Correspondence to M.A.K. m.a.knowles@leeds.ac.uk 2 Preface Urothelial carcinoma of the bladder comprises two long-recognised disease entities with distinct molecular features and clinical outcome. Low-grade, non-muscle-invasive tumours recur frequently but rarely progress to muscle invasion, whereas muscle invasive tumours are usually diagnosed de novo and frequently metastasize. Recent genome-wide expression and sequencing studies identify genes and pathways that are key drivers of urothelial cancer and reveal a more complex picture with multiple molecular subclasses that cut across conventional grade and stage groupings. This improved understanding of molecular features, disease pathogenesis and heterogeneity provides new opportunities for prognostic application, disease monitoring and personalised therapy.Bladder cancer is the most common cancer of the urinary tract with approximately 380,000 new cases and 150,000 deaths per year worldwide 1 . It ranks fifth among cancers in men in Western countries.Epidemiological studies identify a range of environmental risk factors, many of which reflect exposure to excreted carcinogenic molecules (BOX 1). Recent genome-wide association studies have also identified germline variants that contribute to risk 2 .In Europe and North America, more than 90% of bladder cancers are urothelial carcinoma. These tumours are staged using the Tumour Nodes Metastasis (TNM) system 3 , which describes the extent of invasion (Tis-T4), and graded according to their cellular characteristics. Two classification systems are in current use 4, 5 .At diagnosis the majority of bladder cancers (~ 60%) are non-muscle-invasive (NMIBC) (stage Ta) NMIBCs frequently recur (50-70%) but infrequently progress to invasion (10-15%) 6 and five-year survival is ~90%. These patients are monitored by cystoscopy and may have multiple resections over many years.Improved monitoring is needed, ideally via urine analysis, which could reduce the morbidity and costs associated with cystoscopy. Although risk tables provide a prognostic tool 7 , no molecular biomarkers accurately predict disease progression. For these patients, localised therapies to remove residual neoplastic and preneoplastic cells post-resection may have major impacts on both quality of life and in health economic terms. MIBCs (>stage T2) have less favourable prognosis with five-year survival <50% and common progression to metastasis (BOX1). Treatment has not advanced for several decades and new approaches to systemic therapy are needed 8 .Improved treatment requires detailed understanding of urothelial carcinoma pathogenesis and molecular biology. A model has evolved, taking into account both histopathological and molecular features. This socalled 'two-pathway' model proposes that papillary NMIBC develops via epithelial hyperplasia and recruitment of a branching vasculature. MIBCs are proposed to develop via flat dysplasia and carcinoma in situ (CIS). The molecular characteristics of MIBC and NMIBC are highly distinct (Tables 1 and 2). Whilst 3 m...

The protein-kinase family is the most frequently mutated gene family found in human cancer and faulty kinase enzymes are being investigated as promising targets for the design of antitumour therapies. We have sequenced the gene encoding the transmembrane protein tyrosine kinase ERBB2 (also known as HER2 or Neu) from 120 primary lung tumours and identified 4% that have mutations within the kinase domain; in the adenocarcinoma subtype of lung cancer, 10% of cases had mutations. ERBB2 inhibitors, which have so far proved to be ineffective in treating lung cancer, should now be clinically re-evaluated in the specific subset of patients with lung cancer whose tumours carry ERBB2 mutations.

Bladder cancer is a highly prevalent disease and is associated with substantial morbidity, mortality and cost. Environmental or occupational exposures to carcinogens, especially tobacco, are the main risk factors for bladder cancer. Most bladder cancers are diagnosed after patients present with macroscopic haematuria, and cases are confirmed after transurethral resection of bladder tumour (TURBT), which also serves as the first stage of treatment. Bladder cancer develops via two distinct pathways, giving rise to non-muscle-invasive papillary tumours and non-papillary (solid) muscle-invasive tumours. The two subtypes have unique pathological features and different molecular characteristics. Indeed, The Cancer Genome Atlas project identified genetic drivers of muscle-invasive bladder cancer (MIBC) as well as subtypes of MIBC with distinct characteristics and therapeutic responses. For non-muscle-invasive bladder cancer (NMIBC), intravesical therapies (primarily Bacillus Calmette-Guérin (BCG)) with maintenance are the main treatments to prevent recurrence and progression after initial TURBT; additional therapies are needed for those who do not respond to BCG. For localized MIBC, optimizing care and reducing morbidity following cystectomy are important goals. In metastatic disease, advances in our genetic understanding of bladder cancer and in immunotherapy are being translated into new therapies.

We conducted a multi-stage, genome-wide association study (GWAS) of bladder cancer with a primary scan of 589,299 single nucleotide polymorphisms (SNPs) in 3,532 cases and 5,120 controls of European descent (5 studies) followed by a replication strategy, which included 8,381 cases and 48,275 controls (16 studies). In a combined analysis, we identified three new regions associated with bladder cancer on chromosomes 22q13.1, 19q12 and 2q37.1; rs1014971, (P=8×10−12) maps to a non-genic region of chromosome 22q13.1; rs8102137 (P=2×10−11) on 19q12 maps to CCNE1; and rs11892031 (P=1×10−7) maps to the UGT1A cluster on 2q37.1. We confirmed four previous GWAS associations on chromosomes 3q28, 4p16.3, 8q24.21 and 8q24.3, validated previous candidate associations for the GSTM1 deletion (P=4×10−11) and a tag SNP for NAT2 acetylation status (P=4×10−11), as well as demonstrated smoking interactions with both regions. Our findings on common variants associated with bladder cancer risk should provide new insights into mechanisms of carcinogenesis.

