Whole-Genome Sequencing Identifies Genomic Heterogeneity at a Nucleotide and Chromosomal Level in Bladder Cancer

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 amplification) 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 Between were also TP53mut. Two of the five tumors (cases 16933 and 17802) had very Patients. At one end of the spectrum was a more complex geno- few SVs and SNVs, and were also TP53wt. Patient 17802, although having type, characterized by frequent single-nucleotide variants (SNVs) only one SV, shared in common with the TP53mut group a p16 (CDKN2A) mut and structural variants (SVs), TP53 mutation (TP53 ), CDNK2A DEL. Patient 16933 was negative for p16del and TP53wt status, had no del (p16) deletion (p16 ), frequent mutations in known cancer- mutations in any known cancer-related genes in the Cancer Gene Census, related genes, SV breakpoints that often precisely align with and had a single distinct SV represented by a CTX between the SCN8A gene segmental copy number states indicating chromothripsis, and at 12q14 and the GRIN2A gene at 16p13.2. evidence of subclonal intratumoral heterogeneity (Fig. 1). We found (i) evidence that SV breakpoints can have a unique association with copy number in the context of chromothripsis tumors (cases 18693, 18195, 19685, and 17802) contained mutations that may be related to a process of genomic amplification, (ii) in one or more chromatin remodeling genes, including mutations in complex genomic rearrangements mechanistically use kilobase NSD1, PBRM1, KDM6A, ARID2, APC,andEP300, which were fragments of DNA that we refer to as “stitchers” as part of an identified in all of these tumors, consistent with prior exomic se- NHEJ DNA repair process, and (iii) some cases of TCC-UB do quencing in this tumor type (22). In this same group of four tumors, show intratumoral mutational heterogeneity. At the other end of there were 11 mutated genes present in two or more samples (SI the spectrum, was a simpler genotype, with few SNVs and SVs, Appendix,TableS7), including TP53, CTBP2, ZFHX4, XIRP2, infrequent mutations in any known cancer-related gene in the WDR89, PCMTD1, PABPC3, MCM4, GXYLT1, CDCA7L,and Cancer Gene Census, TP53 WT (TP53wt), p16 WT (p16wt), and no CC2D1A. Genes with a nonsynonymous mutation and coding re- evidence of chromothripsis. In this group, we provide (i)anun- gion deletion (DEL) in one or more samples included ANKRD11 equivocal demonstration that amplified interchromosomal trans- and CC2D1A. In both instances, the mutation and DEL occurred in locations (CTXs) can be found in bladder carcinoma and (ii) the same case. Neither of these two genes has been identified evidence of rare events of translocation and, more frequently, previously as mutated in TCC-UB (22), and reports of mutations in GRIN2A amplification of in a subset of TCC-UB representing other tumor types have been reported only rarely in the Cosmic a potential therapeutic target. Mutation Database (www.sanger.ac.uk/genetics/CGP/cosmic/).

Somatic Mutation Analysis Identifies Intertumoral Genomic Heterogeneity MCM4 and Replication–Licensing Complex Defects. With the excep- attheNucleotideLevel.We obtained 44.8-fold mean sequence cov- tion of TP53, none of the above-listed genes was previously erage for each tumor and 39.5-fold mean sequence coverage for the reported as mutated in an Asian cohort of 97 patients using matching normal tissues (SI Appendix,TableS2). To identify so- whole-exome sequencing (22). Among these 97 patients, MCM3 matic events, we compared the sequencing data of each tumor with was mutated in one tumor. MCM3 is part of a six-gene MCM2–7 that of matched blood using multiple algorithms and filtered the list replication–licensing complex that binds chromatin during the by reference to the Single Nucleotide Polymorphism database (dbSNP) build 130 and 1000 Genomes Project (SI Appendix, G1-phase of the cell cycle and is required for initiation of DNA SI Methods and Tables S3–S6). There was a wide variation in replication in the subsequent S-phase. In our study, two tumors MEDICAL SCIENCES MCM4 the number of somatic mutations per tumor, ranging from 14,256 (cases 18195 and 19685) both showed mutations in the – in case 16933 to 49,889 in case 19685 (average of 29,326). Tumors gene of this replication licensing complex, which were validated mut from two patients (cases 16933 and 17802) had fewer somatic by Sanger sequencing. Both of these tumors were TP53 , had mutations (Fig. 1), including both SNVs and SVs, and were the largest number of SNVs and SVs, showed chromothripsis TP53wt. The tumors of three patients (cases 18698, 18195, and involving multiple chromosomes, and demonstrated intratumor 19685) were TP53mut and contained a much larger number of genomic heterogeneity. As we postulate in our discussion, all of somatic mutations, providing additional intertumoral genomic these events may have a single underlying association through heterogeneity at the nucleotide level. Not unexpectedly, four or five a replication–licensing complex defect (see Fig. 7).

