FGFR3 and TP53 gene mutations define two distinct pathways in urothelial cell carcinoma of the bladder

FGFR3 and TP53 mutations are frequent in superficial papillary and invasive disease, respectively. We used denaturing high-performance liquid chromatography and sequencing to screen for FGFR3 and TP53 mutations in 81 newly diagnosed urothelial cell carcinomas. Tumors were classified as follows: 31 pTa, 1 carcinoma in situ, 30 pT1, and 19 pT2-T4. Tumor grades were as follows: 10 G1, 29 G2, and 42 G3. FGFR3 mutations were associated with low-stage (P < 0.0001), low-grade (P < 0.008) tumors, whereas TP53 mutations were associated with high-stage (P < 0.003), high-grade (P < 0.02) tumors. Mutations in these two genes were almost mutually exclusive. Our results suggest that FGFR3 and TP53 mutations define separate pathways at initial diagnosis of urothelial cell carcinoma.     

Mutations in TP53, but not FGFR3, in urothelial cell carcinoma of the bladder are influenced by smoking: contribution of exogenous versus endogenous carcinogens

Smoking is a major risk factor for urothelial cell carcinoma of the bladder (UCC). Mutations in the FGFR3 and TP53 genes have been shown to define two distinct pathways in superficial papillary and invasive UCC disease, respectively. We investigated the relationship between smoking and these mutations by means of denaturing high performance liquid chromatography and sequencing for 110 primary UCC of the bladder. This study included 48 current smokers, 31 ex-smokers and 31 non-smokers. Thirty-five of the tumors were stage pTa, 40 pT1 and 35 > or =pT2. Fourteen of the tumors were grade 1, 37 were grade 2 and 59 grade 3. Smoking was associated with high stage (P = 0.03) and high grade tumors (P = 0.006). Twenty-two of the 110 tumors studied harbored TP53 mutations (20%) and 43 harbored FGFR3 mutations (39%). Odds ratios (OR) were higher for TP53 mutations in current smokers [OR, 2.25; 95% confidence interval (95% CI), 0.65-7.75] and ex-smokers (OR, 1.62; 95% CI, 0.41-6.42) than in non-smokers. Double TP53 mutations and the A:T-->G:C TP53 mutation pattern was found only in current smokers. Patients with the FGFR3(wild-type)/TP53(mutated) genotype had significantly higher levels of tobacco consumption, as measured in pack-years (P = 0.01). Smoking influenced neither the frequency nor the pattern of FGFR3 mutations. Our results suggest that smoking is associated with invasive and high grade UCCs, at initial presentation, and influenced TP53 or the molecular pathway defined by these mutations. In contrast, FGFR3 mutations are not affected by smoking and probably result from endogenous alterations. These data have potential implications for clinical management and prevention strategies.     

PIK3CA mutations are an early genetic alteration associated with FGFR3 mutations in superficial papillary bladder tumors

Bladder tumors constitute a very heterogeneous disease. Superficial tumors are characterized by a high prevalence of FGFR3 mutations and chromosome 9 alterations. High-grade and muscle-invasive tumors are characterized by Tp53 mutations and aneuploidy. We have analyzed the sequence of exons 9 and 20 of PIK3CA in a panel of bladder tumors covering the whole spectrum of the disease. DNA from formalin-fixed, paraffin-embedded tumor sections was amplified by PCR and products were sequenced. In an unselected panel of tumors representative of the disease, the PIK3CA mutation prevalence was 13% (11 of 87). Mutations occurred mainly at the previously identified hotspots (codons 542, 545, 1007, and 1047). The distribution according to stage was as follows: papillary urothelial neoplasms of uncertain malignant potential (PUNLMP; 11 of 43, 25.6%), T(a) (9 of 57, 16%), T(1) (2 of 10, 20%), and muscle-invasive tumors (0 of 20, 0%; P = 0.019). Mutations were associated with low-grade tumors: grade 1 (6 of 27, 22.2%), grade 2 (3 of 23, 13%), and grade 3 (2 of 37, 5.4%; P = 0.047). Overall, PIK3CA mutations were strongly associated with FGFR3 mutations: 18 of 69 (26%) FGFR3(mut) tumors were PIK3CA(mut), versus 4 of 58 (6.9%) FGFR3(wt) tumors (P = 0.005). Our findings indicate that PIK3CA mutations are a common event that can occur early in bladder carcinogenesis and support the notion that papillary and muscle-invasive tumors arise through different molecular pathways. PIK3CA may constitute a novel diagnostic and prognostic tool, as well as a therapeutic target, in bladder cancer.     

