Urothelial Cell Carcinoma Development Research Articles

Human polyomaviruses JCPyV and MCPyV in urothelial cell carcinoma: a single institution experience.

Urothelial cell carcinoma (UCC) is the most common type of urinary bladder. JCPyV and BKPyV have been detected in the urine and tissue of urothelial cell carcinomas (UCC) in immunocompetent patients. Here, we investigated the presence of several HPyVs in UCC samples using diverse molecular techniques to study the prevalence of HPyVs in UCC. A large single-institution database of urine cytology specimens (UCS; n = 22.867 UCS) has previously been searched for decoy cells (n = 30), suggesting polyomavirus infection. The available urine sediments and formalin-fixed paraffin-embedded (FFPE) tissue samples of UCC patients were tested for the presence of JCPyV-LTAg expression by immunohistochemistry (IHC) labeled with SV40-LTAg antibody (clone: PAb416) and subsequent PCR followed by sequencing. In addition, the presence of the oncogenic Merkel cell polyomavirus (MCPyV) and the presence of human polyomavirus 6 (HPyV6) and 7 (HPyV7) DNA were tested with DNA PCR or IHC. Of the 30 patients harboring decoy cells, 14 were diagnosed with UCC of the urinary bladder (14/30; 46.6%) before presenting with decoy cells in the urine. The SV40-LTAg IHC was positive in all 14 UCC urine sediments and negative in the FFPE tissues. JCPyV-DNA was identified in all five available UCS and in three FFPE samples of UCC (three of 14; 21.4%). Two UCC cases were positive for MCPyV-DNA (two of 14; 14.3%), and one of them showed protein expression by IHC (one of 14; 7.1%). All specimens were HPyV6 and HPyV7 negative. Our findings show the presence of JCPyV in the urine and UCC of immunocompetent patients. Moreover, MCPyV was detected in two UCC cases. In total, five UCC cases showed the presence of either JCPyV or MCPyV. The evidence here supports the hypothesis that these viruses might sporadically be associated with UCC. Further studies are needed to confirm the relevance of JCPyV or MCPyV as a possible risk factor for UCC development.

Impact of tissue inhibitor of metalloproteinases-3 genetic variants on clinicopathological characteristics of urothelial cell carcinoma.

To investigate the distribution of single nucleotide polymorphism (SNP) of tissue inhibitor of metalloproteinases-3 (TIMP-3) in patients with/without urothelial cell carcinoma (UCC), three loci of TIMP-3 SNPs (rs9862 C/T, rs9619311 T/C, rs11547635 C/T) were genotyped via TaqMan allelic discrimination for 424 UCC patients and 848 non-UCC participants. Furthermore, the TIMP-3 mRNA expression and its correlation with clinical characters of urothelial bladder carcinoma was analyzed using The Cancer Genome Atlas database (TCGA). The distribution of all 3 studied SNPs of TIMP-3 was insignificantly different between the UCC and non-UCC groups. However, significantly lower tumor T status was found in TIMP-3 SNP rs9862 CT + TT variant than the wild type (OR: 0.515, 95% CI: 0.289-0.917, P = 0.023). Moreover, the muscle invasive tumor type was significantly correlated to the TIMP-3 SNP rs9619311 TC + CC variant in the non-smoker subgroup (OR: 2.149, 95% CI: 1.143-4.039, P = 0.016). With the TIMP-3 expression data provided in TCGA, significantly higher TIMP-3 mRNA expression was observed in UCC with high tumor stage (P < 0.0001), high tumor T status (P < 0.0001) and high lymph node status (P = 0.0005). In conclusions, TIMP-3 SNP rs9862 variant is associated with lower tumor T status of UCC while TIMP-3 SNP rs9619311 variant is correlated to muscle invasive UCC development in non-smoker.

Abstract 926: IIKKa deletion amplifies renal transitional epithelial stem cells and initiates lethal invasive papillary urothelial cell carcinoma development

