Frequent inactivating mutations of STAG2 in bladder cancer are associated with low tumour grade and stage and inversely related to chromosomal copy number changes.

Inactivating mutations of STAG2 have been reported at low frequency in several cancers. In glioblastoma, the function of STAG2 has been related to maintenance of euploidy via its role in the cohesin complex. In a screen of a large series of bladder tumours and cell lines, we found inactivating mutations (nonsense, frameshift and splicing) in 67 of 307 tumours (21.8%) and 6 of 47 cell lines. Thirteen missense mutations of unknown significance were also identified. Inactivating mutation was associated with low tumour stage (P = 0.001) and low grade (P = 0.0002). There was also a relationship with female patient gender (P = 0.042). Examination of copy number profiles revealed an inverse relationship of mutation with both fraction of genome altered and whole chromosome copy number changes. Immunohistochemistry showed that in the majority of cases with inactivating mutations, STAG2 protein expression was absent. Strikingly, we identified a relatively large subset of tumours (12%) with areas of both positive and negative immunoreactivity, in only four of which a potentially function-altering mutation was detected. Regions of differential expression were contiguous and showed similar morphological phenotype in all cases. Microdissected positive and negative areas from one tumour showed an inactivating mutation to be present only in the negative area, suggesting intra-tumoral sub-clonal genomic evolution. Our findings indicate that loss of STAG2 function plays a more important role in non-invasive than that in muscle-invasive bladder cancer and suggest that cohesin complex-independent functions are likely to be important in these cases.

Prevalence and predictors of germline CDKN2A mutations for melanoma cases from Australia, Spain and the United Kingdom.

BACKGROUND: Mutations in the CDKN2A and CDK4 genes predispose to melanoma. From three case-control studies of cutaneous melanoma, we estimated the prevalence and predictors of these mutations for people from regions with widely differing latitudes and melanoma incidence. METHODS: Population-based cases and controls from the United Kingdom (1586 cases, 499 controls) and Australia (596 early-onset cases, 476 controls), and a hospital-based series from Spain (747 cases, 109 controls), were screened for variants in all exons of CDKN2A and the p16INK4A binding domain of CDK4. RESULTS: The prevalence of mutations for people with melanoma was similar across regions: 2.3%, 2.5% and 2.0% for Australia, Spain and the United Kingdom respectively. The strongest predictors of carrying a mutation were having multiple primaries (odds ratio (OR) = 5.4, 95% confidence interval (CI: 2.5, 11.6) for 2 primaries and OR = 32.4 (95% CI: 14.7, 71.2) for 3 or more compared with 1 primary only); and family history (OR = 3.8; 95% CI:1.89, 7.5) for 1 affected first- or second-degree relative and OR = 23.2 (95% CI: 11.3, 47.6) for 2 or more compared with no affected relatives). Only 1.1% of melanoma cases with neither a family history nor multiple primaries had mutations. CONCLUSIONS: There is a low probability (<2%) of detecting a germline CDKN2A mutation in people with melanoma except for those with a strong family history of melanoma (≥2 affected relatives, 25%), three or more primary melanomas (29%), or more than one primary melanoma who also have other affected relatives (27%).

An integrated genomic, transcriptional and protein investigation of FGFRL1 as a putative 4p16.3 deletion target in bladder cancer.

Loss of heterozygosity (LOH) of chromosome arm 4p is a common event in bladder and other malignancies. At least three distinct regions of deletion have been identified, but the deletion targets have so far remained elusive. In this study, we have identified a novel region of deletion mapping to 4p16.3 spanning 0-2.1 Mb, in 15% of bladder tumors and 24% of bladder cancer cell lines. FGFRL1, which maps within this region, was investigated as putative deletion target. The retained FGFRL1 allele was not mutated in cell lines and tumors with LOH, although in patients heterozygous for the rs4647930 functional polymorphism, the common allele was preferentially lost in tumor tissue. Epigenetic silencing of the retained allele was also excluded as levels of FGFRL1 mRNA and protein were similar in cell lines and tumors with and without 4p16.3 loss. However, while FGFRL1 protein was moderately expressed in all layers of the normal bladder epithelium, the majority of tumors showed areas of downregulation. Overall, average FGFRL1 protein expression was significantly lower in bladder tumors compared to normal tissue, but downregulation was independent from 4p16.3 LOH status, FGFR3 mutation, and tumor grade and stage. In conclusion, although we found no evidence supporting a "two-hit" inactivation of FGFRL1 in bladder carcinogenesis, the effect of heterozygous deletion coupled with functional polymorphisms, and the role of post-transcriptional downregulation deserves further investigation.

