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Fibroblast growth factor receptor 3 (FGFR3) overexpression, point mutations or gene fusions are found in ~80% of non muscle-invasive and ~15% of muscle-invasive bladder cancer. FGFR inhibitors have entered clinical trials in advanced bladder cancer however, as with other targeted therapies, intrinsic and acquired resistance are expected to limit treatment efficacy. We have used an in vitro model to explore possible mechanisms of resistance. The urothelial cancer cell line RT112 expresses an FGFR3-TACC3 fusion protein and is sensitive to FGFR inhibition. Isogenic resistant cell lines, termed R1, R2 and R3, were derived by long-term culture of RT112 in the presence of the FGFR inhibitor PD173074. Compared to parental RT112, R1 and R2 show reduced proliferation and have a more mesenchymal morphology, decreased expression of FGFR3 and increased expression of N-cadherin. R3 has a faster growth rate, more epithelial morphology and lower N-cadherin expression than R1 and R2. The changes in morphology and gene expression between parental and resistant derivatives R1 and R2 were reversed when the resistant cells were cultured without PD173074 for 4 passages. Despite this, the cells retained their resistance when re-exposed to PD173074. Exome sequencing, RNA microarray analysis and phospho-receptor tyrosine kinase array data on the parental cells and resistant derivatives and will be presented. Our data suggests that diverse mechanisms of resistance occur following prolonged FGFR inhibition. Citation Format: Geoffrey A. Pettitt, Helen R. McPherson, Carolyn D. Hurst, Julie E. Burns, Olivia A. Alder, Matthew C. Dunning, Margaret A. Knowles. Mechanisms of resistance to FGFR-targeted therapy in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3145. doi:10.1158/1538-7445.AM2017-3145

Contains fulltext : 138103.pdf (Publisher’s version ) (Closed access) Genome-wide association studies (GWAS) of urinary bladder cancer (UBC) have yielded common variants at 12 loci that associate with risk of the disease. We report here the results of a GWAS of UBC including 1670 UBC cases and 90 180 controls, followed by replication analysis in additional 5266 UBC cases and 10 456 controls. We tested a dataset containing 34.2 million variants, generated by imputation based on whole-genome sequencing of 2230 Icelanders. Several correlated variants at 20p12, represented by rs62185668, show genome-wide significant association with UBC after combining discovery and replication results (OR = 1.19, P = 1.5 x 10(-11) for rs62185668-A, minor allele frequency = 23.6%). The variants are located in a non-coding region approximately 300 kb upstream from the JAG1 gene, an important component of the Notch signaling pathways that may be oncogenic or tumor suppressive in several forms of cancer. Our results add to the growing number of UBC risk variants discovered through GWAS.

Activating mutations of FGFR3 are a common and early event in bladder cancer. Ectopic expression of mutant FGFR3 in normal urothelial cells has both pro-proliferative and antiapoptotic effects at confluence, suggesting that mutant cells are insensitive to cell–cell contact inhibition. Herein, detailed analysis revealed that these cells have reduced cell–cell adhesion, with large intercellular spaces observable at confluence, and diminished cell–substrate adhesion to collagen IV, collagen I, and fibronectin. These phenotypic alterations are accompanied by changes in the expression of genes involved in cell adhesion and extracellular matrix remodeling. Silencing of endogenous mutant FGFR3 in bladder cancer cells induced converse changes in transcript levels of CDH16, PLAU, MMP10, EPCAM, TNC, and HAS3, confirming them as downstream gene targets of mutant FGFR3. Overexpression of EPCAM, HAS3, and MMP10 transcripts was found in a large fraction of primary bladder tumors analyzed, supporting their key role in bladder tumorigenesis in vivo. However, no correlation was found between their protein and/or mRNA expression and FGFR3 mutation status in tumor specimens, indicating that these genes may be targeted by several converging oncogenic pathways. Overall, these results indicate that mutant FGFR3 favors the development and progression of premalignant bladder lesions by altering key genes regulating the cell–cell and cell–matrix adhesive properties of urothelial cells. Implications: The ability of mutant FGFR3 to drive transcriptional expression profiles involved in tumor cell adhesion suggests a mechanism for expansion of premalignant urothelial lesions. Mol Cancer Res; 13(1); 138–48. ©2014 AACR.

Objectives The 8th Annual Bladder Cancer Think Tank (BCAN-TT) brought together a multidisciplinary group of clinicians, researchers, and patient advocates in an effort to advance bladder cancer research. Methods and Materials With the theme of “Collaborating to Move Research Forward,” the meeting included three panel presentations and seven small working groups. Results The panel presentations and interactive discussions focused on three main areas: gender disparities, sexual dysfunction, and targeting novel pathways in bladder cancer. Small working groups also met to identify projects for the upcoming year, including: (1) improving enrollment and quality of clinical trials; (2) collecting data from multiple institutions for future research; (3) evaluating patterns of care for non-muscle-invasive bladder cancer; (4) improving delivery of care for muscle-invasive disease; (5) improving quality of life for survivors; (6) addressing upper tract disease; and (7) examining the impact of health policy changes on research and treatment of bladder cancer. Conclusions The goal of the BCAN-TT is to advance the care of patients with bladder cancer and to promote collaborative research throughout the year. The meeting provided ample opportunities for collaboration among clinicians from multiple disciplines, patients and patient advocates, and industry representatives.

A quantitative real-time polymerase chain reaction method (qPCR) was developed and tested for the detection of Taylorella equigenitalis. It was shown to have an analytical sensitivity of 5 colony-forming units (CFU) of T. equigenitalis when applied to the testing of culture swabs that mimicked field samples, and a high analytical specificity in not reacting to 8 other commensal bacterial species associated with horses. As designed, it could also differentiate specifically between T. equigenitalis and T. asinigenitalis. The qPCR was compared to standard culture in a study that included 45 swab samples from 6 horses (1 stallion, 5 mares) naturally infected with T. equigenitalis in Canada, 39 swab samples from 5 naturally infected stallions in Germany, and 311 swab samples from 87 culture negative horses in Canada. When the comparison was conducted on an individual sample swab basis, the qPCR had a statistical sensitivity and specificity of 100% and 96.4%, respectively, and 100% and 99.1% when the comparison was conducted on a sample set basis. A comparison was also made on 203 sample swabs from the 5 German stallions taken over a span of 4 to 9 mo following antibiotic treatment. The qPCR was found to be highly sensitive and at least as good as culture in detecting the presence of T. equigenitalis in post-treatment samples. The work demonstrates that the qPCR assay described here can potentially be used to detect the presence of T. equigenitalis directly from submitted sample swabs taken from infected horses and also for determining T. equigenitalis freedom following treatment.Une épreuve quantitative de réaction en chaîne par la polymérase en temps réel (qPCR) a été développée et testée pour la détection de