Abstract Urothelial cell carcinoma is the most common cancer of the renal pelvis or ureter in humans. Due to a lack of animal models, its pathogenic causes remain elusive. Here, we generated IkkαΔKsp mice lacking Ikkα specifically in epithelial cells of the developing kidney and genitourinary tract as well as renal tubules. The mutant mice developed invasive papillary urothelial cell carcinomas initiated from the pelvis, leading to hydronephrosis, and the animals eventually died. Depleting neutrophils or antibiotic treatment slowed down the development of phenotypes but did not rescue them, suggesting that epithelial-cell-autonomous aberrant alterations induced by Ikkα loss are critical for the initiation of urothelial cancer. Moreover, We found that the urothelial cell carcinomas in the mutant mice highly expressed cancer stem-cell markers such as Sox2, K15, p63, Nanog, and CD44, as well as FGFR3 and osteopontin (OPN). Thus, we hypothesize that Ikkα ablation-mediated OPN and FGFR3 overexpression regulates and amplifies CD44+ renal cancer-stem cells for tumor initiation. These alterations are shared with human papillary urothelial cell carcinomas. Citation Format: Xin Li, Feng Zhu, Chengfei Jiang, Yongmei Zhao, Bao Tran, Donna Butcher, Xiaolin Wu, Tross Debra, Yinling Hu. IIKKa deletion amplifies renal transitional epithelial stem cells and initiates lethal invasive papillary urothelial cell carcinoma development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 926.

Impact of Gene Polymorphisms in GAS5 on Urothelial Cell Carcinoma Development and Clinical Characteristics.

Urothelial cell carcinoma (UCC) is the commonest malignant tumor of the urinary tract and the second most common kidney cancer malignancy. Growth arrest-specific 5 (GAS5), a long noncoding RNA, is encoded by the GAS5 gene and plays a critical role in cellular growth arrest and apoptosis. In the current study, two single nucleotide polymorphisms (SNPs) in the GAS5 gene, rs145204276 and rs55829688, were selected to investigate correlations between these single SNPs and susceptibility to UCC. A total of 430 UCC cases and 860 ethnically matched healthy controls were included. SNP rs145204276 and SNP rs55829688 were determined using a TaqMan genotyping assay. Logistic regression models demonstrated that female patients with UCC carrying the rs145204276 GAS5 Ins/Del or Del/Del genotype had a 3.037-fold higher risk of larger tumor status (95% confidence interval 1.259–7.324) than did rs145204276 wild type (Ins/Ins) carriers (p = 0.011). The Cancer Genome Atlas validation cohort analysis demonstrated that the expression of GAS5 in female patients with bladder urothelial carcinoma (BLCA) with larger tumor size was much lower than that in patients with a smaller tumor size (p = 0.041). Kaplan-Meier curve analysis and the log–rank test revealed that female patients with BLCA and lower GAS5 expression had poorer overall survival than those with higher GAS5 expression. In conclusion, genetic variations in GAS5 rs145204276 may serve as a critical predictor of the clinical status of female patients with UCC.

Impact of Matrix Metalloproteinases 11 Gene Variants on Urothelial Cell Carcinoma Development and Clinical Characteristics.

Urothelial cell carcinoma (UCC) is one of the lethal causes of cancer mortality of the genitourinary tract. Carcinogenic epidemiological risk factors exposure and age over 65 years old are associated with UCC risk. Matrix metalloproteinase 11 (MMP11) was suggested as a tumor marker of metastasis and predictor of poor survival in urothelial carcinomas. In this study, we focused on the associations of MMP11 single-nucleotide polymorphisms (SNPs) to UCC susceptibility, clinicopathological characteristics, and prognosis. In this study, real-time polymerase chain reaction was used to analyze five SNPs of MMP11 rs738791, rs2267029, rs738792, rs28382575, and rs131451 in 431 patients with UCC and 650 cancer-free controls. The MMP11 rs28382575 polymorphic “CT” genotype were susceptible to UCC (AOR = 2.045, 95% CI = 1.088 − 3.843; p = 0.026). For MMP11 rs131451, a significant association was found in 166 UCC patients among age ≤ 65 years old who carried MMP11 rs131451 polymorphic “CC” genotype, which is associated with lower risk to develop later tumor T status (T1-T4) (OR = 0.375, 95% CI = 0.159 − 0.887; p = 0.026) compared with the (CT + TT) genotype. Furthermore, patients of UCC with rs738792 polymorphic “CC” genotype were observed to have higher free of relapse (FS) (p = 0.035), disease specific survival rate (p = 0.037), and overall survival rate (p = 0.009) compared with the rs738792 (CT + CC) genotype. In conclusion, our results demonstrated that the MMP11 SNPs are associated with UCC susceptibility, clinical status, and disease survival. The MMP11 polymorphisms may have potential to predict UCC susceptibility and prognosis.

Involvement of FGFR4 Gene Variants on the Clinicopathological Severity in Urothelial Cell Carcinoma.