Cytokine receptor IL27RA is an NF-κB-responsive gene involved in CD38 upregulation in multiple myeloma.

Multiple myeloma (MM) shows constitutive activation of canonical and noncanonical nuclear factor κB (NF-κB) signaling via genetic mutations or tumor microenvironment (TME) stimulations. A subset of MM cell lines showed dependency for cell growth and survival on the canonical NF-κB transcription factor RELA alone, suggesting a critical role for a RELA-mediated biological program in MM pathogenesis. Here, we determined the RELA-dependent transcriptional program in MM cell lines and found the expression of the cell surface molecules interleukin-27 receptor-α (IL-27Rα) and the adhesion molecule JAM2 to be responsive to RELA at the messenger RNA and protein levels. IL-27Rα and JAM2 were expressed on primary MM cells at higher levels than on healthy long-lived plasma cells (PCs) in the bone marrow. IL-27 activated STAT1, and to a lesser extent STAT3, in MM cell lines and in PCs generated from memory B cells in an IL-21-dependent in vitro PC differentiation assay. Concomitant activity of IL-21 and IL-27 enhanced differentiation into PCs and increased the cell-surface expression of the known STAT target gene CD38. In accordance, a subset of MM cell lines and primary MM cells cultured with IL-27 upregulated CD38 cell-surface expression, a finding with potential implications for enhancing the efficacy of CD38-directed monoclonal antibody therapies by increasing CD38 expression on tumor cells. The elevated expression of IL-27Rα and JAM2 on MM cells compared with that on healthy PCs may be exploited for the development of targeted therapeutic strategies that modulate the interaction of MM cells with the TME.

The role of microRNA-binding site polymorphisms in DNA repair genes as risk factors for bladder cancer and breast cancer and their impact on radiotherapy outcomes.

MicroRNAs (miRNAs) are involved in post-transcriptional regulation of gene expression through binding to messenger RNAs (mRNA) thereby promoting mRNA degradation or altered translation. A single-nucleotide polymorphism (SNP) located within a miRNA-binding site could thus alter mRNA translation and influence cancer risk and treatment response. The common SNPs located within the 3'-untranslated regions of 20 DNA repair genes were analysed for putative miRNA-binding sites using bioinformatics algorithms, calculating the difference in Gibbs free binding energy (ΔΔG) for each wild-type versus variant allele. Seven SNPs were selected to be genotyped in germ line DNAs both from a bladder cancer case-control series (752 cases and 704 controls) and 202 muscle-invasive bladder cancer radiotherapy cases. The PARP-1 SNP rs8679 was also genotyped in a breast cancer case-control series (257 cases and 512 controls). Without adjustment for multiple testing, multivariate analysis demonstrated an association with increased bladder cancer risk with PARP1 rs8679 (P(trend) = 0.05) while variant homozygotes of PARP1 rs8679 were also noted to have an increased breast cancer risk (P = 0.03). In the radiotherapy cases, carriers of the RAD51 rs7180135 minor allele had improved cancer-specific survival (hazard ratio 0.52, 95% confidence interval 0.31-0.87, P = 0.01). This is the first report of associations between DNA repair gene miRNA-binding site SNPs with bladder and breast cancer risk and radiotherapy outcomes. If validated, these findings may give further insight into the biology of bladder carcinogenesis, allow testing of the RAD51 SNP as a potential predictive biomarker and also reveal potential targets for new cancer treatments.

Melanoma risk for CDKN2A mutation carriers who are relatives of population-based case carriers in Australia and the UK.

BACKGROUND: CDKN2A mutations confer a substantial risk of cutaneous melanoma; however, the magnitude of risk is uncertain. METHODS: The study estimated the hazard ratio (HR) and the average age specific cumulative risk (ie, penetrance) of reported melanoma for CDKN2A mutation carriers in case families using a modified segregation analysis of the first and higher degree relatives of 35 population-based cases. The study sample included 223 relatives of 13 melanoma cases diagnosed when aged 18-39 years from Melbourne, Sydney and Brisbane, Australia, and 322 relatives of 22 melanoma cases diagnosed at any age from Yorkshire, UK. RESULTS: The estimated HR for melanoma for mutation carriers relative to the general population decreased with regions of increasing ambient ultraviolet (UV) irradiance, being higher for the UK than Australia (87, 95% CI 50 to 153 vs 31, 95% CI 20 to 50, p=0.008), and across Australia, 49 (95% CI 24 to 98) for Melbourne, 44 (95% CI 22 to 88) for Sydney, and 9 (95% CI 2 to 33) for Brisbane (p=0.02). Penetrance did not differ by geographic region. It is estimated that 16% (95% CI 10% to 27%) of UK and 20% (95% CI 13% to 30%) of Australian CDKN2A mutation carriers would be diagnosed with melanoma by age 50 years, and 45% (95% CI 29% to 65%) and 52% (95% CI 37% to 69%), respectively, by age 80 years. CONCLUSIONS: Contrary to the strong association between UV radiation exposure and melanoma risk for the general population, CDKN2A mutation carriers appear to have the same cumulative risk of melanoma irrespective of the ambient UV irradiance of the region in which they live.