Activating mutations and/or overexpression of FGFR3 are common in bladder cancer, making FGFR3 an attractive therapeutic target in this disease. In addition, FGFR3 gene rearrangements have recently been described that define a unique subset of bladder tumors. Here, a selective HSP90 inhibitor, ganetespib, induced loss of FGFR3-TACC3 fusion protein expression and depletion of multiple oncogenic signaling proteins in RT112 bladder cells, resulting in potent cytotoxicity comparable with the pan-FGFR tyrosine kinase inhibitor BGJ398. However, in contrast to BGJ398, ganetespib exerted pleiotropic effects on additional mitogenic and survival pathways and could overcome the FGFR inhibitor–resistant phenotype of FGFR3 mutant–expressing 97-7 and MHG-U3 cells. Combinatorial benefit was observed when ganetespib was used with BGJ398 both in vitro and in vivo. Interestingly, two additional FGFR3 fusion-positive lines (RT4 and SW480) retained sensitivity to HSP90 inhibitor treatment by the ansamycins 17-AAG and 17-DMAG yet displayed intrinsic resistance to ganetespib or AUY922, both second-generation resorcinol-based compounds. Both cell lines, compared with RT112, expressed considerably higher levels of endogenous UGT1A enzyme; this phenotype resulted in a rapid glucuronidation-dependent metabolism and subsequent efflux of ganetespib from SW780 cells, thus providing a mechanism to account for the lack of bioactivity. Implications: Pharmacologic blockade of the molecular chaperone HSP90 represents a promising approach for treating bladder tumors driven by oncogenic gene rearrangements of FGFR3. Furthermore, UDP-glucuronosyltransferase enzyme expression may serve as a predictive factor for clinical response to resorcinol-based HSP90 inhibitors. Mol Cancer Res; 12(7); 1042–54. ©2014 AACR.

Yann NEUZILLET*, Laura MERTENS, Amsterdam, Netherlands; Shahrokh SHARIAT, Dallas, TX; Peter BOSTROM, Tuomas MIRTTI, Turku, Finland; Arthur SAGALOWSKY, Raheela ASHFAQ, Dallas, TX; Annegien BROEKS, Michiel VAN, DER HEIJDEN, Dennis PETERS, Chantall CURIAL, Jeroen DE JONG, Simon HORENBLAS, Amsterdam, Netherlands; Carolyn HURST, Darren TOMLINSON, Margaret KNOWLES, Leeds, United Kingdom; Bharati BAPAT, Michael JEWETT, Alexandre ZLOTTA, Toronto, Canada; Joyce SANDERS, Amsterdam, Netherlands; Yair LOTAN, Dallas, TX; Theo VAN DER KWAST, Toronto, Canada; Bas VAN RHIJN, Amsterdam, Netherlands

There is much information on the genetic alterations that contribute to the development of bladder cancer. Because it is hypothesised that the genotype of the cancer cell plays a major role in determining phenotype, this genetic information should impact on clinical practice. To date however, this has not happened. Some of the alterations identified in bladder cancer have clear associations with outcome-for example, mutational inactivation of the cell cycle regulator proteins p53 and the retinoblastoma protein (Rb). However, as single markers, these events have insufficient predictive power to be applied in the management of individual patients. The use of panels of markers is a potential solution to this problem. Examples of suitable panels include those genes/proteins with known impact on specific cell cycle checkpoints or with impact on cellular phenotypes, such as immortalisation, invasion, or metastasis. To evaluate such marker panels, large tumour series will be needed-for example, archival samples from completed clinical trials. The use of these valuable resources will require coordination of sample provision. This might involve central collection and distribution of tissue blocks, sections, or tissue arrays and the provision of patient follow up information to laboratories participating in a study. With the availability of microarray technologies, including cDNA and comparative genomic hybridisation arrays, the transcriptome and genome of transitional cell carcinomas of different phenotypes can be compared and will undoubtedly provide a wealth of information with potential diagnostic and prognostic uses. Although these studies can be initiated using small local tissue collections, high quality collection of fresh tissues from new clinical trials will be crucial for proper evaluation of associations with clinical outcome. Funding for molecular pathological studies to date has been poor. To begin to translate molecular information from the laboratory to the clinic and to make maximum use of valuable urological patient resources in the UK, adequate funding and scientific energy are required. Whereas the latter is not in doubt, present funding for this type of translational research is inadequate.

4p16.3 has previously been identified as a region of non-random LOH in transitional cell carcinoma, suggesting the presence of a tumour suppressor gene. One candidate within this region is fibroblast growth factor receptor 3 (FGFR3). Germline mutations in FGFR3 are known to cause several autosomal dominant skeletal dysplasias, the severity of which depends on the position and nature of the mutation in the protein. We investigated the frequency and nature of FGFR3 mutations in a panel of transitional cell carcinomas and cell lines and studied the possible link between mutation and loss of heterozygosity (LOH) on 4p16.3. FGFR3 coding sequence from 63 transitional cell carcinomas (TCC) of various stages and grades, and 18 cell lines was analysed by fluorescent SSCP. Samples with abnormal migration patterns were sequenced to identify the mutation or polymorphism. Thirty-one of the 63 tumours had previously been assessed to have LOH at 4p16.3. Twenty-six of the 63 tumours (41%) and 4/18 (22%) of the cell lines had missense mutations in FGFR3. All mutations detected in our panel have been reported in the germline where all apart from one cause lethal conditions. One tumour contained K652Q which has recently been identified in less severe cases of skeletal dysplasia. Tumours with and without LOH at 4p16.3 had mutations in FGFR3 suggesting that these two events are not causally linked. The frequency of FGFR3 mutation indicates that this protein plays an important role in TCC.

Transcriptional silencing by CpG island hypermethylation of gene regulatory regions is one mechanism for inactivation of tumour suppressor genes. Chromosome 9q deletion is frequently found in transitional cell carcinoma (TCC) of the bladder and upper urinary tract and one of the putative tumour suppressor loci has been mapped to 9q32-33. A gene designated as DBCCR1 was identified in the candidate region and its mRNA expression is thought to be suppressed by hypermethylation. To understand the role of hypermethylation in TCC, we evaluated the methylation status of 20 CpG sites of the DBCCR1 5’-CpG island region in a total of 69 tumours from 45 patients, 21 normal urothelial specimens, and six bladder cancer cell lines. Aberrant hypermethylation levels were found in 36 (52%) of 69 tumours without any association with tumour grade or stage. Methylation was weakly detected in the normal urothelium in association with ageing. Although recurrent tumours tended to have higher methylation levels than the initial tumours, the methylation pattern was mostly maintained between multifocal TCCs in individual patients. The results suggest that hypermethylation of the DBCCR1 region is one of the earliest alterations in the development of TCCs and there may be an age-related hypermethylation-based field defect in normal urothelium. Methylator or methylation-resistant phenotype seems to be maintained during multifocal development or recurrence of most TCCs. Oncogene (2001) 20, 531‐537.

