Abstract A52: Gene alterations associated with clinical characteristics of bladder cancer
Authors: Nickerson Mike , Im Kate M. , Guo Guangwu , Gui Yaoting , Turan Sevilay , Costello James C. , Zhou Quan , Cai Zhiming , Wu Song , Lucia M. Scott , Moore Lee E. , Dean Michael , Theodorescu Dan 
Published: May 2016

Next-generation sequencing (NGS) of urologic tumor genomes has identified frequently altered cancer genes. We recently examined the exomes of bladder tumors (BCa) from 54 U.S. patients and 99 Chinese patients to identify novel, altered cancer genes associated with clinical characteristics of disease.

Exome capture of DNA from tumor and normal urothelial tissue using probes for ~180,000 protein-coding exons and microRNA loci; DNA fragment libraries were sequenced on a HiSeq 2000 platform (Illumina); and base calls on paired-end, 100 bp reads were generated using the Genome Analyzer Pipeline, v. 1.3 and standard parameters. Gene expression was analyzed using quantitative PCR, telomere length by a standard assay, and KDM6A function using cell-based assays in T24T BCa cells. Copy number variation (CNV), aneuploidy, and CpG methylation were analyzed as recently described.

Telomerase (TERT) was the most frequently altered by somatic sequence changes in 37/54 (69%) of U.S. patient tumors. The TERT promoter was altered by frequent germline nucleotide changes in 30/54 (56%) tumors. Most of the TERT germline variants were novel (19/20) and three variants were confirmed in distinct tumors as either somatic or germline, including the most frequent variant, c. –245 T&gt;C (rs2853669). The somatic TERT promoter alterations did not correlate with other somatic BCa gene alterations, but were associated with increased TERT expression and with significantly shorter telomeres in tumors as compared to matched normal tissue. The stromal antigen 2 (STAG2) gene was the most frequently altered novel BCa gene in Chinese patients, by somatic, deleterious sequence changes in 11% of tumors; CNV deletions (5%); and promoter CpG hypermethylation leading to presumed expression silencing (7/30, 23%). STAG2 encodes a component of the cohesion complex involved in sister chromatid cohesion and segregation. STAG2 mutations were associated with an increased number of chromosomal arm copy number changes corresponding to higher tumor cell aneuploidy, and mutations were associated with a significantly reduced patient survival by Kaplan-Meier survival analysis. In total, 32/99 (32%) of tumors displayed SCCS pathway gene alterations. The most frequently altered epigenetic gene was the histone lysine-specific demethylase 6A (KDM6A/UTX) in 24% of U.S. and 30% of Chinese patient tumors. Experimentally, we show KDM6A loss in human BCa T24T cells enhanced in vitro proliferation and cell migration, and in vivo tumor growth in mice. Re-expression reversed these effects, confirming KDM6A loss drives the BCa phenotype. Somatic KDM6A alterations were associated with somatic BRCA1-associated protein-1 (BAP1) alterations observed in 15% of U.S. tumors. Somatic, deleterious sequence changes occurred preferentially in U.S. Caucasian as compared to Chinese BCa patient tumors; are associated with papillary histologic features in a subset of tumors; and contribute to a significant number of tumors (10/14, 71%) that displayed alteration of genes encoding BRCA DNA repair pathway proteins. Genes encoding BRCA pathway proteins, including BAP1, BRCA1, BRCA2, PALB2, and ATM were altered by a combination of somatic and rare germline variants.

We identified altered genes associated with clinical characteristics of BCa, including ‘immortalization' (TERT), aneuploidy (STAG2), proliferation (KDM6A), and papillary features in tumors (BAP1). STAG2 alterations are associated with a reduced patient survival, indicating a lethal subtype of disease; and altered X chromosome genes (STAG2 and KDM6A) are single copy in males and likely contribute to the cancer gender bias. Finally, we identified BRCA pathway alterations in BCa that are similar to those in breast, ovarian, prostate and kidney tumors, that have a common DNA repair deficiency that can be exploited by therapy targeting poly (ADP ribose) polymerase.

Citation Format: Mike Nickerson, Kate M. Im, Guangwu Guo, Yaoting Gui, Sevilay Turan, James C. Costello, Quan Zhou, Zhiming Cai, Song Wu, M. Scott Lucia, Lee E. Moore, Michael Dean, Dan Theodorescu. Gene alterations associated with clinical characteristics of bladder cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A52.