Whole-genome sequencing identifies genomic heterogeneity at a nucleotide and chromosomal level in bladder cancer Carl D. Morrisona,1,2, Pengyuan Liub,1, Anna Woloszynska-Readc, Jianmin Zhangd, Wei Luoc, Maochun Qine, Wiam Bsharaf, Jeffrey M. Conroya, Linda Sabatinif, Peter Vedellb, Donghai Xiongb, Song Liue, Jianmin Wange, He Shend, Yinwei Lid, Angela R. Omilianf, Annette Hillf, Karen Headf, Khurshid Gurug, Dimiter Kunnevh, Robert Leache, Kevin H. Enge, Christopher Darlaka, Christopher Hoeflicha, Srividya Veerankia, Sean Glennd, Ming Youb, Steven C. Pruitth, Candace S. Johnsonc, and Donald L. Trumpi aCenter for Personalized Medicine and Departments of cPharmacology and Therapeutics, dCancer Genetics, eBiostatistics and Bioinformatics, fPathology, gUrology, hMolecular and Cellular Biology, and iMedicine, Roswell Park Cancer Institute, Buffalo, NY 14263; and bDepartment of Physiology and the Cancer Center, Medical College of Wisconsin, Milwaukee, WI 53226 Edited* by Carlo M. Croce, The Ohio State University, Columbus, OH, and approved January 2, 2014 (received for review July 22, 2013) Using complete genome analysis, we sequenced five bladder tumors earlier studies in melanoma (13) and medulloblastoma (14), ev- accrued from patients with muscle-invasive transitional cell carcinoma idence of the association of TP53 mutations with specific copy of the urinary bladder (TCC-UB) and identified a spectrum of genomic number alterations, referred to as chromothripsis, was noted. The aberrations. In three tumors, complex genotype changes were noted. study in medulloblastoma (14) was particularly intriguing in that All three had tumor protein p53 mutations and a relatively large identification of a molecular subclass with TP53 mutations was number of single-nucleotide variants (SNVs; average of 11.2 per associated with chromothripsis and a more aggressive clinical megabase), structural variants (SVs; average of 46), or both. This group outcome was noted. Chromothripsis, or the shattering of two or was best characterized by chromothripsis and the presence of more chromosomes and their reassembly into derivative chro- subclonal populations of neoplastic cells or intratumoral mutational mosomes, is different from other types of genomic instability, heterogeneity. Here, we provide evidence that the process of chromo- which tend to occur on a genome-wide basis (15, 16). Chromo- thripsis in TCC-UB is mediated by nonhomologous end-joining using thripsis is different in that it includes one to three alternating kilobase, rather than megabase, fragments of DNA, which we refer to copy number states across the derivative chromosome, an asso- “ ” as stitchers, to repair this process. We postulate that a potential uni- ciation with changes in heterozygosity, and numerous genomic fying theme among tumors with the more complex genotype group is – rearrangements in localized chromosomal regions likely occurring a defective replication licensing complex. A second group (two bladder in condensed chromosomes (15). There is evidence to suggest tumors) had no chromothripsis, and a simpler genotype, WT tumor that the primary mechanism of reassembly of the derivative protein p53, had relatively few SNVs (average of 5.9 per megabase) chromosome in chromothripsis is nonhomologous end-joining and only a single SV. There was no evidence of a subclonal population (NHEJ) (14). With the advent of next-generation sequencing of neoplastic cells. In this group, we used a preclinical model of bladder (NGS) allowing for detailed genomic analysis, chromothripsis carcinoma cell lines to study a unique SV (translocation and amplifica- tion) of the gene glutamate receptor ionotropic N-methyl D-aspertate as a potential new therapeutic target in bladder cancer. Significance next-generation sequencing | tumor heterogeneity | GRIN2A | replication Genetic alterations are frequently observed in bladder cancer. In this study, we demonstrate that bladder tumors can be ransitional cell carcinoma arising in the urinary bladder (TCC- classified into two different types based on the spectrum of TUB) is a frequent cause of morbidity and mortality, and among genetic diversity they confer. In one class of tumors, we ob- patients in the United States, it is one of the most costly cancers to served tumor protein p53 mutations and a large number of treat (1, 2). The traditional somatic genetic basis of TCC-UB is single-nucleotide and structural variants. Another characteristic a distinct division of low-grade papillary tumors from high-grade of this group was chromosome shattering, known as chromo- invasive tumors. Low-grade papillary superficial tumors are gener- thripsis, and mutational heterogeneity. The other two bladder ally characterized by constitutive activation