The most frequent genetic alteration in transitional cell carcinoma of the urinary bladder (TCC) is loss of chromosome 9 which targets CDKN2A on 9p. The targets on 9q are not confirmed. Here, 81 advanced TCC specimens were investigated for loss of heterozygosity (LOH) and homozygous deletions (HD) on chromosome 9q using multiplex analysis of microsatellite markers. 41/81 tumours (51%) showed LOH on 9q, with LOH at all markers in 33 cases. Eight partial losses involved three regions in 9q12, 9q22.3, and 9q33– 9q34. No mutations were identified in the candidate tumour suppressor gene DBCCR1 in three tumours showing restricted LOH at 9q32-33. 22% of the specimens had HD at CDKN2A, but no HD was found on 9q. Two tumours had lost 9p only and five 9q only. 9q LOH was not related to tumour grade or stage and present or absent with equal frequency in recurrent TCC. LOH on 9q correlated with the extent of genome-wide hypomethylation (P < 0.0001) which extended into satellite sequences located in 9q12 juxtacentromeric heterochromatin. While the high frequency of chromosome 9q loss in TCC may reflect destabilization of the chromosome related to hypomethylation of repetitive DNA, the data are compatible with the existence of tumour suppressor genes on this chromosome arm. http://www.bjcancer.com © 2001 Cancer Research Campaign

The urological malignancies, renal, bladder and prostate cancer, account for approximately 16% of all cancer cases. Unfortunately 5-year survival rates are relatively poor, largely a result of many cases not being diagnosed before the tumour has metastasised. There is a clear need for the identification of markers which will allow earlier detection of disease, and predict prognosis and response to therapy. In addition, they may be of use as therapeutic targets. Current advances in molecular biology are allowing the identification of a number of tumour-associated changes which could be of clinical use in the future. However, with the rapid technological advances being made in the field of proteomics, this approach could be integrated with genomics providing a complementary alternative, overcoming disparities between mRNA levels and protein production, and additionally allowing the identification of tumour-associated post-translational modifications. These approaches have already been used to identify novel genes and other cancer-related changes involved in the pathogenesis of urological malignancies. This review describes current progress in the genomic and proteomic study of urological malignancies, and highlights the potential of using proteomic technologies in the study of this group of diseases.

Loss of heterozygosity (LOH) on chromosome arm 9q is the most frequent genetic alteration found in superficial and invasive transitional cell carcinoma (TCC) in a previous microsatellite-based deletion mapping study of the bladder and upper urinary tract, indicating the presence of one or more important tumor suppressor genes (TSGs). One of the putative tumor suppressor loci on 9q (DBC1) was mapped to 9q32–33 and the candidate region was localized within a single YAC. We report here a case of superficial papillary TCC, which showed a homozygous deletion encompassing this candidate tumor suppressor region. The region of homozygous deletion spanned the interval between D9S275 and AFMA239XA9 at 9q32–33, and was estimated to be ≤6 cM. Although homozygous deletion mapping did not narrow down the candidate tumor suppressor region, this case provides further support for the presence of a TSG for TCC in this chromosomal region. To our knowledge, this is the first report to show homozygous deletion at 9q32–33 in TCC or any other type of human tumor. Genes Chromosomes Cancer 26:171–175, 1999. © 1999 Wiley-Liss, Inc.

In Egypt and other regions of the Middle East where the trematode Schistosoma haematobium is endemic, bladder cancer is the most common adult cancer. Unlike bladder cancers in Western countries, which are predominantly transitional-cell carcinoma (TCC), these schistosomiasis-associated bladder cancers are predominantly squamous-cell carcinoma (SCC). Our aim was to assess a large series of schistosomiasis-associated bladder tumours for genetic alterations commonly found in TCC in the United Kingdom and the United States. We have carried out a partial allelotype of 70 tumours from patients with schistosomiasis. LOH was found on all chromosome arms studied (3p, 4p, 4q, 8p, 9p, 9q, 11p, 11q, 13q, 14q, 17p, 18q). The most frequent regions of LOH were 9p (65%), 17p (58%), 3p (40%), 9q (39%) and 8p (37%). LOH on 17p, where the TP53 gene is located, was more common in Egyptian TCC than in SCC. Similarly, 8p LOH was more common in TCC than SCC. The most striking difference between this group of tumours and TCCs from the United Kingdom and the United States was the high frequency of 9p LOH in the region of the CDKN2 gene (65%) and the relatively low frequency of 9q LOH (39%); 15 of 43 tumours with LOH of at least one marker on chromosome 9 showed LOH of 9p only. This suggests that a 9p gene, possibly CDKN2, may contribute to the development of the majority of schistosomiasis-associated bladder tumours but that genes on 9q play a much less important role. Int. J. Cancer 80:656–661, 1999. © 1999 Wiley-Liss, Inc.

Many genetic alterations have recently been identified in transitional cell carcinoma (TCC) of the bladder. These include alterations to known proto-oncogenes and tumour suppressor genes and the identification of multiple sites of nonrandom chromosomal deletion which are predicted to define the location of as yet unidentified tumour suppressor genes. This review summarises recent efforts to define the location of novel bladder tumour suppressor genes using loss of heterozygositiy (LOH) and homozygous deletion analyses and to isolate the genes targeted by these deletions. For three of the four regions of deletion on chromosome 9, the most frequently deleted chromosome in TCC, candidate genes have been identified. It is anticipated that the identification of the genes and/or genetic regions which are frequently altered in TCC will provide useful tools for diagnosis, prediction of prognosis, patient monitoring and novel therapies.

We have investigated interstitial deletions of chromosome 8 in 70 colorectal carcinomas and 11 colonic adenomas using 11 microsatellite markers, including eight spanning the centromeric region of chromosome 8p (p11.2-p12). Allelic loss or imbalance was observed in 38 (54%) cancers and four (36%) adenomas. Twenty-eight (40%) of the cancers had deletions of 8p11.2-p12. Two distinct and independent regions of interstitial loss were found within this region. Fluorescent in situ hybridization, using an alpha satellite repeat probe to the centromere of 8p and two probes to the P1 region, was performed in four tumours that demonstrated allelic imbalance. Localized heterozygous deletions were confirmed in all four tumours. Eleven (16%) cancers had localized deletion in the region ANK-1 to D8S255 (P1) and a further eleven (16%) cancers had a less well localized deletion in the region defined by the markers D8S87 to D8S259 (P2). Loss of both centromeric loci was identified in a further six (9%) tumours. A functional significance for these two deletion regions was sought by correlation with primary and secondary tumour characteristics. Isolated P2 deletion was associated with 'early' T1 cancers (2p=0.0002), and were also identified in 3/11 adenomas. Conversely, interstitial deletions of the P1 locus were more frequently seen in 'locally invasive' T3/4 cancers (2p=0.015), and isolated P1 deletions were also associated with the presence of liver metastases (2p=0.016). Our data provide evidence of at least two genes within the 8p11.2-p12 region, mutations in which may confer different and independent roles in the pathogenesis of colorectal cancer.