FGFR3 and Tp53 mutations in T1G3 transitional bladder carcinomas: independent distribution and lack of association with prognosis.

FGFR3 and Tp53 mutations have been proposed as defining two alternative pathways in the pathogenesis of transitional bladder cancer. FGFR3 mutations are associated with low-grade tumors and a favorable prognosis. Tp53 alterations are associated with advanced tumors and, possibly, with a poor prognosis. We focus here on the subgroup of T1G3 superficial tumors because they are a major clinical challenge. Patients (n = 119) were identified from a prospective study of 1,356 cases. Mutations in FGFR3 (exons 7, 10, and 15) and Tp53 (exons 4-9) were analyzed using PCR and direct sequencing. All cases were followed for recurrence and death. Survival was analyzed using Kaplan-Meier curves and multivariable Cox regression. FGFR3 mutations were detected in 20 (16.8%) tumors; 100 mutations in Tp53 were found in tumors from 78 (65.5%) cases. Multiple alterations in Tp53 were present in 19 tumors (16%). Inactivating mutations were present in 58% of tumors. The combined mutation distribution (FGFR3/Tp53) was: wt/wt (34.5%), mut/wt (7.6%), wt/mut (48.7%), and mut/mut (9.2%), indicating that the presence of either mutation did not depend on the other (P value = 0.767). FGFR3 and Tp53 mutations were not associated with clinicopathologic characteristics of patients and did not predict, alone or in combination, recurrence or survival. Taking the risk of the wt/wt group as reference, the mutation-associated risks of cancer-specific mortality were: mut/wt 1.42 (0.15-13.75), wt/mut 0.67 (0.19-2.31), mut/mut 1.62 (0.27-9.59). These molecular features support the notion that T1G3 tumors are at the crossroads of the two main molecular pathways proposed for bladder cancer development and progression.     

Molecular characterization of early-stage bladder carcinomas by expression profiles, FGFR3 mutation status, and loss of 9q

We used gene expression profiling, mutation analyses of FGFR3 and TP53, and LOH analyses of chromosome 9 and the TP53 region on chromosome arm 17p, to molecularly characterize 75 Ta and T1 bladder carcinomas. We identified four major cellular processes related to cell cycle, protein synthesis, immune response, and extra cellular components that contribute to the expressional heterogeneity of early-stage urothelial cell carcinoma (UCC). Activating FGFR3 mutations were found at the highest frequency in G1 tumors (80%), and showed a strong correlation with FGFR3 expression. In contrast, G3 tumors displayed mutations in less than 10% of the cases and a low level of FGFR3 expression. Even though LOH on chromosome 9 was not associated with any specific expression pattern, our data indicate that loss of chromosome 9 is associated with tumor development rather than initiation. The combined analyses suggest the existence of two types of UCC tumors, one which is characterized by FGFR3 mutation or expression, high expression of protein synthesis genes, and low expression of cell cycle genes. Furthermore, the presented data underscore FGFR3 receptor involvement in urothelial cell transformation as the presence of FGFR3 mutations has a major impact on the global gene expression profile of bladder carcinomas.     

Molecular grading of urothelial cell carcinoma with fibroblast growth factor receptor 3 and MIB-1 is superior to pathologic grade for the prediction of clinical outcome.