Fibroblast growth factor receptor 4 (FGFR4) plays a prominent role in cell proliferation and cancer progression. This study explored the effect of FGFR4 single-nucleotide polymorphisms (SNPs) on the clinicopathological characteristics of urothelial cell carcinoma (UCC). This study was conducted to survey the possible correlation of the polymorphism of FGFR4 to the risk and clinicopathologic characteristics of UCC. Four loci of FGFR4 (rs2011077 T > C, rs351855 G > A, rs7708357 G>A, and rs1966265 A > G) were genotyped via the TaqMan allelic discrimination approach in 428 UCC cases and 856 controls. The results indicated that UCC subjects who carried the SNP rs2011077 TC+CC genotypes were significantly related to a higher tumor stage (odds ratio (OR): 1.751, 95% confidence interval (CI): 1.078–2.846), primary tumor size (OR: 1.637, 95% CI: 1.006–2.662), and histopathologic grading (OR: 1.919, 95% CI: 1.049–3.511). Moreover, the SNP rs1966265 AG+GG genotypes were prominently related to a higher tumor stage (OR: 1.769, 95% CI: 1.082–2.891), primary tumor size (OR: 1.654, 95% CI: 1.011–2.706), and histopathologic grading (OR: 2.006, 95% CI: 1.096–3.674) compared to individuals with AA homozygotes. In conclusion, our data reveal association of FGFR4 polymorphisms with UCC clinicopathologic characteristics. FGFR4 polymorphisms may serve as a marker or therapeutic target in UCC development.

Effects of Long Noncoding RNA H19 Polymorphisms on Urothelial Cell Carcinoma Development.

Urothelial cell carcinoma (UCC) is one of the major malignancies of the genitourinary tract, and it is induced by carcinogenic epidemiological risk factors. H19 is one of the most crucial long noncoding RNAs (lncRNAs) and is involved in various types of bladder cancer. In this study, we examined H19 single-nucleotide polymorphisms (SNPs) to investigate UCC susceptibility and clinicopathological characteristics. Using real-time polymerase chain reaction, we analyzed five SNPs of H19 in 431 UCC patients and 431 controls without cancer. The results showed that patients with UCC carrying the H19 rs217727 CT + TT and rs2107425 CT + TT genetic variants had a high risk of developing muscle invasive tumors (pT2–T4) (p = 0.030; p = 0.025, respectively). With a median follow up of 39 months, CT+TT polymorphisms of rs2107425 were associated with worse disease-specific survival (adjusted hard ratio (AHR) = 2.043, 95% confidence interval (CI) = 1.029-4.059) in UCC patients aged older than 65 years. In conclusion, our results indicate that patients with UCC carrying the H19 rs217727 CT + TT and rs2107425 CT + TT genetic variants have a high risk of developing muscle invasive tumors. Thus, H19 polymorphisms may be applied as a marker or therapeutic target in UCC treatment.

Impact of endothelial nitric oxide synthase polymorphisms on urothelial cell carcinoma development

ObjectivesUrothelial cell carcinoma (UCC), a major malignancy of the genitourinary tract, is induced through carcinogenic etiological factors. Endothelial nitric oxide synthase (eNOS) is one of the major isoforms of nitric oxide synthase and is involved in various pathophysiologic and physiologic processes. In this study, eNOS single-nucleotide polymorphisms were investigated to evaluate UCC susceptibility and clinicopathological characteristics. Materials and methodsTwo single-nucleotide polymorphisms of eNOS in 431 patients with UCC and 862 controls without cancer were analyzed using real-time polymerase chain reaction. ResultsThe results showed that 272 men with UCC having eNOS 894 G > T rs1799983 “GT + TT” variants had a high risk of developing a large tumor (T1–T4, P = 0.038). Furthermore, a correlation was observed between the expressions of eNOS and invasive tumor, metastasis and poor survival in urothelial carcinoma in The Cancer Genome Atlas data set. ConclusionOur results indicated that male patients with UCC carrying eNOS 894 G > T rs1799983 “GT + TT” genetic variants have a high risk of developing a large tumor, and eNOS polymorphisms may serve as a marker or therapeutic target in UCC treatment.

Impacts of AURKA Genetic Polymorphism on Urothelial Cell Carcinoma Development.