In a search for candidate tumor suppressor genes within a 30-kb common region of deletion previously identified in bladder cancer cell lines, we isolated a 2.4-kb cDNA clone comprising 13 exons that spanned approximately 16 kb of genomic DNA. Mutation analysis was carried out by single-strand conformation polymorphism analysis on DNA from 12 bladder carcinoma cell lines and 26 bladder tumors with LOH on chromosome 4p. Direct sequencing of the transcript in 4 bladder carcinoma cell lines with deletions in this region was also carried out. Two polymorphisms in exons 2 and 5 were identified, but no tumor-specific mutations were found. Sequence analysis identified a high degree of homology with the mouse sh3bp2 gene, which is abl-binding, suggesting that this gene is the human homologue. The predicted amino acid sequence of the putative gene product contains a Src homology 2 domain, a Src homology 3 binding domain, and a pleckstrin homology domain, suggesting a possible role in signal transduction. No evidence was found to indicate that SH3BP2 is the tumor suppressor gene at 4p16.3 involved in bladder cancer. However, this study has identified an interesting human gene that is a potential negative regulator of the abl oncogene.

Loss of heterozygosity (LOH) on chromosome 9q is the most frequent genetic alteration in transitional cell carcinoma (TCC) of the bladder, implicating the presence of a tumour suppressor gene or genes on 9q. To define the location of a tumour suppressor locus on 9q in TCC, we screened 156 TCCs of the bladder and upper urinary tract by detailed deletion mapping using 31 microsatellite markers on 9q. Partial deletions of 9q were found in 10 TCCs (6%), and LOH at all informative loci on 9q was found in 77 TCCs (49%). In five low grade superficial bladder tumours, the partial deletion was localized to D9S195 located at 9q32-33, with retention of heterozygosity at all other informative loci including D9S103, D9S258, D9S275 and GSN. We constructed a yeast artificial chromosome (YAC) contig covering the deleted region in these five tumours and placed another four unmapped microsatellite markers on this contig map. Using these markers, we further defined the common deleted region to the interval between D9S1848 and AFMA239XA9. The region is covered by a single YAC (852e11), whose size is estimated to be 840 kb. Our data should expedite further fine mapping and identification of the candidate tumour suppressor gene at 9q32-33.

The Fifth International workshop on chromosome 9 comprised a gathering of 36 scientists from seven countries and included a fairly even distribution of interests along chromosome 9 as well as a strong input from more global activities and from comparative mapping. At least eight groups had participated in the goal set at the previous workshop which was to improve the fine genetic mapping in different regions of chromosome 9 by meiotic breakpoint mapping in allocated regions and this has resulted in some greatly improved order information. Excellent computing facilities were available and all contributed maps were entered not only into SIGMA (and thence submitted to GDB) but also into a dedicated version of ACEDB which can be accessed on the Web in the form of one of 28 slices into which the chromosome has been arbitrarily divided. It was generally agreed that the amount of data is now overwhelming and that the integration and validation of all data is not only unrealistic in a short meeting but probably impossible until the whole chromosome has been sequenced and fully annotated. Sequence-ready contigs presented at the meeting totalled about 3 MB which is about one fiftieth of the estimated length. The single biggest barrier to integration of maps is the problem of non-standard nomenclature of loci. In the past 2 workshops efforts have been made to compare traditional 'consensus' maps made by human insight (still probably best for small specific regions) with those generated with some computer assistance (such as SIGMA) and those generated objectively by defined computer algorithms such as ldb. Since no single form of map or representation is entirely satisfactory for all purposes the maps reproduced in the published version of the report are confined to one of the genetic maps, in which Genethon and older markers have been incorporated, a Sigma map of the genes as symbols together with a listing of known 'disease' genes on chromosome 9, and a revised assessment of the mouse map together with a list of mouse loci predicted to be on human chromosome 9. One of the 28 ACEDB slices is also shown to illustrate strengths and weaknesses of this approach. Workshop files include not only all maps available at the time but also details of loci and details of the meiotic breakpoints in the CEPH families (http:/(/)www.gene.ucl.ac.uk/scw9db.shtml) .

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.

Activation of fibroblast growth factor receptors (FGFR) is a common oncogenic event. Little is known about the determinants of sensitivity to FGFR inhibition and how these may vary between different oncogenic FGFRs. Using parallel RNA interference (RNAi) genetic screens, we show that the EGF receptor (EGFR) limits sensitivity to FGFR inhibition in FGFR3-mutant and -translocated cell lines, but not in other FGFR-driven cell lines. We also identify two distinct mechanisms through which EGFR limits sensitivity. In partially FGFR3-dependent lines, inhibition of FGFR3 results in transient downregulation of mitogen-activated protein kinase signaling that is rescued by rapid upregulation of EGFR signaling. In cell lines that are intrinsically resistant to FGFR inhibition, EGFR dominates signaling via repression of FGFR3, with EGFR inhibition rescued by delayed upregulation of FGFR3 expression. Importantly, combinations of FGFR and EGFR inhibitors overcome these resistance mechanisms in vitro and in vivo. Our results illustrate the power of parallel RNAi screens in identifying common resistance mechanisms to targeted therapies. Significance: Our data identify a novel therapeutic approach to the treatment of FGFR3-mutant cancer, emphasizing the potential of combination approaches targeting both FGFR3 and EGFR. Our data extend the role of EGFR in mediating resistance to inhibitors targeting a mutant oncogene, showing that EGFR signaling can repress mutant FGFR3 to induce intrinsic resistance to FGFR targeting. Cancer Discov; 3(9); 1058–71. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 953

Radiotherapy is a major treatment modality used to treat muscle-invasive bladder cancer, with patient outcomes similar to surgery. However, radioresistance is a significant factor in treatment failure. Cell-free extracts of muscle-invasive bladder tumors are defective in nonhomologous end-joining (NHEJ), and this phenotype may be used clinically by combining radiotherapy with a radiosensitizing drug that targets homologous recombination, thereby sparing normal tissues with intact NHEJ. The response of the homologous recombination protein RAD51 to radiation is inhibited by the small-molecule tyrosine kinase inhibitor imatinib. Stable RT112 bladder cancer Ku knockdown (Ku80KD) cells were generated using short hairpin RNA technology to mimic the invasive tumor phenotype and also RAD51 knockdown (RAD51KD) cells to show imatinib's pathway selectivity. Ku80KD, RAD51KD, nonsilencing vector control, and parental RT112 cells were treated with radiation in combination with either imatinib or lapatinib, which inhibits NHEJ and cell survival assessed by clonogenic assay. Drug doses were chosen at approximately IC40 and IC10 (nontoxic) levels. Imatinib radiosensitized Ku80KD cells to a greater extent than RAD51KD or RT112 cells. In contrast, lapatinib radiosensitized RAD51KD and RT112 cells but not Ku80KD cells. Taken together, our findings suggest a new application for imatinib in concurrent use with radiotherapy to treat muscle-invasive bladder cancer. Cancer Res; 73(5); 1611–20. ©2012 AACR.

