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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 a

Center for Personalized Medicine and Departments of cPharmacology and Therapeutics, dCancer Genetics, eBiostatistics and Bioinformatics, fPathology, Urology, 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 g

Using complete genome analysis, we sequenced five bladder tumors accrued from patients with muscle-invasive transitional cell carcinoma of the urinary bladder (TCC-UB) and identified a spectrum of genomic aberrations. In three tumors, complex genotype changes were noted. All three had tumor protein p53 mutations and a relatively large number of single-nucleotide variants (SNVs; average of 11.2 per megabase), structural variants (SVs; average of 46), or both. This group was best characterized by chromothripsis and the presence of subclonal populations of neoplastic cells or intratumoral mutational heterogeneity. Here, we provide evidence that the process of chromothripsis in TCC-UB is mediated by nonhomologous end-joining using kilobase, rather than megabase, fragments of DNA, which we refer to as “stitchers,” to repair this process. We postulate that a potential unifying theme among tumors with the more complex genotype group is a defective replication–licensing complex. A second group (two bladder tumors) had no chromothripsis, and a simpler genotype, WT tumor protein p53, had relatively few SNVs (average of 5.9 per megabase) and only a single SV. There was no evidence of a subclonal population of neoplastic cells. In this group, we used a preclinical model of bladder 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. next-generation sequencing

earlier studies in melanoma (13) and medulloblastoma (14), evidence of the association of TP53 mutations with specific copy number alterations, referred to as chromothripsis, was noted. The study in medulloblastoma (14) was particularly intriguing in that identification of a molecular subclass with TP53 mutations was associated with chromothripsis and a more aggressive clinical outcome was noted. Chromothripsis, or the shattering of two or more chromosomes and their reassembly into derivative chromosomes, is different from other types of genomic instability, which tend to occur on a genome-wide basis (15, 16). Chromothripsis is different in that it includes one to three alternating copy number states across the derivative chromosome, an association with changes in heterozygosity, and numerous genomic rearrangements in localized chromosomal regions likely occurring in condensed chromosomes (15). There is evidence to suggest that the primary mechanism of reassembly of the derivative chromosome in chromothripsis is nonhomologous end-joining (NHEJ) (14). With the advent of next-generation sequencing (NGS) allowing for detailed genomic analysis, chromothripsis Significance

| tumor heterogeneity | GRIN2A | replication

Genetic alterations are frequently observed in bladder cancer. In this study, we demonstrate that bladder tumors can be classified into two different types based on the spectrum of genetic diversity they confer. In one class of tumors, we observed tumor protein p53 mutations and a large number of single-nucleotide and structural variants. Another characteristic of this group was chromosome shattering, known as chromothripsis, and mutational heterogeneity. The other two bladder tumors did not show these profound genetic aberrations, but we found a novel translocation and amplification of the gene glutamate receptor ionotropic N-methyl D-aspertate, a potentially druggable target. Advancements in bladder cancer treatment have been slow. Understanding the genetic landscape of bladder cancer may therefore help to identify new therapeutic targets and bolster management of this disease.

T

ransitional cell carcinoma arising in the urinary bladder (TCCUB) is a frequent cause of morbidity and mortality, and among patients in the United States, it is one of the most costly cancers to treat (1, 2). The traditional somatic genetic basis of TCC-UB is a distinct division of low-grade papillary tumors from high-grade invasive tumors. Low-grade papillary superficial tumors are generally characterized by constitutive activation of the receptor tyrosine kinase–Ras pathway, and they have activating mutations in the HRAS and fibroblast growth factor receptor 3 (FGFR3) genes (3–6). In contrast, high-grade invasive TCC-UB is characterized by alterations in the tumor protein p53 (TP53) and retinoblastoma 1 (RB1) pathways. These genes normally regulate the cell cycle by interacting with the Ras–mitogen-activated protein kinase signal transduction pathway (7, 8). Both low-grade papillary and high-grade invasive tumors frequently have loss of chromosome 9. This loss presumably inactivates the p16 gene and is an early event in the initiation of TCC-UB (9, 10) Although TP53, cyclin-dependent kinase inhibitor 2A (p16), RB1, HRAS, and FGFR3 abnormalities have been well described in TCCUB, there are limited data on the more complete genomic analysis of TCC-UB (11). A recent study focusing on genome-wide copy number analysis showed extensive heterogeneity across all subtypes of TCC-UB to such an extent that precise molecular groupings were difficult to define (12). In this study, similar to www.pnas.org/cgi/doi/10.1073/pnas.1313580111

Author contributions: C.D.M., W.B., K.G., M.Y., C.S.J., and D.L.T. designed research; C.D.M., 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 S.V. contributed new reagents/analytic tools; C.D.M., P.L., J.Z., M.Q., J.M.C., P.V., D.X., 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., P.L., A.W.-R., J.Z., J.M.C., S.C.P., C.S.J., and D.L.T. wrote the paper. The authors declare no conflict of interest. *This Direct Submission article had a prearranged editor. 1

C.D.M. and P.L. contributed equally to this work.

2

To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1313580111/-/DCSupplemental.