In vitro functional effects of XPC gene rare variants from bladder cancer patients.

The XPC gene is involved in repair of bulky DNA adducts formed by carcinogenic metabolites and oxidative DNA damage, both known bladder cancer risk factors. Single nucleotide polymorphisms (SNPs) in XPC have been associated with increased bladder cancer risk. Recently, rarer genetic variants have been identified but it is difficult to ascertain which are of functional importance. During a mutation screen of XPC in DNA from 33 bladder tumour samples and matched blood samples, we identified five novel variants in the patients' germ line DNA. In a case-control study of 771 bladder cancer cases and 800 controls, c.905T>C (Phe302Ser), c.1177C>T (Arg393Trp), c.*156G>A [3' untranslated region (UTR)] and c.2251-37C>A (in an intronic C>G SNP site) were found to be rare variants, with a combined odds ratio of 3.1 (95% confidence interval 1.0-9.8, P=0.048) for carriage of one variant. The fifth variant was a 2% minor allele frequency SNP not associated with bladder cancer. The two non-synonymous coding variants were predicted to have functional effects using analytical algorithms; a reduced recruitment of GFP-tagged XPC plasmids containing either c.905T>C or c.1177C>T to sites of 408 nm wavelength laser-induced oxidative DNA damage was found in vitro. c.*156G>A appeared to be associated with reduced messenger RNA stability in an in vitro plasmid-based assay. Although the laser microbeam assay is relevant to a range of DNA repair genes, our 3' UTR assay based on Green fluorescent protein(GFP) has widespread applicability and could be used to assess any gene. These assays may be useful in determining which rare variants are functional, prior to large genotyping efforts.

High-resolution analysis of genomic alteration on chromosome arm 8p in urothelial carcinoma.

Loss of chromosome arm 8p, sometimes in combination with amplification of proximal 8p, is found in urothelial carcinoma (UC) and other epithelial cancers and is associated with more advanced tumor stage. We carried out array comparative genomic hybridization on 174 UC and 33 UC cell lines to examine breakpoints and copy number. This was followed by a detailed analysis of the cell lines using fluorescence in situ hybridization (FISH) and, in some cases, M-FISH, to refine breakpoints and determine translocation partners, heterozygosity analysis, and analysis of expression of selected genes. We showed an overall pattern of 8p loss with reduced heterozygosity and reduced gene expression. Amplification was seen in some samples and shown in the cell line JMSU1 to correlate with overexpression of ZNF703, ERLIN2, PROSC, GPR124, and BRF2. Apart from the centromere, no single breakpoint was overrepresented, and we postulate that frequent complex changes without consistent breakpoints reflect the need for alterations of combinations of genes. The region around 2 Mb, which was homozygously deleted in one cell line and includes the gene ARHGEF10 and the micro-RNA hsa-mir-596, is one candidate tumor suppressor gene region.

Mutation scanning using high-resolution melting.

Mutation scanning techniques are used to detect sequence variants without the need for prior knowledge of the identity or precise location of the variant, in contrast with genotyping techniques, which determine the status of a specific variant. High-resolution melting is a recently developed method that shows great potential as a mutation scanning technique. Sensitivity and specificity for mutation detection are extremely high and the technique also has advantages of cost and throughput. Practical considerations for successful mutation scanning by high-resolution melting are also discussed in this review.

Heart allograft acceptance induced by anti-CD3 antibody in high-responder rats: effect on foxp3 and cytokine expression and graft infiltration.