An allelotype analysis of transitional cell carcinoma of the bladder identified loss of heterozygosity (LOH) on chromosome arm 4p in 22% of tumours. In a more detailed LOH study of 178 bladder carcinomas, a 750 kb common region of deletion was identified between the markers D4S43 and D4S127 just telomeric to the Huntington disease locus. To refine this region of deletion at 4p16.3, we have carried out detailed fluorescence in situ hybridisation (FISH) analysis of 12 bladder cancer cell lines by using a chromosome 4 centromeric probe combined with a series of cosmid probes from contigs spanning the 750 kb region of deletion. A common 30 kb region of deletion was identified at 4p16.3 in over one-third of the bladder cancer cell lines analysed. The present study has refined the localisation of the critical region of deletion from 750 kb to approximately 30 kb, providing a precise starting point for positional cloning of the gene(s) involved in bladder cancer from within a very gene-rich region on chromosome band 4p16.3. This study demonstrates that FISH can be used for fine deletion mapping of potential tumour suppressor gene regions. The utilisation of FISH analysis to map chromosomal deletions should facilitate positional cloning of other genes as bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC) contigs of the human genome are established.

Previous studies have suggested the involvement of tumour-suppressor genes on chromosomes 8p, 22q and 18q (DCC) in prostate cancer. The aim of this study was to further characterize these regions. We investigated 20 polymorphic regions on the 3 chromosome arms in 43 cancers and 10 cases of benign prostatic hyperplasia (BPH). Allelic loss was observed in 72% of cancers on 8p, 16% on 22q and 24% at DCC. For BPH, loss was observed in 20% on 8p and in 12% at DCC. The low incidence of LOH on 22q implies that this locus has no significant role in prostate carcinogenesis. At DCC, although the overall incidence was low, tumours with LOH were mostly of high grade or had metastases, suggesting a role for this gene in prostate cancer progression. On chromosome 8p, 29% of cancers had deletions at the LPL locus on 8p22 and 60% had deletions within a region flanked by the markers D8S339 and ANKI on 8p 11.1-p21.1. Within this region, 2 distinct areas of allelic loss were observed, at one or both ANKI and D8S255, and in the region defined by the markers D8S259-D8S505. For the regions 8p22 and ANKI-D8S255, tumours with metastases had a greater frequency of LOH compared to non-metastasizing tumours, suggesting the presence of putative metastasis-suppressor genes in these regions.

We have screened 33 ovarian tumours of various grades and stages for the loss of heterozygosity (LOH) of markers on chromosome 9. LOH was detected in 26 cases (79%). Eleven tumours (33%) showed LOH of all informative markers. The remaining 15 cases had partial deletions. Of these, six (18%) had losses on 9p only, three (9%) had LOH confined to 9q and six (18%) had losses on both chromosome arms, four of which had a retention of hetereozygosity in between. There was no association between tumour grade stage or histopathology and any losses. High-density deletion mapping was carried out in 12 selected cases that had partial deletions of 9p and/or 9q. The deleted region on 9p included the cyclin-dependent kinase inhibitor 2 (CDKN2) locus and one tumour was found to have a homozygous deletion of CDKN2. LOH on 9q extended over a larger region. We found evidence for two regions of deletion on 9q, one at 9q34 and the other encompassing the nevoid basal cell carcinoma (Gorlin) syndrome locus on proximal 9q. Images Figure 2

Non-muscle-invasive bladder cancers (NMIBC) and muscle-invasive bladder cancers (MIBC) show distinct molecular and clinical features and are considered to follow different pathogenesis pathways. MIBC commonly metastasise (>50%) and have poor prognosis whereas NMIBC, particularly low-stage low-grade tumors, rarely progress to invade muscle (T transitions, followed by C>G transversions (24.8%). Similar to MIBC, APOBEC mutagenesis was the strongest source of mutation in NMIBC with 75% of samples showing up to 5-fold enrichment of the APOBEC mutation signature. Significantly mutated genes included genes implicated in epigenetic regulation and several genes that have not previously been reported as significantly mutated in bladder cancer. Comparison of mutation frequencies in NMIBC with those found in MIBC revealed both known differences (absence of TP53 mutations and high frequency of FGFR3, PIK3CA and STAG2 mutations in NMIBC) and novel findings including a higher frequency of mutations in chromatin-state regulators in NMIBC. Notably CDKN1A, RB1, ERCC2, ERBB3 and FBXW7, which are mutated in >10% of MIBC were not significantly mutated in TaG2 tumors, further delineating the distinct pathogenesis pathways of NMIBC and MIBC. Whole-genome expression array profiling and immunohistochemistry using a panel of markers were carried out to further examine the molecular features of NMIBC samples and define potentially clinically relevant subtypes. These data provide a detailed view of the genomic landscape of NMIBC that highlights chromatin modification as a key area for consideration in the development of potential future therapeutic approaches to the treatment of patients with non-muscle-invasive disease. Citation Format: Carolyn D. Hurst, Olivia Alder, Fiona M. Platt, Alastair Droop, Lucy F. Stead, Julie E. Burns, George J. Burghel, Sunjay Jain, Leszek J. Klimczak, Helen Lindsay, Jo-An Roulson, Claire F. Taylor, Helene Thygesen, Angus J. Cameron, Anne J. Ridley, Helene R. Mott, Dmitry A. Gordenin, Margaret A. Knowles. The genomic landscape of non-muscle-invasive bladder cancer: implications for molecular classification and treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-323.

Deletions of the short arm of chromosome II have been identified by both cytogenetic and molecular criteria in bladder and other types of solid tumor, indicating the presence of one or more suppressor loci in this region. To localize the II p deletion target(s) more precisely and to screen for loss of heterozygosity (LOH) on the long arm of the chromosome, I00 bladder tumors were analyzed for LOH on chromosome II using restriction fragment length polymorphism (RFLPs) and microsatellite markers mapped to both II p and II q. Thirty-four tumors were found to have LOH at I or more loci. Of these, I7 had LOH restricted to II p, I3 had LOH of both II p and II q, and 4 had LOH of II q only. Eight tumors showed LOH at all informative loci indicating probable loss of an entire copy of chromosome II. A common region of deletion was defined on II p between DIIS922(IIpI5.5) and DlIS569 (IIpI5.I-I5.2). This region does not include the HRAS or WTI loci (at IIpI5.5and IIp13, respectively). Seventeen tumors had LOH on II q, 4 of which had LOH on II q only. The common region of deletion on II q was between FGF3 and DI IS490 (IIq I3-q23.2). Two tumors showed LOH on both II p and II q with a clear region of retention of heterozygosity between, indicatinn the existence of two deletion targets on chromosome II. © 1995 Wiley-Liss, Inc.