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Edited* by Carlo M. Croce, The Ohio State University, Columbus, OH, and approved January 2, 2014 (received for review July 22, 2013)

has been identified more frequently (17). Additionally, the presence of these complex genomic events and their potential association with TP53 mutations may contribute to a better understanding of cancer, including TCC-UB. NGS technologies provide other evidence of complex genomic heterogeneity, such as the recent identification of subclonal populations of cells with mutations distinct from the dominant clonal population of cells within one tumor or between a primary, recurrent, or metastatic tumor from one patient (18–20). Importantly, a recent study in chronic lymphocytic leukemia (CLL) (21) showed how selective pressures on cancer cells, such as chemotherapy, select for these subclonal populations to become the dominant clone contributing to genomic heterogeneity. It is not yet certain whether broad measurements of genomic heterogeneity will have an impact on molecular classification of cancer, but it is likely that they will significantly contribute to biological differences, and therefore have an impact on patient outcomes. To evaluate the spectrum of genomic heterogeneity in TCCUB, we performed complete genome sequencing of five highgrade muscle-invasive tumors and matching germ-line blood (SI Appendix, Table S1), and validated a subset of our findings in more than 300 bladder cancer specimens. Our overall results showed a great deal of genomic heterogeneity at either extreme of a spectrum of genomic complexity. Results Overview of Somatic Alterations Reveals Heterogeneity Between Patients. At one end of the spectrum was a more complex geno-

type, characterized by frequent single-nucleotide variants (SNVs) and structural variants (SVs), TP53 mutation (TP53mut), CDNK2A (p16) deletion (p16del), frequent mutations in known cancerrelated genes, SV breakpoints that often precisely align with segmental copy number states indicating chromothripsis, and 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 that may be related to a process of genomic amplification, (ii) complex genomic rearrangements mechanistically use kilobase fragments of DNA that we refer to as “stitchers” as part of an NHEJ DNA repair process, and (iii) some cases of TCC-UB do show intratumoral mutational heterogeneity. At the other end of the spectrum, was a simpler genotype, with few SNVs and SVs, infrequent mutations in any known cancer-related gene in the Cancer Gene Census, TP53 WT (TP53wt), p16 WT (p16wt), and no evidence of chromothripsis. In this group, we provide (i) an unequivocal demonstration that amplified interchromosomal translocations (CTXs) can be found in bladder carcinoma and (ii) evidence of rare events of translocation and, more frequently, amplification of GRIN2A in a subset of TCC-UB representing a potential therapeutic target. Somatic Mutation Analysis Identifies Intertumoral Genomic Heterogeneity at the Nucleotide Level. We obtained 44.8-fold mean sequence cov-

erage for each tumor and 39.5-fold mean sequence coverage for the matching normal tissues (SI Appendix, Table S2). To identify somatic events, we compared the sequencing data of each tumor with that of matched blood using multiple algorithms and filtered the list by reference to the Single Nucleotide Polymorphism database (dbSNP) build 130 and 1000 Genomes Project (SI Appendix, SI Methods and Tables S3–S6). There was a wide variation in the number of somatic mutations per tumor, ranging from 14,256 in case 16933 to 49,889 in case 19685 (average of 29,326). Tumors from two patients (cases 16933 and 17802) had fewer somatic mutations (Fig. 1), including both SNVs and SVs, and were TP53wt. The tumors of three patients (cases 18698, 18195, and 19685) were TP53mut and contained a much larger number of somatic mutations, providing additional intertumoral genomic heterogeneity at the nucleotide level. Not unexpectedly, four or five 2 of 10 | www.pnas.org/cgi/doi/10.1073/pnas.1313580111

100 90

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TP53mut/p16wt

80 70

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60

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50 40 30 20 10

TP53mut/p16del TP53wt/p16wt

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TP 53/p16 wild type Single structural variant (SV) Fewer single nucleotide variants (SNVs) TP53 wild type Chromothripsis absent No evidence of subclonal populations

18698

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19685

TP53 mutant p16 deletion Frequent structural variants (SVs) Frequent single nucleotide variants (SNVs) TP53 mutant Chromothripsis involving multiple chromosomes Subclonal populations identified in 2 of 3

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 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 were also TP53mut. Two of the five tumors (cases 16933 and 17802) had very few SVs and SNVs, and were also TP53wt. Patient 17802, although having only one SV, shared in common with the TP53mut group a p16 (CDKN2A) DEL. Patient 16933 was negative for p16del and TP53wt status, had no mutations in any known cancer-related genes in the Cancer Gene Census, and had a single distinct SV represented by a CTX between the SCN8A gene at 12q14 and the GRIN2A gene at 16p13.2.