of the receptor tyrosine tumors did not show these profound genetic aberrations, but kinase–Ras pathway, and they have activating mutations in the we found a novel translocation and amplification of the gene , HRAS and fibroblast growth factor receptor 3 (FGFR3) genes glutamate receptor ionotropic N-methyl D-aspertate a poten- (3–6). In contrast, high-grade invasive TCC-UB is characterized tially druggable target. Advancements in bladder cancer by alterations in the tumor protein p53 (TP53) and retino- treatment have been slow. Understanding the genetic land- blastoma 1 (RB1) pathways. These genes normally regulate the scape of bladder cancer may therefore help to identify new cell cycle by interacting with the Ras–mitogen-activated protein therapeutic targets and bolster management of this disease. kinase signal transduction pathway (7, 8). Both low-grade papillary Author contributions: C.D.M., W.B., K.G., M.Y., C.S.J., and D.L.T. designed research; C.D.M., and high-grade invasive tumors frequently have loss of chromosome J.Z., W.L., J.M.C., L.S., A.R.O., A.H., and K.H. performed research; M.Q., C.D., C.H., and 9. This loss presumably inactivates the p16 geneandisanearly S.V. contributed new reagents/analytic tools; C.D.M., P.L., J.Z., M.Q., J.M.C., P.V., D.X., event in the initiation of TCC-UB (9, 10) S.L., J.W., H.S., Y.L., D.K., R.L., K.H.E., C.D., C.H., S.V., and S.G. analyzed data; and C.D.M., Although TP53, cyclin-dependent kinase inhibitor 2A (p16), RB1, P.L., A.W.-R., J.Z., J.M.C., S.C.P., C.S.J., and D.L.T. wrote the paper. HRAS,andFGFR3 abnormalities have been well described in TCC- The authors declare no conflict of interest. UB, there are limited data on the more complete genomic analysis *This Direct Submission article had a prearranged editor. of TCC-UB (11). A recent study focusing on genome-wide copy 1C.D.M. and P.L. contributed equally to this work. number analysis showed extensive heterogeneity across all sub- 2To whom correspondence should be addressed. E-mail: [email protected]. types of TCC-UB to such an extent that precise molecular This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. groupings were difficult to define (12). In this study, similar to 1073/pnas.1313580111/-/DCSupplemental. E672–E681 | PNAS | Published online January 27, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1313580111 Downloaded by guest on September 27, 2021 has been identified more frequently (17). Additionally, the 100 PNAS PLUS presence of these complex genomic events and their potential 90 SNV/MbNV/Mb Structural Variants TP53mut/p16wt association with TP53 mutations may contribute to a better un- 80 derstanding of cancer, including TCC-UB. 70 Increasing genomic complexity NGS technologies provide other evidence of complex genomic 60 TP53mut/p16del heterogeneity, such as the recent identification of subclonal pop- 50 ulations of cells with mutations distinct from the dominant clonal 40 population of cells within one tumor or between a primary, re- 30 – TP53mut/p16del current, or metastatic tumor from one patient (18 20). Importantly, 20 TP53wt/p16wt TP53wt/p16del a recent study in chronic lymphocytic leukemia (CLL) (21) showed 10 how selective pressures on cancer cells, such as chemotherapy, se- 0 lect for these subclonal populations to become the dominant 16933 17802 18698 18195 19685 clone contributing to genomic heterogeneity. It is not yet certain TP53 mutant whether broad measurements of genomic heterogeneity will TP 53/p16 have an impact on molecular classification of cancer, but it is wild type p16 deletion likely that they will significantly contribute to biological differ- Single structural variant Frequent structural variants (SVs) ences, and therefore have an impact on patient outcomes. (SV) Frequent single nucleotide variants To evaluate the spectrum of genomic heterogeneity in TCC- Fewer single nucleotide (SNVs) UB, we performed complete genome sequencing of five high- variants (SNVs) TP53 mutant SI TP53 wild type Chromothripsis involving multiple grade muscle-invasive tumors and matching germ-line blood ( Chromothripsis absent chromosomes Appendix, Table S1), and validated a subset of our findings in No evidence of subclonal Subclonal populations identified in 2 more than 300 bladder cancer specimens. Our overall results populations of 3 showed a great deal of genomic heterogeneity at either extreme of a spectrum of genomic complexity. Fig. 1. Number of SNVs per megabase (Mb) of DNA and total number of validated SVs for each of five patients with muscle-invasive TCC-UB used for Results whole-genome sequencing. Three of the five tumors (cases 18195, 18698, and 19685) had many more SVs and SNVs than the other two tumors and Overview of Somatic Alterations Reveals Heterogeneity