Urothelial cell carcinoma (UCC) is the most common primary malignancy of the urinary system and the second-most common type of renal cell carcinoma. Aurora kinase A (AURKA), a serine/threonine kinase, has a critical role in centrosome duplication, spindle assembly checkpoint, and cytokinesis. Here, we determined the correlation between UCC susceptibility and AURKA polymorphisms. We used real-time polymerase chain reaction to compare the genotype distributions and allelic frequencies of four single-nucleotide polymorphisms (SNPs) of AURKA, namely rs1047972, rs2273535, rs2064863, and rs6024836, between 431 UCC cases and 862 healthy controls. Logistic regression models demonstrated that the G allele of rs2064863, a genetic polymorphism of AURKA, exhibited a significant protective effect against UCC among the 862 nonsmokers. Moreover, patients with rs2064863 G allele exhibited a slightly lower risk of lymph node metastasis and those with rs6024836 G allele exhibited a lower risk of distant metastases. Our study suggested that several variants of AURKA SNPs rs2064863 and rs6024836 may serve as critical predictors for the clinical status of UCC.

Dopamine receptor D2 genetic variations is associated with the risk and clinicopathological variables of urothelial cell carcinoma in a Taiwanese population.

Dopamine receptor D2 (DRD2) is overexpressed in several kinds of cancers and was correlated with the prognosis of these cancers. Polymorphisms within the DRD2 gene were shown to be associated with lung and colon cancers. The purpose of this study was to explore effects of DRD2 gene polymorphisms on the susceptibility to and clinicopathological characteristics of urothelial cell carcinoma (UCC). In total, 369 patients diagnosed with UCC and 738 healthy controls were enrolled to analyze DRD2 genotypes at four loci (rs1799732, -141C>del; rs1079597, TaqIB; rs6277, 957C>T; and rs1800497, TaqIA) using a TaqMan-based real-time polymerase chain reaction (PCR). We found a significantly lower risk for UCC in individuals with the DRD2 rs6277 CT genotype compared to those with the wild-type CC genotype (adjusted odds ratio (AOR)=0.405, 95% confidence interval (CI): 0.196~0.837, p=0.015). In 124 younger patients (aged < 65 years) of the recruited UCC cohort, patients who carried at least one T allele of DRD2 rs1800497 were at higher risk (AOR=2.270, 95% CI: 1.060~4.860, p=0.033) of developing an invasive stage (pT2~pT4). In 128 female patients of the recruited UCC cohort, patients who carried at least one deletion allele of DRD2 rs1799732 showed a higher incidence of having an invasive stage (AOR=2.585, 95% CI: 1.066~6.264, p=0.032) and a large tumor (AOR=2.778, 95% CI: 1.146~6.735, p=0.021). Further analyses of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets revealed correlations of the expression of DRD2 with an invasive tumor, tumor metastasis, and the lower survival rate in patients with UCC. Our findings suggest that DRD2 levels might affect the progression of UCC, and the polymorphisms rs6277, rs1800497, and rs1799732 of DRD2 are probably associated with the susceptibility and clinicopathologic development of UCC in a Taiwanese population.

Genetic Variations of Melatonin Receptor Type 1A are Associated with the Clinicopathologic Development of Urothelial Cell Carcinoma.

Melatonin counteracts tumor occurrence and tumor cell progression in several cancer types in vitro and in vivo. It acts predominantly through its melatonin receptor type 1A (MTNR1A), and genetic variations of MTNR1A affect the susceptibility several diseases and cancer. The purpose of this study was to explore the effect of MTNR1A gene polymorphisms on the susceptibility to and clinicopathological characteristics of urothelial cell carcinoma (UCC). We recruited 272 patients with UCC and 272 normal controls to analyze three common single-nucleotide polymorphisms (SNPs) (rs2119882, rs13140012, and rs6553010) of MTNR1A related to cancer risk and clinicopathological relevance according to a TaqMan-based real-time polymerase chain reaction (PCR). We found that these three SNPs of MTNR1A were not associated with UCC susceptibility. However, patients with UCC who had at least one G allele of MTNR1A rs6553010 (in intron 1) were at higher risk (1.768-fold, 95% confidence interval: 1.068~1.849) of developing an invasive stage (p < 0.026), compared to those patients with AA homozygotes. In conclusion, polymorphic genotypes of rs6553010 of MTNR1A might contribute to the ability to predict aggressive phenotypes of UCC. This is the first study to provide insights into risk factors associated with intronic MTNR1A variants in the clinicopathologic development of UCC in Taiwan.

Impacts of ICAM-1 gene polymorphisms on urothelial cell carcinoma susceptibility and clinicopathologic characteristics in Taiwan.