PURPOSE: Fibroblast growth factor receptor 3 (FGFR3) mutations were recently found at a high frequency in well-differentiated urothelial cell carcinoma (UCC). We investigated the relationship between FGFR3 status and three molecular markers (MIB-1, P53, and P27kip1) associated with worse prognosis and determined the reproducibility of pathologic grade and molecular variables. PATIENTS AND METHODS: In this multicenter study, we included 286 patients with primary (first diagnosis) UCC. The histologic slides were reviewed. FGFR3 status was examined by polymerase chain reaction-single strand conformation polymorphism and sequencing. Expression levels of MIB-1, P53, and P27kip1 were determined by immunohistochemistry. Mean follow-up was 5.5 years (range, 0.4 to 18.4 years). RESULTS: FGFR3 mutations were detected in 172 (60%) of 286 UCCs. Grade 1 tumors had an FGFR3 mutation in 88% of patient samples and grade 3 tumors in 16% of patient samples. Conversely, aberrant expression patterns of MIB-1, P53, and P27kip1 were seen in 5%, 2%, and 3% of grade 1 tumors and in 85%, 60%, and 56% of grade 3 tumors, respectively. In multivariate analysis with recurrence rate, progression, and disease-specific survival as end points, the combination of FGFR3 and MIB-1 proved independently significant in all three cases. By using these two molecular markers, three molecular grades (mGs) could be identified: mG1 (mutation; normal expression), favorable prognosis; mG2 (two remaining combinations), intermediate prognosis; and mG3 (no mutation; high expression), poor prognosis. The molecular variables were more reproducible than pathologic grade (85% to 100% v 47% to 61%). CONCLUSION: The FGFR3 mutation represents the favorable molecular pathway of UCC. Molecular grading provides a new, simple, and highly reproducible tool for clinical decision making in UCC patients.     

Fibroblast growth factor receptor 3 protein expression in urothelial carcinoma of the urinary bladder, exhibiting no association with low-grade and/or non-invasive lesions

Activating mutations of fibroblast growth factor receptor 3 (FGFR3), found in autosomal dominant human skeletal dysplasia, were reported to be involved in tumorigenesis and correlate with low-grade and superficial lesions of urothelial carcinoma. FGFR3 protein expression was immunohistochemically investigated in 126 cases of urothelial carcinoma of the urinary bladder to evaluate the role of this receptor in tumor behavior. p53 expression and the proliferating activity of tumor cells, assessed by Ki-67 expression, were also analyzed in parallel. Cytoplasmic and/or membrane immunostaining for FGFR3 was observed in 62 (49.2%) cases, including 20 (15.9%) cases of intense staining and 42 (33.3%) of moderate staining. p53 expression and Ki-67 labeling index (LI) were significantly correlated with high tumor grade (p=0.0093 and <0.0001, respectively) and invasion (p=0.0041 and <0.0001, respectively). Although there were two groups of interesting cases: low-grade and non-invasive tumors negative for p53 but positive for FGFR3, and high-grade and invasive tumors positive for p53 but negative for FGFR3, no statistically significant relationship was found between FGFR3 expression and tumor grade, invasion, p53 expression or Ki-67 LI. These results suggest that FGFR3 protein expression in bladder cancer is unlikely to affect tumor behavior as a unique single factor.     

p53 gene mutation and ink4a-arf deletion appear to be two mutually exclusive events in human glioblastoma

P16 and P14ARF are two tumor suppressors encoded by the locus ink4a-arf which is frequently deleted in human tumors. Recent experiments performed with mouse embryonic fibroblasts have shown that P14ARF is an upstream regulator of the P53 pathway. This raises the question as to whether in human tumors the loss of p14arf and mutation of p53 are mutually exclusive events which segregate with genetic alterations at other loci. To examine this question we performed a multigenic analysis on 29 gliomas. We analysed p53 and p14arf in relation with five other genetic loci encoding the most frequently mutated genes in human gliomas: cdkn2a, mdm2, egfr, pten and the chromosomal regions 10q23.3 and 10q25-26. Our study shows for the first time that p53 mutations and p14arf deletions appear mutually exclusive in human glioblastoma, suggesting that they may be functionally redundant in glioma tumorigenesis. The P53 pathway is, therefore, disrupted in 81.8% of malignant gliomas (WHO grades III and IV), either by mutation of the p53 gene (31.8%) or by p14arf deletion (54.5%). These tumors further showed MDM2 overexpression (9.1%), egfr oncogene amplification/egfr overexpression (50%), pten mutations (27.3%) and loss of heterozygosity (LOH) at the chromosomal regions 10q23.3 (86.4%) and 10q25-26 (100%). These alterations did not segregate with p53 mutations or p14arf deletions, while p14arf and cdkn2a were always deleted.     

