Carolyn D. Hurst

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.

Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity

Urothelial carcinoma of the bladder comprises two long-recognized 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 traverse conventional grade and stage groupings. This improved understanding of molecular features, disease pathogenesis and heterogeneity provides new opportunities for prognostic application, disease monitoring and personalized therapy.

Oncogenic FGFR3 gene fusions in bladder cancer

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.

Spectrum of Phosphatidylinositol 3-Kinase Pathway Gene Alterations in Bladder Cancer

Purpose: The phosphatidylinositol 3-kinase (PI3K) pathway can be activated by alterations affecting several pathway components. For rational application of targeted therapies, detailed understanding of tumor biology and approaches to predict efficacy in individual tumors are required. Our aim was to assess the frequency and distribution of pathway alterations in bladder cancer. Experimental Design: We examined the pathway components (PIK3CA, PTEN, TSC1, RHEB, and LKB1) and putative upstream regulators (FGFR3 and RAS genes) for mutation, allelic loss, copy number alteration, and expression in bladder tumors and cell lines. Results: No mutations were found in RHEB and only a single mutation in LKB1. PIK3CA mutations were detected in 25% of tumors and 26% of cell lines with a significant excess of helical domain mutations (E542K and E545K). There was over-representation but not amplification of the gene. Loss of heterozygosity of the PTEN region and homozygous deletion were found in 12% and 1.4% of tumors, and reduced expression in 49%. Forty-six percent of cell lines showed alterations that implicated PTEN. Sixteen percent of tumors and 11% of cell lines showed TSC1 mutation, and 9q loss of heterozygosity was common (57%). Pathway alterations were independently distributed, suggesting that the mutation of two pathway members may have additive or synergistic effects through noncanonical functions. Conclusions: PI3K pathway alterations are common in bladder cancer. The lack of redundancy of alterations suggests that single-agent PI3K-targeted therapy may not be successful in these cancers. This study provides a well-characterized series of cell lines for use in preclinical studies of targeted agents. (Clin Cancer Res 2009;15(19):6008–17)

Gene Expression Signatures Predict Outcome in Non–Muscle-Invasive Bladder Carcinoma: A Multicenter Validation Study

Purpose: Clinically useful molecular markers predicting the clinical course of patients diagnosed with non–muscle-invasive bladder cancer are needed to improve treatment outcome. Here, we validated four previously reported gene expression signatures for molecular diagnosis of disease stage and carcinoma in situ (CIS) and for predicting disease recurrence and progression. Experimental Design: We analyzed tumors from 404 patients diagnosed with bladder cancer in hospitals in Denmark, Sweden, England, Spain, and France using custom microarrays. Molecular classifications were compared with pathologic diagnosis and clinical outcome. Results: Classification of disease stage using a 52-gene classifier was found to be highly significantly correlated with pathologic stage (P < 0.001). Furthermore, the classifier added information regarding disease progression of Ta or T1 tumors (P < 0.001). The molecular 88-gene progression classifier was highly significantly correlated with progression-free survival (P < 0.001) and cancer-specific survival (P = 0.001). Multivariate Cox regression analysis showed the progression classifier to be an independently significant variable associated with disease progression after adjustment for age, sex, stage, grade, and treatment (hazard ratio, 2.3; P = 0.007). The diagnosis of CIS using a 68-gene classifier showed a highly significant correlation with histopathologic CIS diagnosis (odds ratio, 5.8; P < 0.001) in multivariate logistic regression analysis. Conclusion: This multicenter validation study confirms in an independent series the clinical utility of molecular classifiers to predict the outcome of patients initially diagnosed with non–muscle-invasive bladder cancer. This information may be useful to better guide patient treatment.

High-resolution analysis of genomic copy number alterations in bladder cancer by microarray-based comparative genomic hybridization

We have screened 22 bladder tumour-derived cell lines and one normal urothelium-derived cell line for genome-wide copy number changes using array comparative genomic hybridization (CGH). Comparison of array CGH with existing multiplex-fluorescence in situ hybridization (M-FISH) results revealed excellent concordance. Regions of gain and loss were defined more accurately by array CGH, and several small regions of deletion were detected that were not identified by M-FISH. Numerous genetic changes were identified, many of which were compatible with previous results from conventional CGH and loss of heterozygosity analyses on bladder tumours. The most frequent changes involved complete or partial loss of 4q (83%) and gain of 20q (78%). Other frequent losses were of 18q (65%), 8p (65%), 2q (61%), 6q (61%), 3p (56%), 13q (56%), 4p (52%), 6p (52%), 10p (52%), 10q (52%) and 5p (43%). We have refined the localization of a region of deletion at 8p21.2-p21.3 to an interval of approximately 1 Mb. Five homozygous deletions of tumour suppressor genes were confirmed, and several potentially novel homozygous deletions were identified. In all, 15 high-level amplifications were detected, with a previously reported amplification at 6p22.3 being the most frequent. Real-time PCR analysis revealed a novel candidate gene with consistent overexpression in all cell lines with the 6p22.3 amplicon.

