Promoter hypermethylation is associated with tumor location, stage, and subsequent progression in transitional cell carcinoma.

PURPOSE: Transitional cell carcinoma (TCC) is a pan-urothelial disease characterized by multiplicity. Although little is known about the molecular events in upper-tract TCC, similar carcinogenic mechanisms are thought to occur throughout the urinary tract. However, we have previously shown that distinct patterns of microsatellite instability occur in upper and lower urinary tract TCC, suggesting biologic differences between these tumors. Here we investigate the extent of promoter hypermethylation in TCC throughout the urinary tract. PATIENTS AND METHODS: Tissue was obtained from 280 patients (median follow-up, 56 months) whose tumors comprised 116 bladder and 164 upper-tract tumors (UTT). Analysis for hypermethylation at 11 CpG islands, using methylation-sensitive polymerase chain reaction and bisulfite sequencing, was performed for each sample and compared with the tumor's clinicopathologic details, microsatellite instability status, and subsequent behavior. RESULTS: Promoter methylation was present in 86% of TCC and occurred both more frequently and more extensively in UTT (94%) than in bladder tumors (76%; P < .0001). Methylation was associated with advanced tumor stage (P = .0001) and higher tumor progression (P = .03) and mortality rates (P = .04), when compared with tumors without methylation. Multivariate analysis revealed that methylation at the RASSF1A and DAPK loci, in addition to tumor stage and grade, were associated with disease progression (P < .04). CONCLUSION: Despite morphologic similarities, there are genetic and epigenetic differences between TCC in the upper and lower urinary tracts. Methylation occurs commonly in urinary tract tumors, may affect carcinogenic mechanisms, and is a prognostic marker and a potential therapeutic target.

Distinct patterns of microsatellite instability are seen in tumours of the urinary tract.

To date, two forms of microsatellite instability (MSI) have been described in human cancer. MSI typical of hereditary nonpolyposis colon cancer (HNPCC), is due to deficient DNA mismatch repair (MMR) and is defined with mono- and dinucleotide repeat microsatellites. A second variety of instability is best seen at selective tetranucleotide repeats (EMAST; elevated microsatellite alterations at select tetranucleotides). While MSI occurs infrequently in bladder cancers, EMAST is common. Sporadic tumours with the largest proportion showing MSI are those found most frequently in HNPCC kindreds. While bladder cancer is not frequently seen in HNPCC, upper urinary tract tumours (UTTs) are. Having previously found a low frequency of MSI in bladder cancer, we sought to determine the relative levels of MSI and EMAST in transitional cell carcinoma (TCC) of the upper and lower urinary tracts. Microsatellite analysis was performed at 10 mono- and dinucleotide and eight tetranucleotide loci, in 89 bladder and 71 UTT TCC. Contrasting patterns of instability were seen in urinary tumours. In bladder cancer, MSI was rare and EMAST was common. The presence of EMAST was not related to tumour grade, stage, subsequent outcome or immunohistochemical expression of the MMR proteins. In UTT, while MSI occurred frequently, EMAST was seen less frequently than in bladder cancer. When TCC of the upper and lower urinary tracts are compared, MSI-H is more frequent in UTT and EMAST more frequent in bladder cancer. Our findings show that, as for colorectal cancer, the pattern of MSI varies with location in the urinary tract. In addition, we have confirmed that MSI and EMAST are discrete forms of MSI, and that the presence of EMAST does not affect tumour phenotype.

Tissue microarray analysis of the prognostic value of E-cadherin, Ki67, p53, p27, survivin and MSH2 expression in upper urinary tract transitional cell carcinoma.

OBJECTIVES: Invasive upper urinary tract transitional cell carcinoma (UUT-TCC) has a poor prognosis (survival <50% at 5 years), and tumor stage and grade often fail to predict outcome. Our purpose was to establish whether the expression of Ki67, p53, p27, E-cadherin, survivin or MSH2 can provide prognostic information in UUT-TCC. METHODS: The following data from the files of 62 patients treated for UUT-TCC over 12 years were collated: age at diagnosis, prior history of cancer, tobacco consumption, tumor stage (including surgical margins) and grade, and disease progression. Immunohistochemistry (IHC) for Ki67, p53, p27, E-cadherin, survivin and MSH2 was performed on tissue microarray sections from tumor tissue. RESULTS: Overall, 31 patients died with metastasis from UUT-TCC. Mean survival was 20+/-16 months (range 2-83). In a univariate analysis, advanced age (>68 years), high stage, and loss of E-cadherin and high Ki67 expression were associated with a poor prognosis and disease recurrence. In a multivariate analysis, the independent factors of prognosis and recurrence were E-cadherin (p=0.001; p=0.004), age (p=0.022; p=0.008), and high stage (p=0.023; p=0.008). CONCLUSIONS: E-cadherin is a useful independent prognostic factor in UUT-TCC, for use in addition to age and tumor stage. It is of particular interest to predict recurrence in patients with low grade non-invasive tumors. Ki67 expression is informative but less significant. Survivin, p53, p27 and MSH2 have no prognostic value.