tumors (cases 18693, 18195, 19685, and 17802) contained mutations in one or more chromatin remodeling genes, including mutations in NSD1, PBRM1, KDM6A, ARID2, APC, and EP300, which were identified in all of these tumors, consistent with prior exomic sequencing in this tumor type (22). In this same group of four tumors, there were 11 mutated genes present in two or more samples (SI Appendix, Table S7), including TP53, CTBP2, ZFHX4, XIRP2, WDR89, PCMTD1, PABPC3, MCM4, GXYLT1, CDCA7L, and CC2D1A. Genes with a nonsynonymous mutation and coding region deletion (DEL) in one or more samples included ANKRD11 and CC2D1A. In both instances, the mutation and DEL occurred in the same case. Neither of these two genes has been identified previously as mutated in TCC-UB (22), and reports of mutations in other tumor types have been reported only rarely in the Cosmic Mutation Database (www.sanger.ac.uk/genetics/CGP/cosmic/). MCM4 and Replication–Licensing Complex Defects. With the exception of TP53, none of the above-listed genes was previously reported as mutated in an Asian cohort of 97 patients using whole-exome sequencing (22). Among these 97 patients, MCM3 was mutated in one tumor. MCM3 is part of a six-gene MCM2–7 replication–licensing complex that binds chromatin during the G1-phase of the cell cycle and is required for initiation of DNA replication in the subsequent S-phase. In our study, two tumors (cases 18195 and 19685) both showed mutations in the MCM4 gene of this replication–licensing complex, which were validated by Sanger sequencing. Both of these tumors were TP53mut, had the largest number of SNVs and SVs, showed chromothripsis involving multiple chromosomes, and demonstrated intratumor genomic heterogeneity. As we postulate in our discussion, all of these events may have a single underlying association through a replication–licensing complex defect (see Fig. 7). Morrison et al.

allele frequencies in an individual case provide evidence of intratumoral heterogeneity and support the existence of multiple clones of neoplastic cells with different genotypes within one tumor (20, 21, 23–26). By itself, the presence of multiple neoplastic clones within a single tumor implies a more complex genome, and recent evidence supports an evolution toward a more aggressive phenotype (26). To assess intratumoral clonality, the frequency data of tumor variant alleles for all identified somatic mutations were input into an R function “density” to estimate the empirical probable density function of allele frequencies (19) (Fig. 2). Estimates of clonality were determined using a kernel density analysis of tumor variant allele frequency, which was performed separately for each tumor. The two bladder tumors with the simpler genotype (cases 16933 and 17802) showed a Gaussian distribution of variant frequencies without evidence of subclonal populations of neoplastic cells. In contrast, cases 18195 and 19685, with a more complex genotype, showed an obvious skew of the normal distribution. The kernel density analysis plot for case 18195 shows at least two neoplastic clones centered on variant allelic frequencies of 20% and 40%. Case 19685 showed a similar skewed distribution but without obvious peaks of variant allelic frequencies, perhaps reflecting the resolution of coverage with wholegenome sequencing (Fig. 2). The last case, 18698, although TP53mt and having a relatively large number of mutations, did not show subclonal populations by this analysis, implying there is some continuum across these groups. Larger numbers of patients are necessary to determine if the biological importance of these

SV Analysis Identifies Genomic Heterogeneity at the Chromosomal Level. To detect chromosomal rearrangements, we searched for

fragments in which the sequence from the paired-end read mapped discordantly to the reference genome and further refined them by de novo assembly (SI Appendix, SI Methods). A total of 263 putative somatic rearrangements were predicted. To assess the accuracy of these predictions, PCR was performed across the putative breakpoints for both the tumor and germ-line DNA. We confirmed, by either PCR or FISH, 150 (57%) of these predicted SVs as true somatic rearrangements (SI Appendix, Table S8), 6 (2%) as germ-line, and 59 (22%) as false, and the remaining 48 (19%) failed to produce a PCR in either the tumor or germ-line DNA. The number of somatic SVs per tumor varied greatly (range: 1–79) with a median of 20 (average of 30) per tumor (SI Appendix, Fig. S1 and Table S9). Tumors with a higher number of SNVs also showed more SVs (Fig. 1). DELs were the most common SV identified [58 (39%) of 150], followed by inversions [INVs; 56 (37%) of 150] and CTXs [34 (23%) of 150] (SI Appendix, Fig. S2 and Table S10). Intrachromosomal translocations [2 (1%) of 150] were infrequent and only identified in two of the five tumors. Interestingly, both tumors with the simpler genotype (cases 16933 and 17802) had only a single SV; in case 17802, the SV was a p16del. The remaining tumors (cases 18698, 18195, and 19685) had the more complex genotype, showed an average of 49 (median of 49) SVs, and were evaluated further. 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 primarily 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.

Fig. 2. Kernel density analysis plots of tumor variant allele frequency (Freq) to assess intratumoral heterogeneity. Shown are plots for all five tumors representing either end of the spectrum of genomic complexity. Each plot graphs the variant allelic frequency (x axis) vs. the density of the variant allelic frequencies (y axis). Plots with a single peak (cases 16933, 17802, and 18698) represent clusters of mutations with similar variant allelic frequencies and no evidence of subclonal populations of neoplastic cells. Plots with two or more peaks (cases 18195 and 19685) represent tumors with subclonal populations of neoplastic cells and a more complex genome.

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subclonal populations of neoplastic cells portend a worse prognosis, as has been previously described in leukemia (21).