A sequence variant at 4p16.3 confers susceptibility to urinary bladder cancer

Previously, we reported germline DNA variants associated with risk of urinary bladder cancer (UBC) in Dutch and Icelandic subjects. Here we expanded the Icelandic sample set and tested the top 20 markers from the combined analysis in several European case-control sample sets, with a total of 4,739 cases and 45,549 controls. The T allele of rs798766 on 4p16.3 was found to associate with UBC (odds ratio = 1.24, P = 9.9 × 10−12). rs798766 is located in an intron of TACC3, 70 kb from FGFR3, which often harbors activating somatic mutations in low-grade, noninvasive UBC. Notably, rs798766[T] shows stronger association with low-grade and low-stage UBC than with more aggressive forms of the disease and is associated with higher risk of recurrence in low-grade stage Ta tumors. The frequency of rs798766[T] is higher in Ta tumors that carry an activating mutation in FGFR3 than in Ta tumors with wild-type FGFR3. Our results show a link between germline variants, somatic mutations of FGFR3 and risk of UBC.

Knockdown by shRNA identifies S249C mutant FGFR3 as a potential therapeutic target in bladder cancer

More than 60% of low-grade non-invasive papillary urothelial cell carcinomas contain activating point mutations of fibroblast growth factor receptor 3 (FGFR3). The phenotypic consequences of constitutive activation of FGFR3 in bladder cancer have not been elucidated and further studies are required to confirm the consequences of inhibiting receptor activity in urothelial cells. We measured FGFR3 transcript levels and demonstrated that transcript levels were significantly more abundant in low-stage and grade tumours. We identified a tumour cell line, 97-7, expressing the most common FGFR3 mutation (S249C) at similar FGFR3 transcript levels to low-stage and grade tumours. In these cells, S249C FGFR3 protein formed stable homodimers and was constitutively phosphorylated. We used retrovirus-mediated delivery of shRNA to knockdown S249C FGFR3. This induced cell flattening, decreased cell proliferation and reduced clonogenicity on plastic and in soft agar. However, no effects of knockdown of wild-type FGFR3 were observed in telomerase immortalized normal human urothelial cells, indicating possible dependence of the tumour cell line on mutant FGFR3. Re-expression of S249C FGFR3 in shRNA-expressing 97-7 cells resulted in a reversal of phenotypic changes, confirming the specificity of the shRNA. These results indicate that targeted inhibition of S249C FGFR3 may represent a useful therapeutic approach in superficial bladder cancer.

Phosphatidylinositol 3-kinase (PI3K) pathway activation in bladder cancer.

The phosphatidylinositol 3-kinase (PI3K) pathway is a critical signal transduction pathway that regulates multiple cellular functions. Aberrant activation of this pathway has been identified in a wide range of cancers. Several pathway components including AKT, PI3K and mTOR represent potential therapeutic targets and many small molecule inhibitors are in development or early clinical trials. The complex regulation of the pathway, together with the multiple mechanisms by which it can be activated, make this a highly challenging pathway to target. For successful inhibition, detailed molecular information on individual tumours will be required and it is already clear that different tumour types show distinct combinations of alterations. Recent results have identified alterations in pathway components PIK3CA, PTEN, AKT1 and TSC1 in bladder cancer, some of which are significantly related to tumour phenotype and clinical behaviour. Co-existence of alterations to several PI3K pathway genes in some bladder tumours indicates that these proteins may have functions that are not related solely to the known canonical pathway.

Two Multiplex Assays That Simultaneously Identify 22 Possible Mutation Sites in the KRAS, BRAF, NRAS and PIK3CA Genes

Recently a number of randomized trials have shown that patients with advanced colorectal cancer do not benefit from therapies targeting the epidermal growth factor receptor when their tumors harbor mutations in the KRAS, BRAF and PIK3CA genes. We developed two multiplex assays that simultaneously screen 22 nucleotides in the KRAS, NRAS, BRAF and PIK3CA genes for mutations. The assays were validated on 294 tumor DNA samples from patients with advanced colorectal cancer. In these samples 119 KRAS codon 12 and 13 mutations had been identified by sequence analysis, 126 tumors were wild-type for KRAS and the analysis failed in 49 of the 294 samples due to poor DNA quality. The two mutation assays detected 130 KRAS mutations, among which were 3 codon 61 mutations, and in addition 32 PIK3CA, 13 BRAF and 6 NRAS mutations. In 19 tumors a KRAS mutation was found together with a mutation in the PIK3CA gene. One tumor was mutant for both PIK3CA and BRAF. In summary, the mutations assays identified 161 tumors with a mutation, 120 were wild-type and the analysis failed in 13. The material cost of the 2 mutation assays was calculated to be 8-fold lower than the cost of sequencing required to obtain the same data. In addition, the mutation assays are less labor intensive. We conclude that the performance of the two multiplex mutation assays was superior to direct sequencing. In addition, these assays are cheaper and easier to interpret. The assays may also be of use for selection of patients with other tumor types.