4521Background: gemcitabine (G) plus cisplatin (C) is a standard-of-care, combination chemotherapy regimen for patients (pts) with bladder cancer (BC), in both neoadjuvant and palliative settings. ...

The p16 gene has been identified as a candidate tumour suppressor gene at 9p21, a region commonly deleted in bladder cancer. We screened 140 bladder tumours and 16 cell lines for deletions and sequence variants of p16. Eight cell lines showed homozygous deletion of p16 and two had small sequence variations. All 13 tumours with small defined deletions of 9p21, 18/31 (58%) of tumours with monosomy 9 and 9/91 (10%) of tumours with no chromosome 9 loss of heterozygosity had homozygous deletion of p16. No tumour-specific sequence variants were identified. Deletion mapping revealed a nested set of deletions focused on p16. Six deletions involved p16 but not the related and adjacent gene p15 and one tumour had an intragenic deletion of p16. All other deletions involved both p16 and p15. We conclude that p16 represents the major target for deletion at 9p21 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.

Although activating mutations of PIK3CA are frequent in urothelial carcinoma (UC), no information is available on their specific effects in urothelial cells or the basis for the observed mutation spectrum, which has a large excess of helical domain mutations. We investigated the phenotypic and signaling consequences of hotspot and UC-specific rare PIK3CA mutations in immortalized normal human urothelial cells (NHUC) and mouse fibroblasts (NIH3T3). Our results indicate that in NHUC, rare mutant forms and all three hotspot mutant forms of PIK3CA can activate the PI3K/AKT pathway. The relative frequency at which helical domain and kinase domain mutations are found in UC is related to their potency in inducing signaling downstream of AKT and to the phenotypic effects induced in this cell type (E545K>E542K>H1047R). Helical domain mutations E542K and E545K conferred a significant proliferative advantage at confluence and under conditions of nutrient depletion, and increased cellular resistance to anoikis. Both helical and kinase domain mutants induced increased NHUC cell motility and migration towards a chemoattractant, though no significant differences were found between the mutant forms. In NIH3T3 cells, the kinase domain mutant H1047R induced high levels of AKT activation, but helical domain mutants were significantly less potent and this was reflected in their relative abilities to confer anchorage-independent growth. Our findings indicate that the effects of mutant PIK3CA are both cell type- and mutation-specific. Helical domain mutations in PIK3CA may confer a selective advantage in the urothelium in vivo by overcoming normal contact-mediated inhibitory signals and allowing proliferation in nutrient-limiting conditions. Mutant forms of PIK3CA may also stimulate intraepithelial cell movement, which could contribute to spread of cells within the urothelium.

Tumour invasion and metastasis is the most common cause of death from cancer. For epithelial cells to invade surrounding tissues and metastasise, an epithelial-mesenchymal transition (EMT) is required. We have demonstrated that FGFR1 expression is increased in bladder cancer and that activation of FGFR1 induces an EMT in urothelial carcinoma (UC) cell lines. Here, we created an in vitro FGFR1-inducible model of EMT, and used this model to identify regulators of urothelial EMT. FGFR1 activation promoted EMT over a period of 72 hours. Initially a rapid increase in actin stress fibres occurred, followed by an increase in cell size, altered morphology and increased migration and invasion. By using site-directed mutagenesis and small molecule inhibitors we demonstrated that combined activation of the mitogen activated protein kinase (MAPK) and phospholipase C gamma (PLCγ) pathways regulated this EMT. Actin stress fibre formation was regulated by PLCγ activation, and was also important for the increase in cell size, migration and altered morphology. MAPK activation regulated migration and E-cadherin expression, indicating that combined activation of PLCγ and MAPK is required for a full EMT. We used expression microarrays to assess changes in gene expression downstream of these signalling cascades. COX-2 was transcriptionally upregulated by FGFR1 and caused increased intracellular prostaglandin E(2) levels, which promoted migration. In conclusion, we have demonstrated that FGFR1 activation in UC cells lines promotes EMT via coordinated activation of multiple signalling pathways and by promoting activation of prostaglandin synthesis.

summary The Fifth International workshop on chromosome 9 comprised a gathering of 36 scientists from seven countries and included a fairly even distribution of interests along chromosome 9 as well as a strong input from more global activities and from comparative mapping. At least eight groups had participated in the goal set at the previous workshop which was to improve the fine genetic mapping in dierent regions of chromosome 9 by meiotic breakpoint mapping in allocated regions and this has resulted in some greatly improved order information. Excellent computing facilities were available and all contributed maps were entered not only into SIGMA (and thence submitted to GDB) but also into a dedicated version of ACEDB which can be accessed on the Web in the form of one of 28 slices into which the chromosome has been arbitrarily divided. It was generally agreed that the amount of data is now overwhelming and that the integration and validation of all data is not only unrealistic in a short meeting but probably impossible until the whole chromosome has been sequenced and fully annotated. Sequence-ready contigs presented at the meeting totalled about 3 MB which is about one fiftieth of the estimated length. The single biggest barrier to integration of maps is the problem of non-standard nomenclature of loci. In the past 2 workshops eorts have been made to compare traditional ‘consensus’ maps made by human insight (still probably best for small specific regions) with those generated with some computer assistance (such as SIGMA) and those generated objectively by defined computer algorithms such as ldb. Since no single form of map or representation is entirely satisfactory for all purposes the maps reproduced in the published version of the report are confined to one of the genetic maps, in which Genethon and older markers have been incorporated, a Sigma map of the genes as symbols together with a listing of known ‘disease’ genes

The mutational spectrum for the TP53 gene was investigated in a large series of bladder tumors and bladder tumor cell lines. Tumors and cell lines were screened for the presence of TP53 point mutations by single-strand conformational polymorphism analysis followed by direct sequencing. Mutations were detected in 16 of 88 (18%) tumors and 4 of 14 cell lines (28%). In total, twelve missense mutations, one nonsense mutation, three deletions, and two insertions were identified by direct sequencing. Of the thirteen point mutations sequenced, only one was a transition at a CpG site, whereas five G:C-->T:A transversions were found, suggesting a major role for exogenous mutagens in bladder tumorigenesis. Tumors were also examined for loss of heterozygosity (LOH) on chromosome arm 17p. LOH of one or more markers on 17p was detected in 31% of tumors. All eight tumors with a TP53 mutation from patients informative at TP53 had LOH, whereas nine tumors with LOH at TP53 did not have an identified mutation. Three tumors had LOH on 17p at sites distal to the TP53 locus but retained both TP53 alleles, suggesting the involvement of another tumor suppressor gene on 17p in bladder tumorigenesis in some tumors.