Morrison et al. PNAS | Published online January 27, 2014 | E673 Downloaded by guest on September 27, 2021 Mutated Subclones Contribute to Intratumoral Genomic Heterogeneity subclonal populations of neoplastic cells portend a worse prog- at the Nucleotide Level. Clusters of mutations with dissimilar variant nosis, as has been previously described in leukemia (21). allele frequencies in an individual case provide evidence of intratumoral heterogeneity and support the existence of mul- SV Analysis Identifies Genomic Heterogeneity at the Chromosomal tiple clones of neoplastic cells with different genotypes within Level. To detect chromosomal rearrangements, we searched for one tumor (20, 21, 23–26). By itself, the presence of multiple fragments in which the sequence from the paired-end read mapped discordantly to the reference genome and further re- neoplastic clones within a single tumor implies a more complex SI Appendix SI Methods genome, and recent evidence supports an evolution toward a more fined them by de novo assembly ( , ). A total of 263 putative somatic rearrangements were predicted. To aggressive phenotype (26). To assess intratumoral clonality, the assess the accuracy of these predictions, PCR was performed frequency data of tumor variant alleles for all identified somatic “ ” across the putative breakpoints for both the tumor and germ-line mutations were input into an R function density to estimate the DNA. We confirmed, by either PCR or FISH, 150 (57%) of empirical probable density function of allele frequencies (19) (Fig. these predicted SVs as true somatic rearrangements (SI Appen- 2). Estimates of clonality were determined using a kernel density dix, Table S8), 6 (2%) as germ-line, and 59 (22%) as false, and analysis of tumor variant allele frequency, which was performed the remaining 48 (19%) failed to produce a PCR in either the separately for each tumor. The two bladder tumors with the sim- tumor or germ-line DNA. The number of somatic SVs per tumor pler genotype (cases 16933 and 17802) showed a Gaussian distri- varied greatly (range: 1–79) with a median of 20 (average of 30) bution of variant frequencies without evidence of subclonal per tumor (SI Appendix, Fig. S1 and Table S9). Tumors with populations of neoplastic cells. In contrast, cases 18195 and 19685, a higher number of SNVs also showed more SVs (Fig. 1). DELs with a more complex genotype, showed an obvious skew of the were the most common SV identified [58 (39%) of 150], fol- normal distribution. The kernel density analysis plot for case 18195 lowed by inversions [INVs; 56 (37%) of 150] and CTXs [34 shows at least two neoplastic clones centered on variant allelic (23%) of 150] (SI Appendix, Fig. S2 and Table S10). Intra- frequencies of 20% and 40%. Case 19685 showed a similar skewed chromosomal translocations [2 (1%) of 150] were infrequent distribution but without obvious peaks of variant allelic frequen- and only identified in two of the five tumors. Interestingly, both cies, perhaps reflecting the resolution of coverage with whole- tumors with the simpler genotype (cases 16933 and 17802) had p16del genome sequencing (Fig. 2). The last case, 18698, although TP53mt only a single SV; in case 17802, the SV was a . The re- and having a relatively large number of mutations, did not show maining tumors (cases 18698, 18195, and 19685) had the more subclonal populations by this analysis, implying there is some complex genotype, showed an average of 49 (median of 49) SVs, and were evaluated further. continuum across these groups. Larger numbers of patients are necessary to determine if the biological importance of these Chromothripsis Contributes to Genomic Heterogeneity at the Chromosomal Level. In tumors with the more complex genotype, SVs were not evenly distributed across all chromosomes; 54% (81 of 150) involved chromosomes 4, 5, and 6 in a pattern consistent with chromothripsis (SI Appendix,Figs.S3–S6 and Table S11). The highest number of SVs was identified in chromosome 4 (total of 31), followed closely by chromosomes 5 (total of 27) and 6 (total of 23). Interestingly, when adjusted for SVs per 50 Mb of DNA (SI Appendix,Figs.S7–S11 and Table S12), chromosome 21 (8.6 SVs per 50 Mb) was the most frequently involved chromosome, followed closely by chromosomes 4 (8.3 SVs per 50 Mb), 5 (7.7 SVs per 50 Mb), and 6 (6.6 SVs per 50 Mb). Most of the SVs for chromosome 21 were the result of CTXs with chromosome 5 in a pattern of chromothripsis for case 19685. In addition, the prediction for SVs to involve chromosomes 4, 5, 6, and 21 was pri- marily associated with the occurrence of INVs and CTXs rather than DELs (SI Appendix,Figs.S8–S11). Some chromosomes, such as chromosomes 1, 3, 8, 15, 20, and 22 and chromosome X, showed very few SVs even when adjusted for size.

Chromothripsis Does Not Lead to Functionally Relevant Gene Fusions. Among the 33 CTX events in the three tumors with evidence of chromothripsis, 32 had one or both breakpoints in an intergenic region without the possibility of a gene fusion event. In a similar fashion, none of the 56 INVs resulted in a predicted functional gene fusion event. One CTX event had juxtaposed (intronic– intronic) and appropriately aligned in-frame coding regions predicted to result in a putative productive fusion protein. In this event, the first exon of CDH10 on chromosome 5, encoding a type II classical cadherin that mediates calcium-dependent Fig. 2. Kernel density analysis plots of tumor variant allele frequency (Freq) cell–cell adhesion, was predicted to join with the last three exons to assess intratumoral heterogeneity. Shown are plots for all five tumors of CAB39L, a protein that binds and activates serine/threonine representing either end of the spectrum of genomic complexity. Each plot kinase STK11. A CDH10/CAB39L translocation was validated in graphs the variant allelic frequency (x axis) vs. the density of the variant the index case (case 18195) by PCR but not by FISH. Additional allelic frequencies (y axis). Plots with a single peak (cases 16933, 17802, and 18698) represent clusters of mutations with similar variant allelic frequencies break-apart FISH studies for both CDH10 and CAB39L failed to and no evidence of subclonal populations of neoplastic cells. Plots with two show any other translocations in the validation cohort of 329 or more peaks (cases 18195 and 19685) represent tumors with subclonal bladder cancer samples. We show later that this concept of PCR- populations of neoplastic cells and a more complex genome. positive, FISH-negative translocation events is a common event

E674 | www.pnas.org/cgi/doi/10.1073/pnas.1313580111 Morrison et al. Downloaded by guest on September 27, 2021 in chromothripsis. Similar to the findings in a recent study in of DNA and our probes were purposely designed to allow for PNAS PLUS prostate cancer (27), chromothripsis, although a marker of ge- a 50- to 100-kb error in prediction of the exact breakpoint, this nomic instability, does not lead to recurrent functionally relevant would indicate that the segment of DNA involved in this fusion genes in TCC-UB. stitching process is less than this size.

