conflicts of interest were disclosed by the other authors. The authors thank Dr. W. Marston Linehan, the UOB Tumor Cell Line Repository, National Cancer Institute (Bethesda, MD), for providing the UOK146 and UOK109 cell lines. The authors also thank Ms. Karla Alvarez of Authors' Contributions The Methodist Hospital Research Institute for generating the SNP array Conception and design: G.G. Malouf, F.A. Monzon, B. Escudier, C.G. cytogenomic profiles. Wood, N.M. Tannir Development of methodology: G.G. Malouf, F.A. Monzon, B. Escudier, M. Garcia, N.M. Tannir Grant Support Acquisition of data (provided animals, acquired and managed patients, This study was supported in part by the NIH through MD Anderson’s provided facilities, etc.): G.G. Malouf, F.A. Monzon, B. Escudier, P. Cancer Center Support Grant, 5 P30 CA016672, and in part by the Nelia and Camparo, P. Tamboli, D. Lopez-Terrada, M. Picken, M. Garcia, C.G. Wood, Amadeo Barletta Foundation (to G.G. Malouf). N.M. Tannir The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Analysis and interpretation of data (e.g., statistical analysis, biosta- advertisement tistics, computational analysis): G.G. Malouf, F.A. Monzon, V. Molinie, P. in accordance with 18 U.S.C. Section 1734 solely to indicate Camparo, X. Su, H. Yao, M. Garcia, N.M. Tannir this fact. Writing, review, and/or revision of the manuscript: G.G. Malouf, F.A. Monzon, J. Couturier, V. Molinie, P. Tamboli, M. Garcia, A.S. Multani, S. Received December 17, 2012; revised June 10, 2013; accepted June 12, Pathak, C.G. Wood, N.M. Tannir 2013; published OnlineFirst July 1, 2013.

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Gabriel G. Malouf, Federico A. Monzon, Jérôme Couturier, et al.

Clin Cancer Res Published OnlineFirst July 1, 2013.

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