Fibroblast Growth Factor Receptor 3 Mutations in Bladder Tumors Correlate with Low Frequency of Chromosome Alterations

The aim of this study was to analyze the distribution of FGFR3 mutations in bladder tumors of different grade and stage and determine the relation of mutations to chromosomal alterations detected by comparative genomic hybridization (CGH). One hundred bladder cancer samples served as templates for manual microdissection. DNA was isolated from dissected samples containing at least 80% tumor cells. Mutations in FGFR3 were analyzed by SNaPshot analysis. CGH was carried out according to standard protocols. FGFR3 mutations were detected in 45 of 92 samples (48.9%). Concerning T-category, the following mutation frequencies occurred: pTa, 69%; pT1, 38%; and pT2-3, 0%. The mutation frequency was significantly associated with tumor grade: G1, 72%; G2, 56%; and G3, 4%. In pTaG1 tumors, mutations were found in 74%. A significantly lower number of genetic alterations per tumor detected by CGH was associated with FGFR3 mutations (2 vs 8). This association was also seen in pTaG1 tumors: 2.5 (with mutation) vs 7.5 (without mutation). FGFR3 mutations characterize noninvasive low-risk tumors of low malignancy. The low malignant potential of these tumors is underlined by a low number of genetic alterations per tumor. Therefore, FGFR3 represents a valuable prognostic marker of tumors with low malignant potential and can be used as surrogate marker for the detection of genetically stable bladder tumors.     

p53 Status in Multiple Human Urothelial Cancers: Assessment for Clonality by the Yeast p53 Functional Assay in Combination with p53 Immunohistochemistry

Multifocal synchronous or metachronous tumor development is a common observation in human urothelial cancer cases. However, the underlying mechanism has remained obscure. We have employed a new tool to investigate the p53 gene status, the yeast p53 functional assay, in combination with immunohistochemistry in a total of 50 tumor samples from 32 cases with urothelial cancers, including 8 with multiple synchronous tumor development and 2 demonstrating metachronous tumors. p53 mutations were found in 13 cases (9 with missense mutations, 3 with deletion, 1 with splicing mutation) by the yeast p53 functional assay. p53 protein overexpression was seen in all 9 cases with missense mutations, but in only one of the 4 cases with nonsense mutations. Two tumors without p53 mutation also showed positive p53 immunoreactivity. Overall, p53 abnormalities including mutations and/or protein overexpression were found in 15 (47%) cases. p53 abnormalities were significantly more frequent in non-papillary and in high grade tumors. Loss of the wild type allele in addition to a p53 mutation was suggested in 8 of the 15 (53%) cases. All 4 cases with mutations in multiple synchronous tumors had identical p53 mutations in the separate urothelial cancers, strongly suggestive of monoclonality. The one case with multiple metachronous tumors, in contrast, was characterized by variation in the p53 status, indicative of different clonal origins. In conclusion, combined assessment for p53 status as used here (yeast p53 functional assay plus immunohistochemistry) may provide insights into the molecular mechanisms of urothelial carcinogenesis.     

No mutations of FGFR3 in normal urothelium in the vicinity of urothelial carcinoma of the bladder harbouring activating FGFR3 mutations in patients with bladder cancer