SVs Associated with Chromothripsis Align with Segmental Copy NHEJ Is the Predominant Mechanism of Genomic Rearrangement in Number Changes. Although SVs in the tumors with the complex Bladder Cancer. There is evidence from earlier studies that unique genotype did not have apparent single-gene implications as short stretches of an identical sequence, or microhomology (29), driver mutations in comparison to the simple genotype, the re- located near the breakpoints of DNA double-strand breaks may − lationship of SVs to copy number changes suggests other bi- be critical in a stitching process in mouse Ltk cells (30) similar ologically relevant mechanisms. In this regard, a pattern of CTXs to that reported here. This leads to the creation of localized and INVs closely aligned with segmental copy number states was complex rearrangements. Similar evidence of the importance of characteristic of the complex genotype. To define the genomic microhomology as a general mechanistic model for chromosomal significance of these events better, we developed an enhanced rearrangements and amplification has been provided for human SV viewer similar to Circos but with a linear view and the ca- lymphoma cell lines (31, 32). In these studies, a direct relation- pability to “zoom in” for a more detailed view. This enhanced ship to TP53 mutation status was noted. In three of our tumors linear view showed frequent sharing of breakpoints for INVs and with the complex genotype, microhomology was identified in CTXs. Most interestingly, when the copy number profile across 68% (101 of 148) of the breakpoints. Remarkably, this per- these regions was added to the viewer, an alternating change of centage of microhomology was quite consistent among cases one to a few copies with transitions aligned with shared break- [case 18195, 34 (69%) of 49 breakpoints; case 18698, 15 (79%) of points for both INVs and CTXs consistent with chromothripsis 19 breakpoints; and case 9685, 52 (65%) of 80 breakpoints] and was identified (Fig. 3). Using NGS technology, chromothripsis for subtypes of SV (CTX, 64%; DEL, 75%; and INV, 67%). The has been previously reported in one case of CLL (15) and three results in these three tumors with an average of 2.2 bp of micro- cases of colorectal cancer (17), but this precise alignment of SV homology per SV were similar to those of a recent study (33) of breakpoints and copy number changes was not described. The 95 complete tumor genomes of various histological subtypes, in mechanism of reconstitution of these fragments of DNA into whichanaverageof1.7bpofmicrohomologyperSVwasidenti- a complex, highly rearranged fragment of DNA as either an in- fied. In our study, we also identified nontemplated sequences at tact, cytogenetically recognizable chromosome or double minute the rearrangement junctions in 18 of 148 SVs (SI Appendix,Table has recently been called chromoanagenesis (28) (“chromo,” S14), which, along with microhomology, is considered to be the meaning chromosome, and “anagenesis,” meaning reborn). As signature of a DNA double-strand break repair process (34). Only part of this mechanism, we provide unique sequencing information 20% of all SVs displayed neither microhomology nor nontemplated that small fragments (average size of 50–100 kb) of DNA from sequences, indicating that NHEJ was the predominant DNA dou- chromosomes other than the cytogenetically recognizable chro- ble-strand break repair process. mosome are used to “stitch” such chromosomes together (Fig. 4). This conclusion is based on consistent PCR validation at pre- Recurrent Breakpoints Are Often Amplified in Bladder Cancer. Some dicted interchromosomal breakpoints, and consistently negative of the CTXs and INVs in the group with a more complex genotype findings by FISH. Paired-spectrum orange and green break-apart were further defined by a clustering of breakpoints both within FISH probes were designed on either side of multiple chromo- and between different samples at chr4:180 Mb, chr5:29 Mb, some 4, 5, and 6 CTX and INV breakpoints with a 50- to 100-kb chr5:40 Mb, chr6:10 Mb, chr6:18 Mb, and chr6:24 Mb. These gap (SI Appendix, Table S13) for each probe set. Because the breakpoints were of interest as potential recurrent genomic sensitivity of FISH with interphase nuclei is in the range of 50 kb events in TCC-UB and were subsequently examined in a validation

Fig. 3. Utilization of SV viewer to demonstrate precise alignment of breakpoints for CTXs and INVs with the change in segmental copy number states in a tumor in case 18195. (A) SV viewer with a complete genome view highlighting chromosome 4 (chr4; blue zone) for a tumor in case 18195. CTXs and intrachromosomal translocations (ITXs) are represented as horizontal ticks for each breakpoint, with arcs representing the partner breakpoint. INVs

are represented as solid yellow bars, with each end MEDICAL SCIENCES of the bar representing the two breakpoints. DELs are represented as solid green bars. Small red squares represent tier 1 SNVs. (B) SV viewer highlighting 69 Mb of chr4 from chr4:118,403,673– 187,070,340 and illustrating six CTXs, two INVs, one DEL, and seven SNVs. (C) Copy number is illustrated and shows that all six CTX and three of four INV breakpoints precisely align with five of nine segmental copy number states.

Morrison et al. PNAS | Published online January 27, 2014 | E675 Downloaded by guest on September 27, 2021 A

Fig. 4. Mechanism of creation of complex genomic rearrangement by NHEJ using chromosome-specific stitcher DNA fragments. (A) Chromosome shattering for the 6p amplicon is shown, resulting in seven different megabase pairs in size fragments of DNA B Chr5:40,062,440 CDChr6:18,155,619-18,365,654 Chr6:23,292,635-24,467,711 with one of two segmental copy number states. NGS results predicted a CTX or INV, or both, at each of these changes in segmental copy number state. FISH at each of these breakpoints was consistently PCR- positive but negative for rearrangement. In the process of rejoining these fragments, the resulting reformed chromosome can be linear or circular, may contain inverted segments, and often shows amplification at the breakpoints. (B–D) FISH shows amplification but not translocation at chr5:40 Mb, chr6:18 Mb, and chr6:24 Mb breakpoints in the validation cohort. (Magnification: B–D, 1,000×.)