Constrained binding peptides (peptide aptamers) may serve as tools to explore protein conformations and disrupt protein-protein interactions. The quality of the protein scaffold, by which the binding peptide is constrained and presented, is of crucial importance. SQT (Stefin A Quadruple Mutant-Tracy) is our most recent development in the Stefin A-derived scaffold series. Stefin A naturally uses three surfaces to interact with its targets. SQT tolerates peptide insertions at all three positions. Peptide aptamers in the SQT scaffold can be expressed in bacterial, yeast and human cells, and displayed as a fusion to truncated pIII on phage. Peptides that bind to CDK2 can show improved binding in protein microarrays when presented by the SQT scaffold. Yeast two-hybrid libraries have been screened for binders to the POZ domain of BCL-6 and to a peptide derived from PBP2', specific to methicillin-resistant Staphylococcus aureus. Presentation of the Noxa BH3 helix by SQT allows specific interaction with Mcl-1 in human cells. Together, our results show that Stefin A-derived scaffolds, including SQT, can be used for a variety of applications in cellular and molecular biology. We will henceforth refer to Stefin A-derived engineered proteins as Scannins.

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.

Background XPC is involved in the nucleotide excision repair of DNA damaged by carcinogens known to cause bladder cancer. Individuals homozygous for the variant allele of XPC c.1496C > T (p.Ala499Val) were shown in a large pooled analysis to have an increased bladder cancer risk, and we found two 3'UTR variants, *611T > A and c.*618A > G, to be in strong linkage disequilibrium with c.1496T. Here we determined if these two 3'UTR variants can affect mRNA stability and assessed the impact of all three variants on mRNA and protein expression. Methods In vitro mRNA stability assays were performed and mRNA and protein expression measured both in plasmid-based assays and in lymphocytes and lymphoblastoid cell lines from bladder and breast cancer patients. Results The two 3'UTR variants were associated with reduced protein and mRNA expression in plasmid-based assays, suggesting an effect on mRNA stability and/or transcription/translation. A near-significant reduction in XPC protein expression (p = 0.058) was detected in lymphoblastoid cell lines homozygous for these alleles but no differences in mRNA stability in these lines was found or in mRNA or protein levels in lymphocytes heterozygous for these alleles. Conclusion The two 3'UTR variants may be the variants underlying the association of c.1496C > T and bladder cancer risk acting via a mechanism modulating protein expression.

RB-protein status as determined by immunohistochemical analysis was compared with loss of heterozygosity (LOH) at the RB locus in 68 primary transitional-cell carcinomas of the bladder. Absence of RB-protein expression was found in 15 of 17 tumors in which LOH at the RB locus was identified, whereas 31 of 36 tumors from informative patients which showed no LOH had a normal RB-protein pattern ( p < 0.001). Altered RB-protein expression was also more frequently seen in muscle-invasive and high-grade tumors ( p < 0.003 and

The gene structure and expression of the related peptide regulatory factors TGF beta 1 and TGF beta 2 were studied in a panel of seven urothelial carcinoma cell lines and 40 transitional cell carcinomas. The latter comprised 15 grade 1, 18 grade 2 and 5 grade 3 tumours and two cases of carcinoma in situ. Control tissues included ten matched 'field' biopsies and 17 other biopsies including 11 biopsies of macroscopically normal urothelium, two of which were from patients with no history of bladder cancer. No amplification of rearrangements of either TGF beta 1 or TGF beta 2 were detected in any sample. A complex pattern of expression or the two genes was found in the urothelial cell lines. High, but variable levels of the 2.5 kb TGF beta 1 transcript were detected and lower and more variable levels of the three (4.1 kb, 5.1 kb and 6.5 kb) transcripts of TGF beta 2 were detected. Although those cell lines expressing most TGF beta 1 tended to express less TGF beta 2 transcript there was no clear-cut relationship. In comparison, no TGF beta 2 transcript was identified in any primary transitional cell carcinoma or control tissue. Markedly reduced or undetectable levels of TGF beta 1 transcript were detected in 4/15 (26%) grade 1, 5/18 (28%) grade 2 and 3/5 (60%) grade 3 tumours. There was no clear relationship to tumour stage, lymphocytic infiltration or stromal content of the tumours. Clinical review one year after the 2 year period of tumour collection showed that 6/9 (66%) of patients with tumours with reduced levels of transcript had died or had disease which was not controllable by local resection and 3/9 (33%) had developed tumour re-occurrences. In comparison, in the group with normal levels of expression of TGF beta 1, 3/18 (17%) had disease which was not controllable by local means, 9/18 (50%) had tumour re-occurrence and 6/18 (33%) had no evidence of disease. The association of reduced expression of TGF beta 1 and advanced disease was statistically significant P < 0.02 (Fisher's test). Although the sample size is small, we suggest that the loss of expression of TGF beta 1 may be a potential marker of progressive disease or prognosis in transitional cell carcinoma and warrants further study. Images Figure 1 Figure 2 Figure 3

Objectifs Les mutations activatrices du FGFR3 sont frequemment observees dans les tumeurs de vessie n’infiltrant pas le muscle mais peu etudiee dans les tumeurs infiltrantes. Des etudes in vitro ont montre qu’un traitement anti-FGFR3 reduit la croissance tumorale. Nous avons etudie l’heterogeneite de la mutation de FGFR3 sur des echantillons des parties superficielles et profondes de tumeurs de vessie ainsi que dans les ganglions envahis. Methodes Nous avons identifie les echantillons couples (superficiel et profond) provenant de 61 resection transuretrale de vessie (RTUV) diagnostiques (34 T1 et 27 T ≥ 2) parmi 494 cancers de vessie cN0M0 traites par cystectomie totale (CT) avec curage ganglionnaire. Cent cinquante-cinq sur 494 patients (31 %) etaient pN+. Des echantillons couples provenant de la tumeur vesicale et des ganglions envahis etaient disponibles pour 117 patients (75 %). L’ADN a ete isole a partir du tissu fixe en paraffine et les mutations de FGFR3 ont ete analysees par la technique de SNaPShot. Resultats Une mutation activatrice de FGFR3 a ete objectivee dans 13/34 et 8/27 des echantillons de RTUV T1 et T ≥ 2, respectivement. Dans les tumeurs T1, les memes mutations etaient observees dans les parties superficielles et profondes de la tumeur. Dans les tumeurs T ≥ 2, 4/8 mutations n’ont pas ete detectees dans la partie invasive. Une mutation activatrice de FGFR3 a ete objectivee dans 62/494 echantillons de CT (33 %), parmi lesquels 53 etaient pN0 (85 %). La presence d’une mutation de FGFR3 a ete associe au stade pN0 (p = 0,002). Six sur 117 ganglions envahis (5 %) etaient FGFR3 mutes. La meme mutation a ete detectee dans la tumeur vesicale et dans les ganglions correspondants. Parmi les 111 ganglions envahis, aucun ne provenait de tumeur de vessie porteuse d’une mutation de FGFR3 (specificite = 100 %). Conclusion La mutation de FGFR3 etait tres rare chez les patients ayant des ganglions envahis. Le statut mutationnel de FGFR3 serait utile pour guider la decision d’un traitement adjuvant anti-FGFR3 car il s’est avere homogene dans les tumeurs vesicales et les ganglions envahis. La partie profonde de la tumeur doit etre analysee si un traitement neoadjuvant est envisage.