Mutations in the fibroblast growth factor receptor 3 (FGFR3) gene causing constitutive oncogenic protein activation have been shown to be frequent in papillary noninvasive bladder tumours and are associated with a low risk of progression and a favourable outcome. FGFR3 alterations have also been found in benign urothelial papilloma and flat urothelial hyperplasia suggesting FGFR3 alterations as an early event in bladder tumorigenesis. To date there is no data available on FGFR3 mutations in normal urothelium from patients with bladder cancer. We therefore analysed 64 samples of histopathological unsuspicious normal urothelium from 38 patients with FGFR3 mutated bladder tumours and 15 samples of urothelium from patients (n = 15) without any urothelial malignancy as a control group. Urothelial cells were microdissected from formalin‐fixed, paraffin‐embedded material. After DNA isolation whole genome amplification was done by I‐PEP‐PCR. FGFR3 mutations were detected using SNaPshot analysis. All samples could successfully be investigated. FGFR3 analyses did not reveal any mutation in the urothelium from neither the control group nor the bladder cancer group. All urothelial samples showed a wildtype sequence for FGFR3. These data suggest that mutations in the FGFR3 gene are not the earliest genetic alterations in bladder carcinogenesis and are associated with a hyperproliferative (hyperplastic) phenotype in the urothelium. Chromosomal alterations like deletions on chromosome 9q or aberrant promoter hypermethylation could play more important roles in early urothelial transformation than mutational FGFR3 activation. © 2009 UICC     

p53 status in multiple human urothelial cancers: assessment for clonality by the yeast p53 functional assay in combination with p53 immunohistochemistry.

Multifocal synchronous or metachronous tumor development is a common observation in human urothelial cancer cases. However, the underlying mechanism has remained obscure. We have employed a new tool to investigate the p53 gene status, the yeast p53 functional assay, in combination with immunohistochemistry in a total of 50 tumor samples from 32 cases with urothelial cancers, including 8 with multiple synchronous tumor development and 2 demonstrating metachronous tumors. p53 mutations were found in 13 cases (9 with missense mutations, 3 with deletion, 1 with splicing mutation) by the yeast p53 functional assay. p53 protein overexpression was seen in all 9 cases with missense mutations, but in only one of the 4 cases with nonsense mutations. Two tumors without p53 mutation also showed positive p53 immunoreactivity. Overall, p53 abnormalities including mutations and/or protein overexpression were found in 15 (47%) cases. p53 abnormalities were significantly more frequent in non-papillary and in high grade tumors. Loss of the wild type allele in addition to a p53 mutation was suggested in 8 of the 15 (53%) cases. All 4 cases with mutations in multiple synchronous tumors had identical p53 mutations in the separate urothelial cancers, strongly suggestive of monoclonality. The one case with multiple metachronous tumors, in contrast, was characterized by variation in the p53 status, indicative of different clonal origins. In conclusion, combined assessment for p53 status as used here (yeast p53 functional assay plus immunohistochemistry) may provide insights into the molecular mechanisms of urothelial carcinogenesis.     

Clinicopathological Features of FGFR3 - Mutated Upper Tract Urothelial Carcinoma: A Genomic Database Analysis

BackgroundUpper tract urothelial carcinomas (UTUCs) arise in the renal pelvis or the ureter, accounting for approximately 5% of all urothelial carcinomas. Recent years have witnessed the publication of several studies aimed at assessing the molecular, biologic, and clinical features of UTUC, reporting that FGFR3 mutations are the most observed genetic aberrations; however, several knowledge gaps persist in the understanding of the genomic landscape of this genitourinary malignancy with few treatment options.Patients and MethodsIn the current study, we aimed to comprehensively analyze clinicopathological features of FGFR3-mutated UTUCs patients in public datasets to increase the current knowledge of the molecular and biologic profile of UTUC. Data regarding clinical outcomes, mutational profiles, and copy number alterations in patients affected by UTUC were downloaded from the cBioPortal for Cancer Genomics Database. UTUC data were available from 4 studies, for a total number of 358 patients; among these, 150 UTUC patients presented FGFR3 mutations.ResultsThe current database analysis of the mutational profile of 150 FGFR3-mutated UTUCs suggested that FGFR3 mutations may represent a prognostic factor in this disease, with a statistically longer overall survival compared to wild-type patients treated with radical surgery. In addition, FGFR3 mutations were more frequent in low-grade UTUCs with early-stage disease (pT1, pT2, and pT3).ConclusionGenomic characterization of UTUC is destined to become increasingly important, and more efforts aimed at implementing UTUC genomics analysis are warranted.     