cohort of 343 patients. Using a FISH break-apart approach, we did exon 1 and GRIN2A exon 4 primer set did not delineate this fusion not identify translocations at any of these sites in the validation variant for unexplained reasons. Further analysis of this SV in cohort; however, surprisingly, amplification (Fig. 4 B–D) was a case 16933, using our enhanced linear SV viewer (Fig. 5 B and common event at chr5:40 Mb [32 (9%) of 343 patients], chr6:18 C), showed an unexpected finding. For the SCN8A breakpoint Mb [53 (16%) of 332 patients], and chr6:24 Mb [53 (16%) of 332 at 12q13, the corresponding data from Illumina SNP chips patients]. The gains at chr6:18 Mb and chr6:24 Mb were high-level (HumanOmni1-Quad_v1-0 containing 1,140,419 dbSNP) showed tandem amplifications typical of known oncogenes, such as HER-2, copy number gain centromeric to the breakpoint, corresponding to MYC, CCND1, or MDM2, whereas the gain at chr5:40 Mb was the 5′ UTR and exon 1 region of SCN8A, and a diploid state on the one of low to intermediate amplification with a copy number telomeric side. For the GRIN2A breakpoint at 16p13.2, the cor- consistently ranging from 5 to 10. FISH with additional BAC clones responding results showed copy number gain telomeric to the performed in the validation cohort across the chr18–24 Mb at 1- to breakpoint, corresponding to the CDS and 3′ UTR of GRIN2A, 2-Mb intervals (SI Appendix,TableS13) showed this was one and a diploid state on the centromeric side. Translocation with continuous amplicon. The highest level of amplification and mini- subsequent amplification of the involved genes is unique in cancer mal region of copy number gain was seen at chr6:19.8 Mb (RP11- genetics and has been identified frequently only in the COL1A1 93O13) to chr6:21.4 Mb (RP11-204E9), including the genes E2F3 and PDGFB translocations in dermatofibrosarcoma protuberans and SOX4. Previous studies in multiple tumor types (35), including (37); to the best of our knowledge, this has not been previously bladder cancer (36), have identified increased copy number in a reported in a carcinoma. 6p22 amplicon that is centered between the recurrent breakpoints with a segmental copy number change at chr6:18–24 Mb. FISH Confirms an Amplified Reciprocal SCN8A-GRIN2A Translocation. Recent release of data by the Cancer Genome Atlas Network To interrogate the chr12:52,049,200 breakpoint for SCN8A,we (www.cbioportal.org) shows this 6p22 amplicon containing the designed a break-apart FISH probe set (SI Appendix, Table S13), E2F3, SOX4, PRL,andCDKAL1 genes to be the most common with a SpectrumOrange-labeled probe (RP11-923I11, orange) amplification in TCC-UB. The amplification at chr5:40 Mb was centromeric to the breakpoint representing the translocated 5′ not studied further, but the overall evidence supports the ob- UTR and exon 1 of SCN8A and an FITC-labeled probe (RP11- servation that the large number of SVs in the complex genotype 285E4, green) telomeric to the breakpoint representing the non- is not simply a reflection of random chromosomal instability. translocated portion of this gene. Likewise, to interrogate the chromosome 16:10,035,762 breakpoint for GRIN2A, we designed SV Analysis Identifies a SCN8A-GRIN2A Translocation. As previously a break-apart FISH probe set in the reverse fashion with an FITC- discussed, the single SV in case 17802 was a DEL involving the p16 labeled probe (RP11-895K13, green) telomeric to the breakpoint gene at chromosome 9p21. The single SV identified in case 16933 representing the translocated CDS and 3′ UTR of GRIN2A and was unique in that it predicted an in-frame fusion protein involving a SpectrumOrange-labeled probe (RP11-297M9, orange) centro- the SCN8A gene at 12q13 and the GRIN2A gene at 16p13.2 (Fig. meric to the breakpoint representing the nontranslocated portion 5). This fusion variant was predicted to result in an in-frame fusion of this gene. Based on our sequencing results, we predicted evi- of the SCN8A 5′ UTR and exon 1 with the GRIN2A complete dence of orange-green break-apart at both sites with amplification coding sequence (CDS) and 3′ UTR (Fig. 5A). Subsequent ge- of the orange probe for SCN8A and amplification of the green nomic PCR and capillary sequencing of this tumor using primers probe for GRIN2A.Fig.5D shows the FISH results using the for SCN8A 5′ UTR and GRIN2A exon 1 demonstrated the pre- SCN8A break-apart probe set for case 16933, and, as anticipated, dicted fusion variant in tumor DNA and not in the corresponding multiple single orange signals with no associated green signals germ line (SI Appendix, Figs. S12 and S13). Although RT-PCR were identified. Fig. 5E shows the FISH results using the GRIN2A with a GRIN2A exon 2 and 4 primer set using case 16933 tumor break-apart probe set for case 16933 and, as anticipated, multiple cDNA demonstrated expression of CDS GRIN2A,theSCN8A green signals with no associated orange signals were identified.