The ability of anti-T cell monoclonal antibody G4.18 and polyclonal anti-lymphocyte serum (ALS) to induce long-term graft survival was examined in a high-responder rat heart transplant model. Heterotopic heart allografts were performed from PVG rat strain donors to high-responder Lewis recipients. Immunosuppressive properties of G4.18 and ALS were investigated by immunohistochemistry and PCR analysis. Untreated graft rejection was 8.5 days while treatment with 1 ml ALS prolonged survival to 11.5 days (p=0.01). Treatment with 7 mg/kg G4.18 on days 1 and 3 prolonged survival to >100 days (p=0.002 vs. control and p=0.002 vs. ALS) but did not induce tolerance. Acceptance was associated with marked inhibition of cellular infiltration and inflammatory cytokine expression and only a brief, slight increase in Foxp3:T cell ratio in the graft and no increase in the spleen. In conclusion, G4.18 treatment led to long-term heart transplant survival associated with marked inhibition of early inflammation. Failure to develop tolerance was associated with a lack of early accumulation of Foxp3 cells in the graft or spleen.

Genomic Subtypes of Non-invasive Bladder Cancer with Distinct Metabolic Profile and Female Gender Bias in KDM6A Mutation Frequency.

Bladder cancer incurs a higher lifetime treatment cost than other cancers due to frequent recurrence of non-invasive disease. Improved prognostic biomarkers and localized therapy are needed for this large patient group. We defined two major genomic subtypes of primary stage Ta tumors. One of these was characterized by loss of 9q including TSC1, increased KI67 labeling index, upregulated glycolysis, DNA repair, mTORC1 signaling, features of the unfolded protein response, and altered cholesterol homeostasis. Comparison with muscle-invasive bladder cancer mutation profiles revealed lower overall mutation rates and more frequent mutations in RHOB and chromatin modifier genes. More mutations in the histone lysine demethylase KDM6A were present in non-invasive tumors from females than males.

FGFR3 and Ras gene mutations are mutually exclusive genetic events in urothelial cell carcinoma.

Fibroblast growth factor receptor 3 (FGFR3) mutations are frequent in superficial urothelial cell carcinoma (UCC). Ras gene mutations are also found in UCC. As oncogenic activation of both FGFR3 and Ras is predicted to result in stimulation of the mitogen-activated protein kinase (MAPK) pathway, we hypothesized that these might be mutually exclusive events. HRAS mutation has been widely studied in UCC, but all three Ras gene family members have not been screened for mutation in the same sample series. We screened 98 bladder tumours and 31 bladder cell lines for mutations in FGFR3, HRAS, NRAS and KRAS2. FGFR3 mutations were present in 54 tumours (55%) and three cell lines (10%), and Ras gene mutations in 13 tumours (13%) and four cell lines (13%). These included mutations in all three Ras genes; ten in HRAS, four in KRAS2 and four in NRAS and these were not associated with either tumour grade or stage. In no cases were Ras and FGFR3 mutation found together. This mutual exclusion suggests that FGFR3 and Ras gene mutation may represent alternative means to confer the same phenotype on UCC cells. If these events have biological equivalence, Ras mutant invasive UCC may represent a novel subgroup.

A mutation hotspot at the p14ARF splice site.

Germline mutations of CDKN2A that affect the p16INK4a transcript have been identified in numerous melanoma pedigrees worldwide. In the UK, over 50% of pedigrees with three or more cases of melanoma have been found to carry mutations of CDKN2A. Mutations that affect p14ARF exon 1beta exclusively are very rare. This has led to the suggestion that it is p16INK4a and not p14ARF that plays the critical role in melanoma predisposition. We report the identification of a cluster of five different germline mutations at the p14ARF exon 1beta splice donor site in melanoma pedigrees. All the five splice site variants showed evidence of being causal mutations. Three of the variants were demonstrated to result in aberrant splicing of the p14ARF mRNA, confirming their role in melanoma predisposition. No other point mutations were identified in the coding region of p14ARF. The p14ARF transcript of CDKN2A is clearly important in disease predisposition in a subset of melanoma pedigrees. Curiously, the only mutations so far reported to affect p14ARF exon 1beta exclusively have been knockout mutations. Further investigation into the spectrum of mutations observed in this gene may help clarify the exact role of p14ARF in melanoma predisposition.

The conundrum of claudication.