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 cell–cell adhesion, was predicted to join with the last three exons of CAB39L, a protein that binds and activates serine/threonine kinase STK11. A CDH10/CAB39L translocation was validated in the index case (case 18195) by PCR but not by FISH. Additional break-apart FISH studies for both CDH10 and CAB39L failed to show any other translocations in the validation cohort of 329 bladder cancer samples. We show later that this concept of PCRpositive, FISH-negative translocation events is a common event PNAS Early Edition | 3 of 10

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Mutated Subclones Contribute to Intratumoral Genomic Heterogeneity at the Nucleotide Level. Clusters of mutations with dissimilar variant

in chromothripsis. Similar to the findings in a recent study in prostate cancer (27), chromothripsis, although a marker of genomic instability, does not lead to recurrent functionally relevant fusion genes in TCC-UB.

of DNA and our probes were purposely designed to allow for a 50- to 100-kb error in prediction of the exact breakpoint, this would indicate that the segment of DNA involved in this stitching process is less than this size.

SVs Associated with Chromothripsis Align with Segmental Copy Number Changes. Although SVs in the tumors with the complex

NHEJ Is the Predominant Mechanism of Genomic Rearrangement in Bladder Cancer. There is evidence from earlier studies that unique

genotype did not have apparent single-gene implications as driver mutations in comparison to the simple genotype, the relationship of SVs to copy number changes suggests other biologically relevant mechanisms. In this regard, a pattern of CTXs and INVs closely aligned with segmental copy number states was characteristic of the complex genotype. To define the genomic significance of these events better, we developed an enhanced SV viewer similar to Circos but with a linear view and the capability to “zoom in” for a more detailed view. This enhanced linear view showed frequent sharing of breakpoints for INVs and CTXs. Most interestingly, when the copy number profile across these regions was added to the viewer, an alternating change of one to a few copies with transitions aligned with shared breakpoints for both INVs and CTXs consistent with chromothripsis was identified (Fig. 3). Using NGS technology, chromothripsis has been previously reported in one case of CLL (15) and three cases of colorectal cancer (17), but this precise alignment of SV breakpoints and copy number changes was not described. The mechanism of reconstitution of these fragments of DNA into a complex, highly rearranged fragment of DNA as either an intact, cytogenetically recognizable chromosome or double minute has recently been called chromoanagenesis (28) (“chromo,” meaning chromosome, and “anagenesis,” meaning reborn). As part of this mechanism, we provide unique sequencing information that small fragments (average size of 50–100 kb) of DNA from chromosomes other than the cytogenetically recognizable chromosome are used to “stitch” such chromosomes together (Fig. 4). This conclusion is based on consistent PCR validation at predicted interchromosomal breakpoints, and consistently negative findings by FISH. Paired-spectrum orange and green break-apart FISH probes were designed on either side of multiple chromosome 4, 5, and 6 CTX and INV breakpoints with a 50- to 100-kb gap (SI Appendix, Table S13) for each probe set. Because the sensitivity of FISH with interphase nuclei is in the range of 50 kb

short stretches of an identical sequence, or microhomology (29), located near the breakpoints of DNA double-strand breaks may be critical in a stitching process in mouse Ltk− cells (30) similar to that reported here. This leads to the creation of localized complex rearrangements. Similar evidence of the importance of microhomology as a general mechanistic model for chromosomal rearrangements and amplification has been provided for human lymphoma cell lines (31, 32). In these studies, a direct relationship to TP53 mutation status was noted. In three of our tumors with the complex genotype, microhomology was identified in 68% (101 of 148) of the breakpoints. Remarkably, this percentage of microhomology was quite consistent among cases [case 18195, 34 (69%) of 49 breakpoints; case 18698, 15 (79%) of 19 breakpoints; and case 9685, 52 (65%) of 80 breakpoints] and for subtypes of SV (CTX, 64%; DEL, 75%; and INV, 67%). The results in these three tumors with an average of 2.2 bp of microhomology per SV were similar to those of a recent study (33) of 95 complete tumor genomes of various histological subtypes, in which an average of 1.7 bp of microhomology per SV was identified. In our study, we also identified nontemplated sequences at the rearrangement junctions in 18 of 148 SVs (SI Appendix, Table S14), which, along with microhomology, is considered to be the signature of a DNA double-strand break repair process (34). Only 20% of all SVs displayed neither microhomology nor nontemplated sequences, indicating that NHEJ was the predominant DNA double-strand break repair process. Recurrent Breakpoints Are Often Amplified in Bladder Cancer. Some of the CTXs and INVs in the group with a more complex genotype were further defined by a clustering of breakpoints both within and between different samples at chr4:180 Mb, chr5:29 Mb, chr5:40 Mb, chr6:10 Mb, chr6:18 Mb, and chr6:24 Mb. These breakpoints were of interest as potential recurrent genomic 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 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.