E676 | www.pnas.org/cgi/doi/10.1073/pnas.1313580111 Morrison et al. Downloaded by guest on September 27, 2021 A D PNAS PLUS Chr12:52,049,200 breakpoint SCN8A

GRIN2A RP11-285E4 Chr16:10,036,019 breakpoint SCN8A chr12

Chr12:52,049,20 Chr16:10,036,019 RP11-923I11 SCN8A- 0 GRIN2A gene fusion E Translocation SCN8A 5’UTR and exon 1 Complete GRIN2A CDS and 3’UTR Copy number Chr12:51,049,200-53,049,200 CTX breakpoint

B RP11-297M9 FISH probe RP11-923I11 RP11-285E4 GRIN2A Chr16

SCN8A RP11-895K13 F C Copy number Chr16:9,035,762-11,035,762 CTX breakpoint FISH probe RP11-895K13 RP11-297M9 Chr16 SCN8A RP11-895K13

GRIN2A Chr12 GRIN2A RP11-923I11

Fig. 5. Details of the SCN8A-GRIN2A for the tumor in case 16933. (A, Upper) Illustration of the chr12:52,049,200 breakpoint between the 5′ UTR and exon 1 of SCN8A.(A, Middle) Illustration in reverse orientation of the chr16:10,036,019 breakpoint between the 5′ UTR and exon 1 of GRIN2A.(A, Lower) Illustration of the SCN8A-GRIN2A in-frame translocation using the SCN8A 5′ UTR and exon 1 and the GRIN2A CDS and 3′ UTR. (B) SV viewer highlighting the chr12 breakpoint for SCN8A using a copy number profile of 1 Mb on either side of the breakpoint. Centromeric to the breakpoint copy number gain is identified, whereas telomeric to the breakpoint copy number is diploid. (C) SV viewer highlighting the chr16 breakpoint for GRIN2A using a copy number profile of 1 Mb on either side of the breakpoint. Centromeric to the breakpoint copy number gain is identified, whereas telomeric to the breakpoint copy number is diploid. (D) Break-apart FISH probe for the SNC8A gene shows amplification of the orange probe but not the green probe, consistent with the prediction by the SV viewer. (Magnification: 1,000×.) (E) Break-apart FISH probe for the GRIN2A gene shows amplification of the green probe but not the orange probe, consistent with the prediction by the SV viewer. (Magnification: 1,000×.) (F) Fusion design FISH probe using SCN8A orange probe and GRIN2A green probe shows a green-orange fusion signal indicative of translocation and highly amplified for both partner genes. (Magnification: 1,000×.)

For both SCN8A and GRIN2A, the amplified signals consisted of surprisingly, identified a high-level tandem duplication of the microclusters indicating high-level tandem duplication. To confirm GRIN2A gene in 8% (26 of 333) of tumors. Additional probe sets our findings, we then designed a fusion FISH probe set using the spanning the region telomeric (chr16:8,558,071–8,349,774) and amplified member of the two break-apart FISH probe sets, or centromeric (chr16:11,180,357–11,439,054) to GRIN2A with SpectrumOrange-labeled RP11-923I11 (orange) for SCN8A rep- a CEP16 probe showed the most frequent region of amplification ′ resenting the translocated 5 UTR and exon 1 of this gene and was centered within the region containing the GRIN2A gene. GRIN2A FITC-labeled probe RP11-895K13 (green) for repre- Neither of the two additional samples in the validation cohort ′ F senting the translocated CDS and 3 UTR of this gene. Fig. 5 with GRIN2A translocation showed SCN8A translocation using shows the results of this fusion probe set, displaying multiple GRIN2A-SCN8A

a fusion probe set, and the translocation part- MEDICAL SCIENCES clustered green-orange fusion signals representing an amplified ner of these two GRIN2A translocation-positive samples was not SCN8A-GRIN2A translocation. The configuration of these am- determined due to the lack of a high-quality tumor sample. plified signals is most consistent with translocation and amplifi- GRIN2A cation within a ring chromosome. amplification was identified in none of the 41 TCC- UBs of the low-grade superficial type. Among high-grade su- Grin2A Is Often Amplified in Bladder Cancer. FISH validation in an perficial TCC-UBs, GRIN2A amplification was nearly as fre- additional 333 tumors from patients with bladder cancer using quent [7 (8%) of 87] as in the muscle-invasive bladder cancer a GRIN2A break-apart probe (SI Appendix, Table S15) showed cohort [19 (9%) of 205]. The lack of GRIN2A amplification in infrequent GRIN2A translocation (two of 333 tumors), but, low-grade vs. high-grade superficial or muscle-invasive TCC-UB