Stable claudication has traditionally been treated conservatively by many clinicians as operative therapies involve considerable risk for a condition that is often slowly progressive and non-fatal. The relative safety of less invasive endovascular techniques brings potential survival benefits from the increased exercise tolerance that result. We aimed to revisit and clarify the aetiologies of intermittent claudication in a review of the rarer causes that can mimic atherosclerotic occlusive disease. An extensive search of Medline, Embase and the Cochrane databases was carried out to compile published work addressing the aetiology of claudication and specific non-atherosclerotic causes. The reference lists of these manuscripts were also searched for relevant articles. There are several vasculogenic and neurogenic causes for intermittent claudication, many of which are unrelated to atherosclerosis. Recognition of these rarer syndromes is essential when planning endovascular or operative management strategies. Consideration of non-atherosclerotic differential diagnoses is recommended when assessing the patient with intermittent claudication. This is particularly critical in the young patient whose pattern of symptoms and risk factors may not fit precisely with atherosclerosis.

Expression of hypoxia-inducible factors in human renal cancer: relationship to angiogenesis and to the von Hippel-Lindau gene mutation.

The von Hippel-Lindau tumor suppressor protein acts as the substrate recognition component of a ubiquitin E3 ligase that targets hypoxia-inducible factor (HIF)-alpha subunits for proteolysis. Stabilization of HIF-alpha subunits has been described in VHL-defective cell lines, leading to HIF activation and up-regulation of hypoxia-inducible mRNAs. Mutations of the von Hippel-Lindau tumor suppressor protein are found in most clear cell renal cell carcinomas (CC-RCCs) but not other renal tumors, raising a question about the importance of activation of the HIF pathway in CC-RCC development. To address this question, we have examined the expression of HIF-alpha subunits in 45 primary renal tumors and related this to tumor subtype, the presence of VHL mutations, and measures of angiogenesis. We show that HIF-alpha is up-regulated in the majority of CC-RCCs, and that the pattern of expression is biased toward the HIF-2alpha isoform. Expression of HIF-alpha proteins was associated significantly with up-regulation of VEGF mRNA and protein and increased microvessel density. Up-regulation of HIF-alpha in CC-RCC was found to involve increased mRNA as well as protein expression, suggesting that both VHL-dependent and VHL-independent mechanisms are involved. These results suggest that activation of the HIF pathway is functionally important in CC-RCC development and might provide a new therapeutic target.

Mutation analysis of the 8p candidate tumour suppressor genes DBC2 (RHOBTB2) and LZTS1 in bladder cancer.

Genomic deletions of the short arm of chromosome 8 are common in many human cancers and are frequently associated with a more aggressive tumour phenotype. One of the regions of loss of heterozygosity (LOH) on 8p22 identified in bladder cancer contains two genes, LZTS1 (FEZ1) and DBC2 (RHOBTB2) that have been shown to be mutated at low frequency in other cancers. We screened a panel of bladder tumours and bladder tumour-derived cell lines for mutations in these genes. Forty two percent of the tumours were found to have LOH in the 8p22 region and many of the cell lines have known loss of 8p. Several known polymorphisms and novel polymorphisms were detected. One possible mutation of LZTS1 (G374S) was found in a cell line. The functional significance of this is unknown but the novel serine residue created may represent a novel phosphorylation site. In DBC2, we found a single somatic mutation in a tumour (E349D) that lies in a highly conserved region of the protein. mRNA levels for both genes were reduced in the majority of bladder cancer cell lines. We conclude that neither LZTS1 nor DBC2 is commonly mutated in bladder cancer. However, neither can yet be excluded as the target of 8p22 LOH. The finding of a somatic mutation of DBC2 in a tumour sample and the down-regulation of both gene transcripts in bladder tumour cell lines may indicate that an alternative mechanism of inactivation of the second allele, for example promoter hypermethylation, is more common than mutation and this must now be examined.

Diverticulectomy is inadequate treatment for short Meckel's diverticulum with heterotopic mucosa.