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Chr5:40,062,440

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Chr6:18,155,619-18,365,654

D

Chr6:23,292,635-24,467,711

cohort of 343 patients. Using a FISH break-apart approach, we did not identify translocations at any of these sites in the validation cohort; however, surprisingly, amplification (Fig. 4 B–D) was a common event at chr5:40 Mb [32 (9%) of 343 patients], chr6:18 Mb [53 (16%) of 332 patients], and chr6:24 Mb [53 (16%) of 332 patients]. The gains at chr6:18 Mb and chr6:24 Mb were high-level tandem amplifications typical of known oncogenes, such as HER-2, MYC, CCND1, or MDM2, whereas the gain at chr5:40 Mb was one of low to intermediate amplification with a copy number consistently ranging from 5 to 10. FISH with additional BAC clones performed in the validation cohort across the chr18–24 Mb at 1- to 2-Mb intervals (SI Appendix, Table S13) showed this was one continuous amplicon. The highest level of amplification and minimal region of copy number gain was seen at chr6:19.8 Mb (RP1193O13) to chr6:21.4 Mb (RP11-204E9), including the genes E2F3 and SOX4. Previous studies in multiple tumor types (35), including bladder cancer (36), have identified increased copy number in a 6p22 amplicon that is centered between the recurrent breakpoints with a segmental copy number change at chr6:18–24 Mb. Recent release of data by the Cancer Genome Atlas Network (www.cbioportal.org) shows this 6p22 amplicon containing the E2F3, SOX4, PRL, and CDKAL1 genes to be the most common amplification in TCC-UB. The amplification at chr5:40 Mb was not studied further, but the overall evidence supports the observation that the large number of SVs in the complex genotype is not simply a reflection of random chromosomal instability. SV Analysis Identifies a SCN8A-GRIN2A Translocation. As previously discussed, the single SV in case 17802 was a DEL involving the p16 gene at chromosome 9p21. The single SV identified in case 16933 was unique in that it predicted an in-frame fusion protein involving the SCN8A gene at 12q13 and the GRIN2A gene at 16p13.2 (Fig. 5). This fusion variant was predicted to result in an in-frame fusion of the SCN8A 5′ UTR and exon 1 with the GRIN2A complete coding sequence (CDS) and 3′ UTR (Fig. 5A). Subsequent genomic PCR and capillary sequencing of this tumor using primers for SCN8A 5′ UTR and GRIN2A exon 1 demonstrated the predicted fusion variant in tumor DNA and not in the corresponding germ line (SI Appendix, Figs. S12 and S13). Although RT-PCR with a GRIN2A exon 2 and 4 primer set using case 16933 tumor cDNA demonstrated expression of CDS GRIN2A, the SCN8A Morrison et al.

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 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 PCRpositive 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×.)

exon 1 and GRIN2A exon 4 primer set did not delineate this fusion variant for unexplained reasons. Further analysis of this SV in case 16933, using our enhanced linear SV viewer (Fig. 5 B and C), showed an unexpected finding. For the SCN8A breakpoint at 12q13, the corresponding data from Illumina SNP chips (HumanOmni1-Quad_v1-0 containing 1,140,419 dbSNP) showed copy number gain centromeric to the breakpoint, corresponding to the 5′ UTR and exon 1 region of SCN8A, and a diploid state on the telomeric side. For the GRIN2A breakpoint at 16p13.2, the corresponding results showed copy number gain telomeric to the breakpoint, corresponding to the CDS and 3′ UTR of GRIN2A, and a diploid state on the centromeric side. Translocation with subsequent amplification of the involved genes is unique in cancer genetics and has been identified frequently only in the COL1A1 and PDGFB translocations in dermatofibrosarcoma protuberans (37); to the best of our knowledge, this has not been previously reported in a carcinoma. FISH Confirms an Amplified Reciprocal SCN8A-GRIN2A Translocation.

To interrogate the chr12:52,049,200 breakpoint for SCN8A, we designed a break-apart FISH probe set (SI Appendix, Table S13), with a SpectrumOrange-labeled probe (RP11-923I11, orange) centromeric to the breakpoint representing the translocated 5′ UTR and exon 1 of SCN8A and an FITC-labeled probe (RP11285E4, green) telomeric to the breakpoint representing the nontranslocated portion of this gene. Likewise, to interrogate the chromosome 16:10,035,762 breakpoint for GRIN2A, we designed a break-apart FISH probe set in the reverse fashion with an FITClabeled probe (RP11-895K13, green) telomeric to the breakpoint representing the translocated CDS and 3′ UTR of GRIN2A and a SpectrumOrange-labeled probe (RP11-297M9, orange) centromeric to the breakpoint representing the nontranslocated portion of this gene. Based on our sequencing results, we predicted evidence of orange-green break-apart at both sites with amplification of the orange probe for SCN8A and amplification of the green probe for GRIN2A. Fig. 5D shows the FISH results using the SCN8A break-apart probe set for case 16933, and, as anticipated, multiple single orange signals with no associated green signals were identified. Fig. 5E shows the FISH results using the GRIN2A break-apart probe set for case 16933 and, as anticipated, multiple green signals with no associated orange signals were identified. PNAS Early Edition | 5 of 10

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Chr16:10,036,019 breakpoint

Chr12:52,049,20 Chr16:10,036,019 0 Translocation

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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 microclusters indicating high-level tandem duplication. To confirm our findings, we then designed a fusion FISH probe set using the amplified member of the two break-apart FISH probe sets, or SpectrumOrange-labeled RP11-923I11 (orange) for SCN8A representing the translocated 5′ UTR and exon 1 of this gene and FITC-labeled probe RP11-895K13 (green) for GRIN2A representing the translocated CDS and 3′ UTR of this gene. Fig. 5F shows the results of this fusion probe set, displaying multiple clustered green-orange fusion signals representing an amplified SCN8A-GRIN2A translocation. The configuration of these amplified signals is most consistent with translocation and amplification within a ring chromosome. Grin2A Is Often Amplified in Bladder Cancer. FISH validation in an additional 333 tumors from patients with bladder cancer using a GRIN2A break-apart probe (SI Appendix, Table S15) showed infrequent GRIN2A translocation (two of 333 tumors), but, 6 of 10 | www.pnas.org/cgi/doi/10.1073/pnas.1313580111