Morrison et al. PNAS | Published online January 27, 2014 | E677 Downloaded by guest on September 27, 2021 implies GRIN2A amplification may be an early event in the carcinoma in those who have never smoked (40), with a few mu- progression of bladder cancer to a lethal phenotype. Hence, tations that are likely driven by one or a few driver mutations. GRIN2A amplification may be a driver “event” in bladder cancer Chromothripsis appears to be a relatively common event in and appears to be independent of the more common well-known TCC-UB, but its role as a “passenger” or “driver” in bladder events, such as loss of p16 and TP53 mutations. GRIN2A ampli- cancer progression is not yet determined. A recent study in leu- fication was more common in node-positive TCC-UB [11 (14%) kemia involving a patient with multiple recurrences provides an of 81] compared with node-negative TCC-UB [12 (7%) of 166], example of chromothripsis as a passenger event (41). In that study, further suggesting a role for GRIN2A amplification in the meta- chromothripsis was identified in a specimen from the time of re- static phenotype. Of interest, a higher frequency of GRIN2A am- currence, presumably as part of tumor progression, but it was not plification was noted in patients with evidence of cancer at last present in additional relapses after subsequent intervening che- follow-up [12 (9%) of 127]) vs. those with no evidence of cancer [10 motherapy. It is possible that only a subset of rearrangements in (5%) of 183]. However, there was no apparent association between chromothripsis confers a selective single-cell advantage, much like GRIN2A amplification and survival (P =0.24)(SI Appendix,Table subclonal populations of mutations that are selected through S15). To evaluate the biological mechanisms and significance of therapeutic interventions (21). Analyses of multiple samples of GRIN2A amplification further, we compared the mRNA level of TCC-UB from one patient, preferably primary and metastatic GRIN2A for 20 GRIN2A-amplified patient samples and 20 non- tumors with some period of months to years between the two amplified patient samples (SI Appendix,Fig.S14). Tumors with events, will be required to decipher this potential mechanism. If GRIN2A amplification showed 12-fold increased expression of this process is merely a passenger event, it is more difficult to GRIN2A mRNA (P = 0.005), supporting the hypothesis that explain how evidence in both medulloblastoma (14) and mela- overexpression of GRIN2A occurs by gene amplification. noma (13) would suggest that chromothripsis is associated with a more aggressive clinical course. We provide some evidence that GRIN2A as a Potential Oncogene in Bladder Cancer. GRIN2A enc- complex localized genomic rearrangements may result in some odes the e-1 subunit of the NMDA receptor, which has been competitive advantage for neoplastic cells via gene amplification in reported to confer growth advantage to glioma implants and is at least one tumor (case 18195), where breakpoints precisely lined associated with glutamate release (38). Therefore, we hypothe- up with the well-known 6p22 amplicon containing the E2F3 and sized that GRIN2A expression may also contribute to a growth Sox4 genes (42). advantage in TCC cells. To test this hypothesis, we examined the Another intriguing question is whether chromothripsis evolves mRNA expression of GRIN2A in a collection of 17 human TCC through the same mechanism in different tumor types or is unique cell lines (SI Appendix, Fig. S15). From this list, we chose two to TCC-UB in this regard. In our study, we showed that NHEJ is high Grin2A mRNA-expressing cell lines, 253J and HT-1376, the predominant mechanism, whereas in prostate cancer, Teles and developed a shRNA lentiviral construct specifically to target Alves et al. (27) showed no evidence of microhomology involving the expression of GRIN2A in these TCC cell lines. Knockdown chromosome 5 for the vertibral cancer prostate cell line. This in of GRIN2A was successfully achieved, and reduced expression of vitro finding contrasts to the finding of Drier et al. (33) in 95 GRIN2A decreased cell proliferation of both the 253J and HT-1376 matched tumor/normal samples that included 46 breast carcinoma cell lines (Fig. 6A). Using the HT-1376 tumor model in mice, we samples, 23 multiple myeloma samples, 9 colorectal carcinoma implanted s.c. HT-1376/shGRIN2A and HT-1376/shGFP constructs samples, 7 prostate adenocarcinoma samples, 5 melanoma sam- into the right and left flanks of SCID mice (6–8 wk of age, five mice ples, 3 CLL samples, and 2 head and neck carcinoma samples. In per group). As shown in Fig. 6B, we observed a reduction of HT- these cohorts, chromothripsis was associated with all cancer types 1376/shGRIN2A tumor growth in mice compared with HT-1376/ except CLL. This group gave additional evidence that chromo- shGFP tumors (P < 0.01), where expression of GRIN2A was de- thripsis is associated with replication time, proximity to transcribed creased in HT-1376/shGRIN2A tumor cells compared with HT- genes, and guanine-cytosine content. The association of chromo- 1376/shGFP tumor cells at the time of injection. Similarly, as thripsis with microhomology, replication time, and proximity to a marker of proliferation, Ki-67 was decreased in tumor sections transcribed genes could result from deficiencies in the replication– taken from HT-1376/shGRIN2A tumor cells compared with con- licensing complex that is loaded onto chromatin during the G1- trols (Fig. 6B). These results indicate that that the silencing of phase of the cell cycle and is required for initiation of DNA rep- GRIN2A inhibitsproliferationinvitroandinvivoinabladder lication in the subsequent S-phase. In two of our tumors with tumor cell line model. We also evaluated the oncogenic effect of MCM4 mutations, this association could be defined mechanistically GRIN2A using a SV40 immortalized human urothelial cell line by genomic alteration of the family of MCM2-7 genes (43). Con- (SV-HUC), which is an SV40 immortalized, nontransformed, hu- sistent with this hypothesis, prior studies have shown that Mcm man uroepithelial cell line (39). GRIN2A was overexpressed in the protein deficiencies result in high rates of cancer in mouse models SV-HUCs as shown by mRNA and protein levels (Fig. 6C), and (44, 45), catastrophic chromosomal rearrangements in human consistent with the data in Figs. 6 A and B, overexpression of lymphoblasts in culture (46), and complex chromosomal alterations GRIN2A increased the proliferation and migration of SV-HUCs, at discrete locations that are consistent with chromothripsis (43). suggesting that overexpression of GRIN2A promotes an increase in Previously, we have suggested that the frequent, short intra- cell proliferation and migration of bladder epithelial cells. chromosomal DELs spanning 500 kbp or less that occur in mouse tumors resulting from deficient Mcm2 protein levels Discussion could result from failure to rescue stalled replication forks within These data reveal a spectrum of heterogeneity among sequenced individual replication factories (43). This mechanism may also bladder tumors. Based on our whole-genome sequencing analysis, help to explain the frequent localization of multiple trans- we show evidence that SV breakpoints can have a unique associa- location events seen in individual tumors in the present study. tion with copy number in the context of chromothripsis, possibly For example, Fig. 7 shows the location of all structural alter- related to a process of genomic amplification. Additionally, we ations occurring within a single tumor that additionally harbors demonstrate that complex genomic rearrangements mechanistically a nonsynonymous point mutation within the Mcm N domain of use kilobase fragments of DNA that we call stitchers as part of an MCM4. Multiple translocation events occur between approxi- NHEJ DNA repair process. Furthermore, our results support the mately five and six sites on each of chromosomes 4, 5, and 6, presence of intratumoral mutational heterogeneity in TCC-UB. where the size of sites involved is ∼500 kbp or less. To account Finally, although not related to smoking in our study, we provide for the number of DNA replication forks generated during the evidence of a subset of tumors similar to lung non–small-cell S-phase, a single replication factory must contain 20–200 DNA