Intercellular adhesion molecule (ICAM)-1, a cell adhesion molecule, is reportedly overexpressed in several cancers and may contribute to tumorgenesis and metastasis. The current study explored the effect of ICAM-1 gene polymorphisms on the susceptibility of developing urothelial cell carcinoma (UCC) and the clinicopathological status. A total of 558 participants, including 279 healthy people and 279 patients with UCC, were recruited for this study. Four single-nucleotide polymorphisms of the ICAM-1 gene were assessed by a real-time polymerase chain reaction with the TaqMan assay. After adjusting for other covariants, the individuals carrying at least one G allele at ICAM-1 rs5498 had a 1.603-fold risk of developing UCC than did wild-type (AA) carriers. Furthermore, UCC patients who carried at least one G allele at rs5498 had a higher invasive stage risk (p < 0.05) than did patients carrying the wild-type allele. In conclusion, the rs5498 polymorphic genotypes of ICAM-1 might contribute to the prediction of susceptibility to and pathological development of UCC. This is the first study to provide insight into risk factors associated with ICAM-1 variants in carcinogenesis of UCC in Taiwan.

Impacts of CA9 gene polymorphisms on urothelial cell carcinoma susceptibility and clinicopathologic characteristics in Taiwan.

BackgroundCarbonic anhydrase 9 (CA9) is reportedly overexpressed in several types of carcinomas and is generally considered a marker of malignancy. The current study explored the effect of CA9 gene polymorphisms on the susceptibility of developing urothelial cell carcinoma (UCC) and the clinicopathological status.Methodology and Principal FindingsA total of 442 participants, including 221 healthy people and 221 patients with UCC, were recruited for this study. Four single-nucleotide polymorphisms (SNPs) of the CA9 gene were assessed by a real-time PCR with the TaqMan assay. After adjusting for other co-variants, the individuals carrying at least one A allele at CA9 rs1048638 had a 2.303-fold risk of developing UCC than did wild-type (CC) carriers. Furthermore, UCC patients who carried at least one A allele at rs1048638 had a higher invasive stage risk (p< 0.05) than did patients carrying the wild-type allele. Moreover, among the UCC patients with smoker, people with at least one A allele of CA9 polymorphisms (rs1048638) had a 4.75-fold (95% CI = 1.204–18.746) increased risk of invasive cancer.ConclusionThe rs1048638 polymorphic genotypes of CA9 might contribute to the prediction of susceptibility to and pathological development of UCC. This is the first study to provide insight into risk factors associated with CA9 variants in carcinogenesis of UCC in Taiwan.

Genome-wide methylation profiling and the PI3K-AKT pathway analysis associated with smoking in urothelial cell carcinoma

Urothelial cell carcinoma (UCC) is the second most common genitourinary malignant disease in the USA, and tobacco smoking is the major known risk factor for UCC development. Exposure to carcinogens, such as those contained in tobacco smoke, is known to directly or indirectly damage DNA, causing mutations, chromosomal deletion events and epigenetic alterations in UCC. Molecular studies have shown that chromosome 9 alterations and P53, RAS, RB and PTEN mutations are among the most frequent events in UCC. Recent studies suggested that continuous tobacco carcinogen exposure drives and enhances the selection of epigenetically altered cells in UCC, predominantly in the invasive form of the disease. However, the sequence of molecular events that leads to UCC after exposure to tobacco smoke is not well understood. To elucidate molecular events that lead to UCC oncogenesis and progression after tobacco exposure, we developed an in vitro cellular model for smoking-induced UCC. SV-40 immortalized normal HUC1 human bladder epithelial cells were continuously exposed to 0.1% cigarette smoke extract (CSE) until transformation occurred. Morphological alterations and increased cell proliferation of non-malignant urothelial cells were observed after 4 months (mo) of treatment with CSE. Anchorage-independent growth assessed by soft agar assay and increase in the migratory and invasive potential was observed in urothelial cells after 6 mo of CSE treatment. By performing a PCR mRNA expression array specific to the PI3K-AKT pathway, we found that 26 genes were upregulated and 22 genes were downregulated after 6 mo of CSE exposure of HUC1 cells. Among the altered genes, PTEN, FOXO1, MAPK1 and PDK1 were downregulated in the transformed cells, while AKT1, AKT2, HRAS, RAC1 were upregulated. Validation by RT-PCR and western blot analysis was then performed. Furthermore, genome-wide methylation analysis revealed MCAM, DCC and HIC1 are hypermethylated in CSE-treated urothelial cells when compared with non-CSE exposed cells. The methylation status of these genes was validated using quantitative methylation-specific PCR (QMSP), confirming an increase in methylation of CSE-treated urothelial cells compared to untreated controls. Therefore, our findings suggest that a tobacco signature could emerge from distinctive patterns of genetic and epigenetic alterations and can be identified using an in vitro cellular model for the development of smoking-induced cancer.