surprisingly, identified a high-level tandem duplication of the GRIN2A gene in 8% (26 of 333) of tumors. Additional probe sets spanning the region telomeric (chr16:8,558,071–8,349,774) and centromeric (chr16:11,180,357–11,439,054) to GRIN2A with a CEP16 probe showed the most frequent region of amplification was centered within the region containing the GRIN2A gene. Neither of the two additional samples in the validation cohort with GRIN2A translocation showed SCN8A translocation using a GRIN2A-SCN8A fusion probe set, and the translocation partner of these two GRIN2A translocation-positive samples was not determined due to the lack of a high-quality tumor sample. GRIN2A amplification was identified in none of the 41 TCCUBs of the low-grade superficial type. Among high-grade superficial TCC-UBs, GRIN2A amplification was nearly as frequent [7 (8%) of 87] as in the muscle-invasive bladder cancer cohort [19 (9%) of 205]. The lack of GRIN2A amplification in low-grade vs. high-grade superficial or muscle-invasive TCC-UB Morrison et al.

GRIN2A as a Potential Oncogene in Bladder Cancer. GRIN2A encodes the e-1 subunit of the NMDA receptor, which has been reported to confer growth advantage to glioma implants and is associated with glutamate release (38). Therefore, we hypothesized that GRIN2A expression may also contribute to a growth advantage in TCC cells. To test this hypothesis, we examined the mRNA expression of GRIN2A in a collection of 17 human TCC cell lines (SI Appendix, Fig. S15). From this list, we chose two high Grin2A mRNA-expressing cell lines, 253J and HT-1376, and developed a shRNA lentiviral construct specifically to target the expression of GRIN2A in these TCC cell lines. Knockdown of GRIN2A was successfully achieved, and reduced expression of GRIN2A decreased cell proliferation of both the 253J and HT-1376 cell lines (Fig. 6A). Using the HT-1376 tumor model in mice, we implanted s.c. HT-1376/shGRIN2A and HT-1376/shGFP constructs into the right and left flanks of SCID mice (6–8 wk of age, five mice per group). As shown in Fig. 6B, we observed a reduction of HT1376/shGRIN2A tumor growth in mice compared with HT-1376/ shGFP tumors (P < 0.01), where expression of GRIN2A was decreased in HT-1376/shGRIN2A tumor cells compared with HT1376/shGFP tumor cells at the time of injection. Similarly, as a marker of proliferation, Ki-67 was decreased in tumor sections taken from HT-1376/shGRIN2A tumor cells compared with controls (Fig. 6B). These results indicate that that the silencing of GRIN2A inhibits proliferation in vitro and in vivo in a bladder tumor cell line model. We also evaluated the oncogenic effect of GRIN2A using a SV40 immortalized human urothelial cell line (SV-HUC), which is an SV40 immortalized, nontransformed, human uroepithelial cell line (39). GRIN2A was overexpressed in the SV-HUCs as shown by mRNA and protein levels (Fig. 6C), and consistent with the data in Figs. 6 A and B, overexpression of GRIN2A increased the proliferation and migration of SV-HUCs, suggesting that overexpression of GRIN2A promotes an increase in cell proliferation and migration of bladder epithelial cells.

Discussion These data reveal a spectrum of heterogeneity among sequenced bladder tumors. Based on our whole-genome sequencing analysis, we show evidence that SV breakpoints can have a unique association with copy number in the context of chromothripsis, possibly related to a process of genomic amplification. Additionally, we demonstrate that complex genomic rearrangements mechanistically use kilobase fragments of DNA that we call stitchers as part of an NHEJ DNA repair process. Furthermore, our results support the presence of intratumoral mutational heterogeneity in TCC-UB. Finally, although not related to smoking in our study, we provide evidence of a subset of tumors similar to lung non–small-cell Morrison et al.