E678 | www.pnas.org/cgi/doi/10.1073/pnas.1313580111 Morrison et al. Downloaded by guest on September 27, 2021 PNAS PLUS SV-HUC A 1.2 253J 1.2 HT-1376 C 1 1 3000 0.8 0.8 2500 0.6 0.6 2000 0.4 0.4 1500 0.2 0.2 1000 Relative Expression Relative Expression 0 0 500 0 shGFP shGRIN2A shGFP shGRIN2A Relative Expression 8 7 7 shGFP shGFP 6 vector 6 shGRIN2A shGRIN2A GRIN2A 5 5 4 Vector 4 4 3 Grin2A 3 3 2 2 2 1 1 Proliferation Index 0 Proliferation Index 0 1 HT-1376 1 2 3 4 5 1 2 3 4 5 6 0 Time (Days) Time (Days) Proliferation Index 1 2 3 4 5 6 7 70 Time (days) B 60 1.2 * * 1 1200 shGFP 50 0.8 shGRIN2A * * * 40 800 * 0.6 * * 30 0.4 * * shGFP shGRIN2A 400 * * 0.2 * * * Ki-67 20 Relative Expression 0 Cell Migration per Field 0 (mm3) Volume Tumor 10 4 8 12 16 20 24 Day Post Implantation 0 shGFP vector Grin2A shGRIN2A

Fig. 6. In vitro and in vivo models using a shRNA lentiviral construct specifically to target the expression of GRIN2A in the 253J and HT-1376 TCC lines. (A) In-vitro model using a shRNA lentiviral construct targeting GRIN2A in the 253J and HT-1376 bladder cancer cell lines by real-time RT-PCR and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (B) In vivo model using the HT-1376 cell line with HT-1376/shGRIN2A and HT-1376/shGFP constructs that were validated for GRIN2A expression before injection s.c. into the right and left flanks of SCID mice (6–8wkofage,five mice per group), with Ki-67 staining of these tumors examined on day 24. (Magnification: 20×; magnification of Insets,40×.) (C) Nontransformed bladder epithelial SV-HUCs were transfected with a shRNA lentiviral construct that targets GRIN2A, and expression was determined in RT-PCR and immunoblot assays. SV-HUCs that overexpressed GRIN2A were examined for a change in proliferation and migration using the MTT assay and transwell migration assay, respectively.

replication forks (47), which, assuming ∼50,000 bp between two or more chromosomes to form a highly complex derivative replication origins, span 0.5–5 Mb of DNA. Although it is typi- chromosome may be incomplete. Our study suggests that although cally assumed that replication factories assemble around domains one chromosome provides megabase fragments of DNA, other in- within an individual chromosome, it is also possible that a single volved chromosomes provide only 50- to 100-kb fragments that we factory contains origins from different chromosomes (48), as refer to as stitchers. Although our findings do not fully define this shown in Fig. 7B. In this case, failure to rescue stalled replication mechanism at the current time, a plausible explanation could be that forks could lead to the observed complex recombination events tumors use stitchers in stalled replication forks in the replication– involving multiple chromosomes. It is interesting to note that the licensing complex during the G1-phase of the cell cycle (43). Our other case in our study with the highest number of both SVs and findings provide a framework for further mechanistic investigations. SNVs (case 19685) also had an MCM4 mutation in the same Although chromothripsis and the complex process underlying domain as the tumor in case 18195. this event may not lead to a driver mutation, intratumoral muta- Nonhomologous reciprocal translocations have been identified tional heterogeneity leading to driver events is likely, at least in

in lymphomas and sarcomas; however, these complex rearrange- leukemia (19, 21). In a comparative sense, it could be possible that MEDICAL SCIENCES ments, at least in TCC-UB, are different from the ones seen in intratumoral mutational heterogeneity is a marker of underlying lymphomas and sarcomas. Although this is not surprising, given genomic events, much as we postulate that chromothripsis is related the definition of chromothripsis and current knowledge of this to a defective replication–licensing complex. Intratumoral muta- genomic event, the evidence we present of a stitching process tional heterogeneity may be associated with resistance to chemo- using 50- to 100-kb fragments to reconstitute these interchro- therapy and/or advanced stage at the time of presentation (21). In mosomal events is intriguing and raises questions about current our study, both cases that showed evidence of intratumoral muta- theories regarding this process. The prior concept that chromo- tional heterogeneity were also TP53mut, whereas among the three thripsis results in exchange of megabase fragments of DNA from cases with no evidence of intratumoral mutational heterogeneity,

Morrison et al. PNAS | Published online January 27, 2014 | E679 Downloaded by guest on September 27, 2021 mutated gene in melanoma (49) and a frequently overexpressed gene in ALK-positive lung cancer (50), as well as the recent recognition that glutamate transport and intermediary metab- olism may be important in the etiology of other tumors (glio- blastoma) (38), provide convincing evidence that GRIN2A is of importance in cancer. To conclude, we have provided additional insight into the genomic landscape of muscle-invasive bladder cancer and developed a framework for future whole-genome sequencing studies of TCC-UB to use as a comparison. We have shown a great deal of genomic diversity in a small sample set of TCC-UB that will provide important information in planning for additional studies. Methods Samples and Clinical Data. We studied five tumor samples (cases 16933, 17802, 18195, 18698, and 19685) of chemotherapy-naive muscle-invasive TCC-UB American Joint Committee on Cancer stage III or IV with whole-genome sequencing (SI Appendix, Table S1). There were three males and two females (all Caucasian non-Hispanic), with an average age of 67 y. Three were smokers, two were nonsmokers, and none had a prior history of superficial TCC-UB. We identified tumor-specific somatic DNA alterations by comparing each tumor with its corresponding normal germ-line DNA derived from matching blood. The validation cohort consisted of 333 patients with a history of TCC-UB of the bladder that spanned the gamut of clinical scenarios ranging from low-grade superficial bladder carcinoma to high-grade invasive and noninvasive TCC-UB with and without a prior history of superficial disease (SI Appendix, SI Methods).