Microsatellite instability in urothelial carcinoma of the upper urinary tract.

PURPOSE: Transitional cell carcinoma (TCC) of the upper urinary tract (TCC-UUT) may develop with high frequency in patients with hereditary nonpolyposis colorectal cancer syndrome. Tumors in patients with this syndrome show genomic lesions in DNA mismatch repair genes that are detectable as microsatellite instability (MSI). Because little is known about genetic lesions in TCC-UUT compared with bladder TCC, we determined the genetic profiles (MSI and allelic loss) in a series of 26 upper urinary tract tumors using 5 informative microsatellite markers. MATERIALS AND METHODS: A total of 26 paraffin embedded samples from 24 patients with clinically diagnosed TCC-UUT (renal pelvis and/or ureter) were tested for loss of heterozygosity (LOH) and MSI with the dinucleotide markers D9S171 (9p21) and D5S346 (5q22), and the mononucleotide repeats BAT25 (4q12), BAT26 (2p16) and BAT40 (1p13.1). RESULTS: MSI was detected at 1 or more microsatellite loci in 12 of the 26 tumors (46%). The markers BAT40, BAT25, BAT26, D9S171 and D5S346 showed instability in 7, 4, 4, 2 and 3 tumor samples, respectively. LOH at D9S171 was detected in 58% of the cases and 10 of the 14 tumors showing LOH were superficial. LOH at D5S346 occurred in 27% of the cases and it was a feature of invasive high grade TCC-UUT. CONCLUSIONS: Frequent LOH at D9S171 in TCC-UUT confirms that LOH at 9p21 is not only observed in bladder TCC, but rather in whole urinary tract TCC. Furthermore, our study indicates a high level of MSI in TCC-UUT, although it is a rare event in bladder cancer. The establishment of distinct genetic profiles between upper and lower urinary tract tumors could provide an additional tool to improve diagnosis, disease monitoring and prediction of prognosis.

Expression in bladder transitional cell carcinoma by real-time quantitative reverse transcription polymerase chain reaction array of 65 genes at the tumor suppressor locus 9q34.1-2: identification of 5 candidates tumor suppressor genes.

Frequent deletions on 9q34.1-2 were reported in bladder transitional cell carcinoma. High deletion mapping studies delimited a critical interval between markers D9S61 and D9S66, which is highly susceptible to contain a tumor suppressor gene. Expression level of the 65 genes localized in this region was analyzed by real-time quantitative RT-PCR, comparing tumor to normal urothelium. Five genes exhibited a significantly reduced expression level: C9orf9, KIAA0625, ABL1, LAMC3 and KIAA1857-netrin-G2, which exhibited the most significant downregulation (p=0.0007). KIAA1857-netrin-G2 belongs to the netrins and might then be a tumor suppressor gene in bladder cancer, as netrin1 receptor DCC has been implicated in tumorigenesis.

Non-invasive molecular detection of bladder cancer recurrence.

Transitional cell carcinoma (TCC) is the most common bladder tumor and approximately 90% of bladder TCC are superficial at initial diagnosis. High recurrence rate and possible progression to muscle invasive disease that is eventually indicated for radical cystectomy are established features of these tumors. Therefore, reliable predictors of tumor recurrence are of critical importance for management of superficial bladder TCC. Successful molecular diagnosis of bladder cancer by detecting genetic lesions: loss of heterozygosity (LOH) or microsatellite instability (MSI) in cells exfoliated in urine has been reported by several groups including ours. The aim of our study was to evaluate the predictive potential of microsatellite analysis of cells exfoliated in urine in the detection of superficial bladder TCC recurrence. We studied 47 Caucasian patients with confirmed superficial bladder TCC (37 pTa, 10 pT1) at initial diagnosis. Blood samples were obtained once from every patient whereas urine samples were collected before each cystoscopy (initial and follow-up). Matched DNAs from blood and urine were subjected to microsatellite analysis in a blinded fashion. The follow-up period ranged 12-48 months after tumor resection. Microsatellite analysis correctly identified 94% (44/47) of primary tumors and 92% (12/13) of tumor recurrences. Interestingly enough, 75% (9/12) of tumor recurrences were molecularly detected 1-9 months before cystoscopic evidence of recurrent disease. This study demonstrated clearly that not only urine microsatellite analysis reliably detected superficial bladder tumors, but also was a reliable test for detecting and predicting tumor recurrence in Caucasian patients. These results warrant multicenter randomized trials.