BACKGROUND: Meckel's diverticulum is a vestigial remnant of the vitellointestinal duct that may occasionally contain heterotopic gastric mucosa thought to arise from residual yolk sac cells. This may cause significant rectal bleeding, the source of which may be difficult to identify. The present paper addresses the question of whether the choice of resection technique should depend on the macroscopic appearance of the Meckel's diverticulum. METHODS: A retrospective analysis of patients with resected Meckel's diverticulum at Prince of Wales and Sydney Children's Hospitals between 1992 and May 2003 was performed. The external appearance was expressed as a height-to-diameter ratio (HDR) and the presence or absence of macroscopic thickening was recorded. The morphology was then correlated with the presence and site of the heterotopic gastric mucosa (HGM). RESULTS: Seventy-seven patients were identified with an age range between 1 day and 92 years. Fifty-seven (74%) of the patients were men. Presenting symptoms were gastrointestinal bleeding (11.7%), diverticulitis (15.6%), volvulus (2.6%), intussusception (10%) and umbilical fistula (7.8%). Fifty-seven per cent of the resected Meckel's diverticulae were found incidentally. Eight patients underwent a technetium pertechnate nuclear Meckel's scan. The Meckel's scan detected only two of seven patients with HGM on pathological examination. Twenty-nine (38%) patients underwent diverticulectomy and 48 (62%) small bowel resection. Ectopic mucosa was found in 25 (32.5%) patients. Of the Meckel's diverticula that were defined as long (HDR >or=2.0) and containing HGM, five of five (100%) had the ectopic mucosa in the diverticular tip and body only. Of those that were short (HDR <2.0) there was a wide distribution of HGM sites with 12 (60%) involving the whole diverticulum including the base and eight (40%) involving the tip and body only. The presence or absence of macroscopic thickening was described in 18 resected Meckel's diverticula. Thirteen (72%) were described as thickened in the operation report and six of these 13 (46%) were found to have HGM. One of the seven (14%) Meckel's diverticulae with HGM was thought to be of normal appearance and was therefore undetected. CONCLUSION: Simple transverse resection is not recommended for the short Meckel's diverticulum. A HDR of 2.0 is recommended as the cut-off when deciding on the most appropriate operation. The external appearance of the Meckel's diverticulum does not predict the presence of HGM and is therefore an unreliable indicator to aid resection decisions when presented with an incidental Meckel's diverticulum.

Identification of mutations in distinct regions of p85 alpha in urothelial cancer.

Bladder cancers commonly show genetic aberrations in the phosphatidylinositol 3-kinase signaling pathway. Here we have screened for mutations in PIK3R1, which encodes p85α, one of the regulatory subunits of PI3K. Two hundred and sixty-four bladder tumours and 41 bladder tumour cell lines were screened and 18 mutations were detected. Thirteen mutations were in C-terminal domains and are predicted to interfere with the interaction between p85α and p110α. Five mutations were in the BH domain of PIK3R1. This region has been implicated in p110α-independent roles of p85α, such as binding to and altering the activities of PTEN, Rab4 and Rab5. Expression of these mutant BH-p85α forms in mouse embryonic fibroblasts with p85α knockout indicated that all forms, except the truncation mutants, could bind and stabilize p110α but did not increase AKT phosphorylation, suggesting that BH mutations function independently of p110α. In a panel of 44 bladder tumour cell lines, 80% had reduced PIK3R1 mRNA expression relative to normal urothelial cells. This, along with mutation of PIK3R1, may alter BH domain functioning. Our findings suggest that mutant forms of p85α may play an oncogenic role in bladder cancer, not only via loss of ability to regulate p110α but also via altered function of the BH domain.

Novel tumor subgroups of urothelial carcinoma of the bladder defined by integrated genomic analysis.

PURPOSE: There is a need for improved subclassification of urothelial carcinoma (UC) at diagnosis. A major aim of this study was to search for novel genomic subgroups. EXPERIMENTAL DESIGN: We assessed 160 tumors for genome-wide copy number alterations and mutation in genes implicated in UC. These comprised all tumor grades and stages and included 49 high-grade stage T1 (T1G3) tumors. RESULTS: Our findings point to the existence of genomic subclasses of the "gold-standard" grade/stage groups. The T1G3 tumors separated into 3 major subgroups that differed with respect to the type and number of copy number events and to FGFR3 and TP53 mutation status. We also identified novel regions of copy number alteration, uncovered relationships between molecular events, and elucidated relationships between molecular events and clinico-pathologic features. FGFR3 mutant tumors were more chromosomally stable than their wild-type counterparts and a mutually exclusive relationship between FGFR3 mutation and overrepresentation of 8q was observed in non-muscle-invasive tumors. In muscle-invasive (MI) tumors, metastasis was positively associated with losses of regions on 10q (including PTEN), 16q and 22q, and gains on 10p, 11q, 12p, 19p, and 19q. Concomitant copy number alterations positively associated with TP53 mutation in MI tumors were losses on 16p, 2q, 4q, 11p, 10q, 13q, 14q, 16q, and 19p, and gains on 1p, 8q, 10q, and 12q. Significant complexity was revealed in events affecting chromosome 9. CONCLUSIONS: These findings may lead to improved biologic understanding and the development of prognostic biomarkers. Novel regions of copy number alteration may reveal potential therapeutic targets.