DNA Library Preparation and Massively Parallel Sequencing. Whole-genome sequencing was done using a 500-bp library with 100-bp paired-end reads and, additionally, a 5-kb library with 36-bp paired-end reads (mate pair). Sequencing was carried out for the prepared DNA libraries with a HiSeq 2000 sequencing system (Illumina) following the manufacturer’s standard Fig. 7. Model of microhomology-mediated translocation events occurring protocol using the Illumina cBot and HiSeq paired-end cluster kit, version 1 between two or more chromosomes in an individual replication factory. (A) (SI Appendix, SI Methods). Illustration of multiple translocation events occurring between chr4, chr5, and chr6 for case 18195, with each line denoting an individual CTX. Note Read Mapping and Alignment and Variant Analysis. We recently developed an that many of the translocations between any two chromosomes often show in-house analysis pipeline for cancer genome sequencing data that includes a second breakpoint within a few thousand base pairs as part of a different (i) mapping and alignment, (ii) SNV and indel discovery, and (iii) SNV and translocation, with the third chromosome resulting in this complex web-like indel filtering and annotation (SI Appendix, SI Methods). pattern of rearrangement. (B) DNA replication factory involving portions of chr4, chr5, and chr6, with stalled replication forks indicated by red x marks. Detecting SVs. BreakDancer was used to detect SVs from paired-end Illumina Dashed lines indicate translocation occurring at stalled replication forks, sequencing data. Then, the de novo assembly was performed for all filtered DELs, often with closely adjacent breakpoints involving multiple chromosomes in insertions, and INVs using the newly developed sensitive assembler TIGRA_SV a complicated web-like fashion. (http://genome.wustl.edu/software/tigra_sv) and for translocations using Phrap (www.phrap.org/), followed by extraction of mapped reads using SAMtools (http://samtools.sourceforge.net/)(SI Appendix, SI Methods). one was TP53mut and the other two were TP53wt. Early clonal ex- pansion of TP53-mutated cells would be predicted to lead to in- PCR Validation of SVs. Putative SVs were validated by PCR using R script to creased genetic heterogeneity through lack of sufficient DNA repair select genomic sequences around the de novo assembly-determined break- processes. It is feasible that the final evaluation of this topic points for each SV from the University of California, Santa Cruz genome browser (http://genome.ucsc.edu/)(SI Appendix, SI Methods). can be done with exomic sequencing and will not require sequencing of the entire genome. Progress in developing more Detection of Somatic Copy Number Alteration. To identify somatic copy targeted therapies in TCC-UB will be informed by further number alterations, each tumor and its matched normal DNA were geno- analysis of this genomic event. typed using Illumina HumanOmni1-Quad BeadChips, which contain 1,140,419 In the more “genomically simple” subset of TCC-UB, we iden- SNPs, with a median SNP spacing of 1.2 kb (SI Appendix, SI Methods). tified unique events that included (i) an unequivocal demonstration thatamplifiedCTXscanbefoundincarcinomasand(ii)infrequent FISH for SVs. All SVs in this study were evaluated by FISH using RP11 clones translocation but frequent amplification of GRIN2A in a subset of from the Roswell Park Cancer Institute BAC library. A complete list of all BAC clones and probe designs is provided in SI Appendix, Table S13. Both TCC-UB. Furthermore, our preliminary functional studies in breakpoints of a given SV were evaluated separately using a break-apart bladder carcinoma cell lines support GRIN2A as a candidate on- probe FISH design (SI Appendix, SI Methods). cogene in TCC-UB. Although the first of these unique findings was only demonstrated in the index case (case 16933), it does demon- RT-PCR Analysis for GRIN2A mRNA Expression. Real-time RT-PCR analysis was strate the utility of NGS as a discovery tool and likely portends the done using SYBR Green I as a reporter and ROX (Applied Biosystems) as discovery of additional such examples in bladder cancer and other a reference dye for GRIN2A mRNA expression (SI Appendix, SI Methods). carcinomas as this technology expands. In our study, this initial discovery of an SCN8A-GRIN2A translocation was further noted in Statistical Analysis. The association between clinical/histological covariates and 6p22 amplification was tested using two-sample t tests for the equality the validation cohort. However, thisobservationledustothedis- of proportions. Survival time associations were tested with a log-rank test. covery of a second unique finding, amplification of GRIN2A,in Statistical analysis of data was performed using the SPSS Statistics software a subset of TCC-UB. Previous findings of GRIN2A as a frequently package (IBM). All results are expressed as mean ± SD.

E680 | www.pnas.org/cgi/doi/10.1073/pnas.1313580111 Morrison et al. Downloaded by guest on September 27, 2021 In Vitro Tumor Assays. Human bladder cancer cell lines 253J and HT-1376, as well ACKNOWLEDGMENTS. Biospecimens or research pathology services for PNAS PLUS as SV40 immortalized human uroepithelial SV-HUCs, were cultured, and trans- this study were provided by the Pathology Resource Network, which is fection was performed using X-tremeGENE 9 DNA Transfection Reagent following funded by the National Cancer Institute and is a Roswell Park Cancer Institute Cancer Center Support Grant shared resource. Clinical data the manufacturer’s protocol (Roche). Packaging of retrovirus and lentivirus, delivery and honest broker services for this study were provided by the cell transduction, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Clinical Data Network, which is funded by the National Cancer Institute assays, Western blotting, shRNA knockdown experiments, and migration assays and is a Roswell Park Cancer Institute Cancer Center Support Grant were performed following standard protocols (SI Appendix, SI Methods). shared resource.

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