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carcinoma in those who have never smoked (40), with a few mutations that are likely driven by one or a few driver mutations. Chromothripsis appears to be a relatively common event in TCC-UB, but its role as a “passenger” or “driver” in bladder cancer progression is not yet determined. A recent study in leukemia involving a patient with multiple recurrences provides an example of chromothripsis as a passenger event (41). In that study, chromothripsis was identified in a specimen from the time of recurrence, presumably as part of tumor progression, but it was not present in additional relapses after subsequent intervening chemotherapy. It is possible that only a subset of rearrangements in chromothripsis confers a selective single-cell advantage, much like subclonal populations of mutations that are selected through therapeutic interventions (21). Analyses of multiple samples of TCC-UB from one patient, preferably primary and metastatic tumors with some period of months to years between the two events, will be required to decipher this potential mechanism. If this process is merely a passenger event, it is more difficult to explain how evidence in both medulloblastoma (14) and melanoma (13) would suggest that chromothripsis is associated with a more aggressive clinical course. We provide some evidence that complex localized genomic rearrangements may result in some competitive advantage for neoplastic cells via gene amplification in at least one tumor (case 18195), where breakpoints precisely lined up with the well-known 6p22 amplicon containing the E2F3 and Sox4 genes (42). Another intriguing question is whether chromothripsis evolves through the same mechanism in different tumor types or is unique to TCC-UB in this regard. In our study, we showed that NHEJ is the predominant mechanism, whereas in prostate cancer, Teles Alves et al. (27) showed no evidence of microhomology involving chromosome 5 for the vertibral cancer prostate cell line. This in vitro finding contrasts to the finding of Drier et al. (33) in 95 matched tumor/normal samples that included 46 breast carcinoma samples, 23 multiple myeloma samples, 9 colorectal carcinoma samples, 7 prostate adenocarcinoma samples, 5 melanoma samples, 3 CLL samples, and 2 head and neck carcinoma samples. In these cohorts, chromothripsis was associated with all cancer types except CLL. This group gave additional evidence that chromothripsis is associated with replication time, proximity to transcribed genes, and guanine-cytosine content. The association of chromothripsis with microhomology, replication time, and proximity to transcribed genes could result from deficiencies in the replication– licensing complex that is loaded onto chromatin during the G1phase of the cell cycle and is required for initiation of DNA replication in the subsequent S-phase. In two of our tumors with MCM4 mutations, this association could be defined mechanistically by genomic alteration of the family of MCM2-7 genes (43). Consistent with this hypothesis, prior studies have shown that Mcm protein deficiencies result in high rates of cancer in mouse models (44, 45), catastrophic chromosomal rearrangements in human lymphoblasts in culture (46), and complex chromosomal alterations at discrete locations that are consistent with chromothripsis (43). Previously, we have suggested that the frequent, short intrachromosomal DELs spanning 500 kbp or less that occur in mouse tumors resulting from deficient Mcm2 protein levels could result from failure to rescue stalled replication forks within individual replication factories (43). This mechanism may also help to explain the frequent localization of multiple translocation events seen in individual tumors in the present study. For example, Fig. 7 shows the location of all structural alterations occurring within a single tumor that additionally harbors a nonsynonymous point mutation within the Mcm N domain of MCM4. Multiple translocation events occur between approximately five and six sites on each of chromosomes 4, 5, and 6, where the size of sites involved is ∼500 kbp or less. To account for the number of DNA replication forks generated during the S-phase, a single replication factory must contain 20–200 DNA

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implies GRIN2A amplification may be an early event in the progression of bladder cancer to a lethal phenotype. Hence, GRIN2A amplification may be a driver “event” in bladder cancer and appears to be independent of the more common well-known events, such as loss of p16 and TP53 mutations. GRIN2A amplification was more common in node-positive TCC-UB [11 (14%) of 81] compared with node-negative TCC-UB [12 (7%) of 166], further suggesting a role for GRIN2A amplification in the metastatic phenotype. Of interest, a higher frequency of GRIN2A amplification was noted in patients with evidence of cancer at last follow-up [12 (9%) of 127]) vs. those with no evidence of cancer [10 (5%) of 183]. However, there was no apparent association between GRIN2A amplification and survival (P = 0.24) (SI Appendix, Table S15). To evaluate the biological mechanisms and significance of GRIN2A amplification further, we compared the mRNA level of GRIN2A for 20 GRIN2A-amplified patient samples and 20 nonamplified patient samples (SI Appendix, Fig. S14). Tumors with GRIN2A amplification showed 12-fold increased expression of GRIN2A mRNA (P = 0.005), supporting the hypothesis that overexpression of GRIN2A occurs by gene amplification.

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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 (6–8 wk of age, 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 replication origins, span 0.5–5 Mb of DNA. Although it is typically assumed that replication factories assemble around domains within an individual chromosome, it is also possible that a single factory contains origins from different chromosomes (48), as shown in Fig. 7B. In this case, failure to rescue stalled replication forks could lead to the observed complex recombination events involving multiple chromosomes. It is interesting to note that the other case in our study with the highest number of both SVs and SNVs (case 19685) also had an MCM4 mutation in the same domain as the tumor in case 18195. Nonhomologous reciprocal translocations have been identified in lymphomas and sarcomas; however, these complex rearrangements, at least in TCC-UB, are different from the ones seen in lymphomas and sarcomas. Although this is not surprising, given the definition of chromothripsis and current knowledge of this genomic event, the evidence we present of a stitching process using 50- to 100-kb fragments to reconstitute these interchromosomal events is intriguing and raises questions about current theories regarding this process. The prior concept that chromothripsis results in exchange of megabase fragments of DNA from 8 of 10 | www.pnas.org/cgi/doi/10.1073/pnas.1313580111

two or more chromosomes to form a highly complex derivative chromosome may be incomplete. Our study suggests that although one chromosome provides megabase fragments of DNA, other involved chromosomes provide only 50- to 100-kb fragments that we refer to as stitchers. Although our findings do not fully define this mechanism at the current time, a plausible explanation could be that tumors use stitchers in stalled replication forks in the replication– licensing complex during the G1-phase of the cell cycle (43). Our findings provide a framework for further mechanistic investigations. Although chromothripsis and the complex process underlying this event may not lead to a driver mutation, intratumoral mutational heterogeneity leading to driver events is likely, at least in leukemia (19, 21). In a comparative sense, it could be possible that intratumoral mutational heterogeneity is a marker of underlying genomic events, much as we postulate that chromothripsis is related to a defective replication–licensing complex. Intratumoral mutational heterogeneity may be associated with resistance to chemotherapy and/or advanced stage at the time of presentation (21). In our study, both cases that showed evidence of intratumoral mutational heterogeneity were also TP53mut, whereas among the three cases with no evidence of intratumoral mutational heterogeneity, Morrison et al.