Missense mutation of the hMSH6 and p53 genes in sporadic urothelial transitional cell carcinoma

Functional defects in DNA mismatch repair genes have been shown to be associated mainly with hereditary human malignancies. We examined genomic DNA from 88 sporadic transitional cell carcinomas (TCCs) of the urinary tract for mutations in hMSH6 gene by polymerase chain reaction and direct sequencing analysis. Mutational status of p53 gene was also studied as a potential target of genetic instability secondary to hMSH6 dysfunction. A total of 5 cases (5.7%: 5/88) displayed hMSH6 mutations all consisted of transition and located in exon 4, including three cases with missense mutation and two without change of corresponding amino acid. These three tumors with hMSH6 missense mutation had no microsatellite instability with five microsatellite markers tested. p53 gene mutations were detected in 22 cases (25.0%: 22/88). No tumors with p53 mutation had any hMSH6 missense mutations. Compared to the cases without hMSH6 alterations, the three patients with hMSH6 alterations had more frequent additional primary cancer (P<0.05). These findings provide the first in vivo evidence for the type of alterations and frequency of possible involvement of the hMSH6 mutations in sporadic type urothelial TCCs.     

Molecular changes during progression from nonmuscle invasive to advanced urothelial carcinoma

Molecular changes occurring during invasion and clinical progression of cancer are difficult to study longitudinally in patient-derived material. A unique feature of urothelial bladder cancer (UBC) is that patients frequently develop multiple nonmuscle invasive tumors, some of which may eventually progress to invade the muscle of the bladder wall. Here, we use a cohort of 73 patients that experienced a total of 357 UBC diagnoses to study the stability or change in detected molecular alterations during cancer progression. The tumors were subtyped by gene expression profiling and analyzed for hotspot mutations in FGFR3, PIK3CA and TERT, the most frequent early driver mutations in this tumor type. TP53 alterations, frequent in advanced UBC, were inferred from p53 staining pattern, and potential genomic alterations were inferred by gene expression patterns at regions harboring frequent copy number alterations. We show that early driver mutations were largely preserved in UBC recurrences. Changes in FGFR3, PIK3CA or TERT mutation status were not linked to changes in molecular subtype and aggressive behavior. Instead, changes into a more aggressive molecular subtype seem to be associated with p53 alterations. We analyze changes in gene expression from primary tumors, to recurrences and progression tumors, and identify two modes of progression: Patients for whom progression is preceded by or coincides with a radical subtype shift, and patients who progress without any systematic molecular changes. For the latter group of patients, progression may be either stochastic or depending on factors already present at primary tumor initiation.     

Evidence for oligoclonality and tumor spread by intraluminal seeding in multifocal urothelial carcinomas of the upper and lower urinary tract

Multifocality and recurrence of urothelial carcinoma may result from either the field effect of carcinogens leading to oligoclonal tumors or monoclonal tumor spread. Previous molecular studies, favoring the monoclonality hypothesis, are mostly limited to the urinary bladder. We investigated genetic alterations in a total of 94 synchronous or metachronous multifocal tumors from 19 patients with at least one tumor both in the upper and lower urinary tract. Loss of heterozygosity (LOH) was determined using eight markers on chromosome 9 and one marker on 17p13 (p53). Microsatellite instability was investigated at six loci and protein expression of MSH2 and MLH1 was evaluated by immunohistochemistry. In addition, exons 5-9 of the p53 gene were sequenced. Deletions at chromosome 9 were found in 73% of tumors and at 17p13 in 18% of tumors. There was no significant difference in the frequency of LOH in the upper and lower urinary tract. Deletions at 9p21 were significantly correlated with invasive tumor growth. The pattern of deletion revealed monoclonality of all tumors in nine patients. In five patients there were at least two tumor clones with different genetic alterations. In four of these patients the different clones occurred in the bladder and subsequently in the ureter and renal pelvis. All four patients with p53 mutations revealed identical mutations in all tumors. Thus, multifocal urothelial carcinomas are frequently monoclonal, whereas others show oligoclonality, providing molecular evidence for field cancerization. Intraluminal tumor cell seeding appears to be an important mechanism of multifocal occurrence and recurrence of urothelial carcinomas.     

Combined microsatellite and FGFR3 mutation analysis enables a highly sensitive detection of urothelial cell carcinoma in voided urine.