Abstract 5451: Arsenic induced altered pathways in bladder cancer

Abstract Background: Urine excretion is the primary route for arsenic elimination. The bladder may thus be exposed to higher concentrations of arsenic due to the bioconcentration of urine by the kidneys. The molecular etiology of cancer linked to arsenic exposure is complex and multiple factors have been described as playing a role in this process. Although there is extensive epidemiologic evidence of increased risk for the development of urothelial cell carcinoma (UCC) associated with arsenic exposure, the mechanisms by which arsenic participates in tumorigenesis are not fully elucidated. Materials and Methods: To prepare in vitro model we chronically exposed immortalized normal human bladder cell line (HUC-1) to arsenic. Briefly, HUC1 cells were cultured in 25cm flask in F12K complete medium with or without 1µM As2O3. Medium and arsenic was changed every two days. Cells were sub-cultured as necessary and frozen down each month for future studies. Morphology differences were monitored over the entire treatment schedule, using a light microscope. Cell growth was monitored by MTT assay. Tumorigenic properties were analyzed by soft agar and invasion assay. Expression of several key molecules of PI3K-AKT signaling pathway was determined by western blot analysis. Results: At 6 months arsenic treated HUC1 cells started to became more rounded and had a tendency to pile on to one another. MTT was performed for each month of treatment in order to determine any changes in cell proliferation due to arsenic treatment. As expected the growth of the HUC1 cells was increased in a time dependent manner after arsenic treatment. Anchorage independent growth was evaluated using soft agar assay. Colonies were observed only in arsenic treated cells and the number of colonies was gradually increased with longer period of treatment. Similarly invaded cells were determined only in arsenic treated cells after 8 months. Withdrawal of arsenic for 2.5 months from 8 and 10 months arsenic treated cells does not reverse the tumorigenic properties. Western Blot analysis of AKT pathway genes, PTEN decreased and AKT and mTOR increased in arsenic treated HUC cells. Conclusion: Our study suggests that the exposure of HUC-1 to arsenic rapidly induces a multifaceted dedifferentiation program characterized by increased cell proliferation, anchorage independent growth and invasion, a motive that is also present after the end of the arsenic treatment indicating that such a manifestation seems to be irreversible. Further studies are needed in order to delineate the molecular effect of arsenic exposal to primary urothelial cancer tissue collected from patients exposed to arsenic. Studies are in progress to understand the mechanisms that induce this tumorigenic properties and potential stem cells activities in arsenic exposed urothelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5451. doi:1538-7445.AM2012-5451

Sensitive Allele-Specific PCR Assay Able to Detect FGFR3 Mutations in Tumors and Urine from Patients with Urothelial Cell Carcinoma of the Bladder

Activating somatic point mutations in exons 7, 10, and 15 of the FGFR3 gene are frequently observed in urothelial cell carcinoma (UCC) (1)(2). These mutations have been found primarily in superficial papillary pTa tumors and were absent in carcinoma in situ (3). Given the high frequency of FGFR3 mutations and the possible implication of this receptor in the development of UCC, it was important to develop a simple, fast, and reliable method to identify these mutations in greater detail as a potential tool for the diagnosis and follow-up of UCC patients. Ten different FGFR3 mutations have been described in UCC, but 4 of them (R248C, S249C, G372C, and Y375C) account for >95% of cases (2). These mutations therefore represent an excellent target for assays, such as allele-specific PCR (AS-PCR), that depend on the specific detection of point mutations. The general principle underlying the AS-PCR technique is to design a mutation-specific primer that produces the preferential amplification of a specific mutant allele (4). We compared the results obtained with this assay with those obtained by direct sequencing in a series of 95 DNA samples extracted from UCC tumors. We also analyzed matching tumors and voided urine from 20 patients. Our results demonstrate the sensitivity, specificity, and reliability of this technique for detecting FGFR3 mutations in both tumors and urine from patients with UCC. Ninety-five primary tumor samples were collected from patients admitted to the Department of Urology of Hospitals Henri Mondor (Creteil) and CHRU (Besancon) and who had received no previous treatment. Patients gave written informed consent, and we collected matched tumors and blood samples. Tumors from each patient were snap-frozen and stored at −80 °C. DNA from tumor samples was extracted as described previously (5). DNA was extracted from the blood with use of the QIAamp system …