Abstract 2980: Integrated transcriptome and methylome sequencing of Xp11 translocation renal cell carcinomas reveal uncoupling of DNA methylation and transcriptional profiles.
Authors: Malouf Gabriel G. , Su Xiaoping , Yao Hui , Wood Christopher G. , Lu Yue , Liang Shoudan , Thompson Erika J. , Doss Denaha J. , Jelinek Jaroslav , Issa Jean-pierre , Tannir Nizar M. 
Published: August 2014

Abstract
Background: Xp11 translocation renal cell carcinoma (RCC) is a rare subtype of RCC characterized by the fusion of TFE3 gene to multiple partners. Despite its genetic homogeneity, there is considerable heterogeneity in the clinical course of this disease, probably due to epigenetic events that follow the initial translocation.
Material and methods: We performed whole-transcriptome next-gen paired end sequencing of 7 Xp11 RCC samples, 10 clear-cell RCC and 19 papillary RCC. In addition, we performed genome-wide analysis of DNA methylation of 9 Xp11 samples by Digital Restriction Enzyme analysis of Methylation (DREAM), based on next-generation sequencing.
Results: Using a robust bioinformatics pipelines, we confirmed 6 previously known TFE3 translocation partners, and discovered a novel partner involving the splicing gene LUC7L3 that we validated by RT-PCR. Unsupervised clustering of gene expression profiling revealed genetic homogeneity of Xp11 cases that clustered together, independently from clear-cell and papillary RCC subgroups. Ingenuity analysis confirmed MITF activation. Gene Set Enrichment Analysis (GSEA) showed mTOR pathway activation. In contrast with genetic homogeneity of gene expression, unsupervised clustering analysis of CpG sites located within CpG islands (CGI) revealed: (1) a CpG islands methylator phenotype (CIMP+) subgroup related to patients with metastatic disease (n=3), and (2) a CIMP negative (CIMP-) subgroup related to patients with local disease (n=6). Integrative analysis showed that genes with differentially methylated levels between CIMP+ and CIMP- tumors (SD: +/-20%) and which were down-regulated in CIMP+ subtype as compared to normal kidney, were also down-regulated in CIMP- subgroup . Of note, FZD10 and CCDC8 were found to gain promoter methylation in three cases of Xp11 RCC, but their baseline expression was already low in normal kidney. In contrast, small gain of promoter DNA methylation in cancer samples was associated with gene silencing of putative tumor suppressor genes. Interestingly, GSEA for gene body methylation identified enrichment of MAPK signaling pathway in CIMP+ versus CIMP- cases. Exome-sequencing of 2 out of 3 cases with CIMP+ phenotype identified non-synonymous IDH1 (p.V71I) mutation present in COSMIC database for one case and a frameshift mutation of the chromatin remodeling enzyme INO80D in the second case.
Conclusion: Our data reveal uncoupling of DNA methylation and gene expression profiling in Xp11 RCC. Patients with metastatic disease showed higher methylation level, with CIMP+ phenotype possibly related to IDH1 mutation in one case. These results suggest that additional epigenetic alterations following the initial Xp11 translocation may explain the aggressive biology of some Xp11 RCCs.
Citation Format: Gabriel G. Malouf, Xiaoping Su, Hui Yao, Christopher G. Wood, Yue Lu, Shoudan Liang, Erika J. Thompson, Denaha J. Doss, Jaroslav Jelinek, Jean-pierre Issa, Nizar M. Tannir. Integrated transcriptome and methylome sequencing of Xp11 translocation renal cell carcinomas reveal uncoupling of DNA methylation and transcriptional profiles. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2980. doi:10.1158/1538-7445.AM2013-2980