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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 metabolism may be important in the etiology of other tumors (glioblastoma) (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.

Fig. 7. Model of microhomology-mediated translocation events occurring between two or more chromosomes in an individual replication factory. (A) Illustration of multiple translocation events occurring between chr4, chr5, and chr6 for case 18195, with each line denoting an individual CTX. Note that many of the translocations between any two chromosomes often show a second breakpoint within a few thousand base pairs as part of a different translocation, with the third chromosome resulting in this complex web-like pattern of rearrangement. (B) DNA replication factory involving portions of chr4, chr5, and chr6, with stalled replication forks indicated by red x marks. Dashed lines indicate translocation occurring at stalled replication forks, often with closely adjacent breakpoints involving multiple chromosomes in a complicated web-like fashion.

one was TP53mut and the other two were TP53wt. Early clonal expansion of TP53-mutated cells would be predicted to lead to increased genetic heterogeneity through lack of sufficient DNA repair processes. It is feasible that the final evaluation of this topic can be done with exomic sequencing and will not require sequencing of the entire genome. Progress in developing more targeted therapies in TCC-UB will be informed by further analysis of this genomic event. In the more “genomically simple” subset of TCC-UB, we identified unique events that included (i) an unequivocal demonstration that amplified CTXs can be found in carcinomas and (ii) infrequent translocation but frequent amplification of GRIN2A in a subset of TCC-UB. Furthermore, our preliminary functional studies in bladder carcinoma cell lines support GRIN2A as a candidate oncogene in TCC-UB. Although the first of these unique findings was only demonstrated in the index case (case 16933), it does demonstrate the utility of NGS as a discovery tool and likely portends the discovery of additional such examples in bladder cancer and other carcinomas as this technology expands. In our study, this initial discovery of an SCN8A-GRIN2A translocation was further noted in the validation cohort. However, this observation led us to the discovery of a second unique finding, amplification of GRIN2A, in a subset of TCC-UB. Previous findings of GRIN2A as a frequently Morrison et al.

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 protocol using the Illumina cBot and HiSeq paired-end cluster kit, version 1 (SI Appendix, SI Methods). Read Mapping and Alignment and Variant Analysis. We recently developed an in-house analysis pipeline for cancer genome sequencing data that includes (i) mapping and alignment, (ii) SNV and indel discovery, and (iii) SNV and indel filtering and annotation (SI Appendix, SI Methods). Detecting SVs. BreakDancer was used to detect SVs from paired-end Illumina sequencing data. Then, the de novo assembly was performed for all filtered DELs, insertions, and INVs using the newly developed sensitive assembler TIGRA_SV (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). PCR Validation of SVs. Putative SVs were validated by PCR using R script to select genomic sequences around the de novo assembly-determined breakpoints for each SV from the University of California, Santa Cruz genome browser (http://genome.ucsc.edu/) (SI Appendix, SI Methods). Detection of Somatic Copy Number Alteration. To identify somatic copy number alterations, each tumor and its matched normal DNA were genotyped using Illumina HumanOmni1-Quad BeadChips, which contain 1,140,419 SNPs, with a median SNP spacing of 1.2 kb (SI Appendix, SI Methods). FISH for SVs. All SVs in this study were evaluated by FISH using RP11 clones 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 breakpoints of a given SV were evaluated separately using a break-apart probe FISH design (SI Appendix, SI Methods). RT-PCR Analysis for GRIN2A mRNA Expression. Real-time RT-PCR analysis was done using SYBR Green I as a reporter and ROX (Applied Biosystems) as a reference dye for GRIN2A mRNA expression (SI Appendix, SI Methods). Statistical Analysis. The association between clinical/histological covariates and 6p22 amplification was tested using two-sample t tests for the equality of proportions. Survival time associations were tested with a log-rank test. Statistical analysis of data was performed using the SPSS Statistics software package (IBM). All results are expressed as mean ± SD.

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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).

In Vitro Tumor Assays. Human bladder cancer cell lines 253J and HT-1376, as well as SV40 immortalized human uroepithelial SV-HUCs, were cultured, and transfection was performed using X-tremeGENE 9 DNA Transfection Reagent following the manufacturer’s protocol (Roche). Packaging of retrovirus and lentivirus, cell transduction, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, Western blotting, shRNA knockdown experiments, and migration assays were performed following standard protocols (SI Appendix, SI Methods).

ACKNOWLEDGMENTS. Biospecimens or research pathology services for this study were provided by the Pathology Resource Network, which is funded by the National Cancer Institute and is a Roswell Park Cancer Institute Cancer Center Support Grant shared resource. Clinical data delivery and honest broker services for this study were provided by the Clinical Data Network, which is funded by the National Cancer Institute and is a Roswell Park Cancer Institute Cancer Center Support Grant shared resource.

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Morrison et al.