The Second International Workshop on Human Chromosome 9 was held in Chatham, Massachusetts on April 18--20, 1993. Fifty-three abstracts were received and the data presented on posters. The purpose of the meeting was to bring together all interested investigators working on the map of chromosome 9, many of whom had disease-specific interests. After a brief presentation of interests and highlighted results, the meeting broke up into the following subgroups for production of consensus maps: 9p; 9cen-q32; 9q32 ter. A global mapping group also met. Reports of each of these working groups is presented in the summary.

Therapeutic decisions for muscle-invasive bladder cancer (MIBC) are largely based on histopathologic characteristics. In this issue of Cancer Cell, Choi and colleagues report three molecular subtypes of MIBC with the potential to guide prognosis, patient stratification, and treatment.

We conducted a multi-stage, genome-wide association study of bladder cancer with a primary scan of 591,637 SNPs in 3,532 affected individuals (cases) and 5,120 controls of European descent from five studies followed by a replication strategy, which included 8,382 cases and 48,275 controls from 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 previously identified genome-wide 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), and found interactions with smoking in both regions. Our findings on common variants associated with bladder cancer risk should provide new insights into the mechanisms of carcinogenesis.

Fibroblast growth factor receptors (FGFRs) play key roles in proliferation, differentiation, and tumorigenesis. Previously, we demonstrated that FGFR1 expression is increased in urothelial carcinoma cell lines and tumors, which promotes proliferation and survival via activation of the mitogen-activated protein kinase (MAPK) pathway. Here we examined splice variants of FGFR1 in both urothelial carcinoma cell lines and tumors. Two known FGFR1 IIIc splice variants (FGFR1α and FGFR1β) were expressed. FGFR1β lacks exon 3 of FGFR1α, removing the first Ig loop of the extracellular domain. Both isoforms were expressed at similar levels in normal urothelial cells, but FGFR1β was expressed at higher levels in most tumor cell lines. In tumor tissues, expression levels were higher than in controls, and the FGFR1β:FGFR1α ratio was significantly increased in association with tumor stage and grade. When FGFR1α and FGFR1β were expressed in urothelial cells, no differences in signaling were observed. FGFR1-induced proliferation paralleled MAPK pathway activation. The relative activation of FGFR1β and FGFR1α by all known mammalian FGFs was examined. Both isoforms were activated by the same FGFs, but the level of activation differed. FGFR1β showed higher affinity for low concentrations of FGF1, leading to enhanced signaling and increased proliferation. An FGFR1α-to-FGFR1β isoform switch and increased FGF1-induced activation of FGFR1β may result in a proliferative advantage that plays a key role during bladder tumor progression.

Radical radiotherapy and surgery achieve similar cure rates in muscle-invasive bladder cancer, but the choice of which treatment would be most beneficial cannot currently be predicted for individual patients. The primary aim of this study was to assess whether expression of any of a panel of DNA damage signaling proteins in tumor samples taken before irradiation could be used as a predictive marker of radiotherapy response, or rather was prognostic. Protein expression of MRE11, RAD50, NBS1, ATM, and H2AX was studied by immunohistochemistry in pretreatment tumor specimens from two cohorts of bladder cancer patients (validation cohort prospectively acquired) treated with radical radiotherapy and one cohort of cystectomy patients. In the radiotherapy test cohort (n = 86), low tumor MRE11 expression was associated with worse cancer-specific survival compared with high expression [43.1% versus 68.7% 3-year cause-specific survival (CSS), P = 0.012] by Kaplan-Meier analysis. This was confirmed in the radiotherapy validation cohort (n = 93; 43.0% versus 71.2%, P = 0.020). However, in the cystectomy cohort (n = 88), MRE11 expression was not associated with cancer-specific survival, commensurate with MRE11 being a predictive marker. High MRE11 expression in the combined radiotherapy cohort had a significantly better cancer-specific survival compared with the high-expression cystectomy cohort (69.9% versus 53.8% 3-year CSS, P = 0.021). In this validated immunohistochemistry study, MRE11 protein expression was shown and confirmed as a predictive factor associated with survival following bladder cancer radiotherapy, justifying its inclusion in subsequent trial designs. MRE11 expression may ultimately allow patient selection for radiotherapy or cystectomy, thus improving overall cure rates. Cancer Res; 70(18); 7017–26. ©2010 AACR.

Despite the fact that the current histopathologic classification for bladder cancer has led to improved concepts for the clinical management of the disease, key questions with regard to assessment of risk for recurrence and/or progression to invasive disease remain. In addition, response to specific therapies cannot be predicted accurately. Bladder tumors comprise a heterogeneous group with respect to both histopathology and clinical behavior. Thus, it is anticipated that a thorough knowledge and interpretation of the molecular alterations involved in tumor development and progression will lead to greater prognostic and predictive power. This may not only lead to better comprehension of the biology of the disease, but may also lead to the development of novel individualized therapies. Novel means of stratification are urgently needed to provide a new subclassification of urothelial lesions. This review discusses and summarizes the genetic alterations that have been reported in bladder cancer and relates these to the current 2-pathway model for tumor development. The molecular pathogenesis of high-grade noninvasive papillary tumors and of T1 tumors is not yet clear, and possibilities are discussed.

FGFR3 mutations have recently been identified in several benign epidermal skin lesions such as seborrheic keratosis, epidermal nevus and solar lentigo. The functional consequences of these mutations in human skin are as yet unknown. In this study we analyzed the functional effects of the most common FGFR3 mutation in benign skin tumors, the R248C FGFR3 hotspot mutation, in human HaCaT keratinocytes. The cells were stably transduced with either the R248C or wildtype FGFR3 IIIb cDNA using a retroviral vector system. FGFR3 mutant and wildtype cells showed similar growth rates at subconfluence. However, at confluence FGFR3 mutant keratinocytes revealed a significantly higher cell number than wildtype cells. Furthermore, FGFR3 mutant cells showed significantly lower levels of apoptosis and decreased attachment to fibronectin compared with FGFR3 wildtype cells. Expression of mutant FGFR3 did not alter migration and senescence. Microarray analysis revealed only a few differentially expressed genes between FGFR3 mutant and wildtype keratinocytes. Enhanced phosphorylation of ERK1/2 was observed in confluent R248C mutant HaCaT cells compared with wildtype keratinocytes. Our results suggest that an increased cell number at confluence along with a decreased apoptosis may contribute to the development of acanthotic tumors in FGFR3 mutant skin in vivo.