PURPOSE: Fibroblast growth factor receptor 3 (FGFR3) mutations were reported recently at a high frequency in low-grade urothelial cell carcinoma (UCC). We investigated the feasibility of combining microsatellite analysis (MA) and the FGFR3 status for the detection of UCC in voided urine. EXPERIMENTAL DESIGN: In a prospective setting, 59 UCC tissues and matched urine samples were obtained, and subjected to MA (23 markers) and FGFR3 mutation analysis (exons 7, 10, and 15). In each case, a clinical record with tumor and urine features was provided. Fifteen patients with a negative cystoscopy during follow-up served as controls. RESULTS: A mutation in the FGFR3 gene was found in 26 (44%) UCCs of which 22 concerned solitary pTaG1/2 lesions. These mutations were absent in the 15 G3 tumors. For the 6 cases with leukocyturia, 46 microsatellite alterations were found in the tumor. Only 1 of these was also detected in the urine. This was 125 of 357 for the 53 cases without leukocyte contamination. The sensitivity of MA on voided urine was lower for FGFR3-positive UCC (15 of 21; 71%) as compared with FGFR3 wild-type UCC (29 of 32; 91%). By including the FGFR3 mutation, the sensitivity of molecular cytology increased to 89% and was superior to the sensitivity of morphological cytology (25%) for every clinical subdivision. The specificity was 14 of 15 (93%) for the two (molecular and morphological) cytological approaches. CONCLUSIONS: Molecular urine cytology by MA and FGFR3 mutation analysis enables a highly sensitive and specific detection of UCC. The similarity of molecular profiles in tumor and urine corroborate their clonal relation.     

Discovery of TP53 splice variants in two novel papillary urothelial cancer cell lines

Background

Using a novel cell culture technique, we established two new cell lines, BC44 and BC61, from papillary urothelial carcinoma and analyzed them for genetic changes typical of this tumor type.

Methods and results

Karyotyping revealed aneuploid karyotypes with loss of chromosome 9 and rearranged chromosome 5p. Molecular analysis showed CDKN2A deletions but wild-type PIK3CA. BC61 contained a G372C FGFR3 mutation. TP53 was not mutated in either cell line and BC61 expressed normal full-length protein. In contrast, BC44 exclusively expressed cytoplasmic and nuclear p53??40 and 133 isoforms from the alternative promoter P2 as revealed by Western blotting, immunocytochemistry and PCR. The only discernible difference in TP53 in BC44 was homozygosity for the deletion allele of the rs17878362 polymorphism in the P2 promoter. Expression of p53 isoforms was also detected in a few other urothelial carcinoma cell lines and tumor cultures and in 4 out of 28 carcinoma tissues.

Conclusion

In urothelial cancers, TP53 is typically inactivated by mutations in one allele and loss of the wildtype allele and more frequently in invasive compared to papillary carcinomas. We show that some urothelial carcinomas may predominantly or exclusively express isoforms which are not detected by commonly used antibodies to epitopes located in the p53 TA amino-terminal region. Expression of these isoforms may constitute a further mode of p53 inactivation in urothelial carcinoma. Our findings raise the question to which extent this mechanism may compromise wildtype p53 function in papillary tumors in particular, where point mutations in the gene are rare.     

Novel therapeutic targets in bladder cancer: mutation and expression of FGF receptors

Bladder cancer presents several challenges in clinical management. For the large group of noninvasive tumors, key problems are the development of multiple recurrences that require long-term surveillance and a lack of effective therapies to prevent recurrence. For the smaller group of poor prognosis patients with invasive disease, novel therapies are urgently needed. The identification of mutations of FGF receptor 3 (FGFR3) in most noninvasive bladder tumors and the recent finding of overexpression of this receptor not only in superficial tumors but also in many invasive bladder cancers has generated optimism that therapies targeting this receptor tyrosine kinase may have major application in the treatment of urothelial cancers. There is little information on the other members of this receptor family apart from FGFR2, which is implicated as a tumor suppressor. Recent preclinical evaluations of FGFR3 as a therapeutic target have provided a strong impetus for the development of targeted agents for clinical use.