FGF receptor 3 (FGFR3) is activated by mutation or over-expression in many bladder cancers. Here, we identify an additional mechanism of activation via chromosomal re-arrangement to generate constitutively activated fusion genes. FGFR3–transforming acid coiled coil 3 (TACC3) fusions resulting from 4p16.3 re-arrangements and a t(4;7) that generates a FGFR3-BAI1-associated protein 2-like 1 (BAIAP2L1) fusion were identified in 4 of 43 bladder tumour cell lines and 2 of 32 selected tissue samples including the tumour from which one of the cell lines was derived. These are highly activated and transform NIH-3T3 cells. The FGFR3 component is identical in all cases and lacks the final exon that includes the phospholipase C gamma 1 (PLCγ1) binding site. Expression of the fusions in immortalized normal human urothelial cells (NHUC) induced activation of the mitogen-activated protein kinase pathway but not PLCγ1. A protein with loss of the terminal region alone was not as highly activated as the fusion proteins, indicating that the fusion partners are essential. The TACC3 fusions retain the TACC domain that mediates microtubule binding and the BAIAP2L1 fusion retains the IRSp53/MIM domain (IMD) that mediates actin binding and Rac interaction. As urothelial cell lines with FGFR3 fusions are extremely sensitive to FGFR-selective agents, the presence of a fusion gene may aid in selection of patients for FGFR-targeted therapy.

Protein kinases are frequently mutated in human cancer and inhibitors of mutant protein kinases have proven to be effective anticancer drugs. We screened the coding sequences of 518 protein kinases (f1.3 Mb of DNA per sample) for somatic mutations in 26 primary lung neoplasms and seven lung cancer cell lines. One hundred eighty-eight somatic mutations were detected in 141 genes. Of these, 35 were synonymous (silent) changes. This result indicates that most of the 188 mutations were ''passenger'' mutations that are not causally implicated in oncogenesis. However, an excess of f40 nonsynonymous substitutions compared with that expected by chance (P = 0.07) suggests that some nonsynonymous mutations have been selected and are contributing to oncogenesis. There was considerable variation between individual lung cancers in the number of mutations observed and no mutations were found in lung carcinoids. The mutational spectra of most lung cancers were characterized by a high proportion of C:G > A:T transversions, compatible with the mutagenic effects of tobacco carcinogens. However, one neuroendocrine cancer cell line had a distinctive mutational spectrum reminiscent of UV-induced DNA damage. The results suggest that several mutated protein kinases may be contributing to lung cancer development, but that mutations in each one are infrequent. (Cancer Res 2005; 65(17): 7591-5)

FGFR3 is frequently activated by mutation in urothelial carcinoma (UC) and represents a potential target for therapy. In multiple myeloma, both over-expression and mutation of FGFR3 contribute to tumour development. To define the population of UC patients who may benefit from FGFRtargeted therapy, we assessed both mutation and receptor over-expression in primary UCs from a population of new patients. Manual or laser capture microdissection was used to isolate pure tumour cell populations. Where present, non-invasive and invasive components in the same section were microdissected. A screen of the region of highest tumour stage in each sample yielded a mutation frequency of 42%. Mutations comprised 61 single and 5 double mutations, all in hotspot codons previously identified in UC. There was a significant association of mutation with low tumour grade and stage. Subsequently, non-invasive areas from the 43 tumours with both non-invasive and invasive components were analysed separately. Eighteen of these had mutation in at least one region, including 9 with mutation in all regions examined, 8 with mutation in only the non-invasive component and one with different mutations in different regions. Of the 8 with mutation in only the non-invasive component, 6 were predicted to represent a single tumour and 2 showed morphological dissimilarity of fragments within the block, indicating possible presence of distinct tumour clones. Immunohistochemistry showed over-expression of FGFR3 protein in many tumours compared to normal bladder and ureteric controls. Increased expression was associated with mutation (85% of mutant tumours showed high-level expression). Overall, 42% of tumours with no detectable mutation showed over-expression including many muscle invasive tumours. This may represent a non-mutant subset of tumours in which FGFR3 signalling contributes to the transformed phenotype and which may benefit from FGFR-targeted therapies.

We conducted a genome wide SNP association study on 1,803 Urinary Bladder Cancer (UBC) cases and 34,336 controls from Iceland and the Netherlands and follow up studies in seven additional case control groups (2,165 cases and 3,800 controls). The strongest association was observed with allele T of rs9642880 on chromosome 8q24, 30kb upstream of the c-Myc gene (allele specific OR=1.22; P=9.34×10−12). Approximately 20% of individuals of European ancestry are homozygous for rs9642880 (T) and their estimated risk of developing UBC is 1.49 times that of non-carriers with population attributable risk (PAR) of 17%. No association was observed between UBC and the four 8q24 variants previously associated with prostate, colorectal and breast cancers, nor did rs9642880 associate with any of these three cancers. A weaker signal, but nonetheless of genome wide significance, was captured by rs710521 (A) located near the TP63 gene on chromosome 3q28 (allele specific OR=1.19; P=1. 15× 10−7).