Search

Urothelial carcinoma (UC) is the 10 th most common cancer globally with an almost 4 times higher prevalence in men. The main risk factors for development of urothelial carcinoma are advanced age, smoking, arsenic contamination, exposure to carcinogens. Metastatic urothelial carcinoma (mUC) has overall poor prognosis with a 5-year overall survival rate of only < 5%. The standard of care comprises of platinum-based chemotherapy, but the responses are often not sustained. A working group was established with an objective to discuss the most recent clinical data on the genitourinary tumors of interest and comprised of experts across Latin America, Emerging Asia (except China, Japan, and South Korea), Africa, and the Middle East (known as Emerging Markets or EM). There is an evident disparity in terms of uneven mortality and incidence rate distribution among various regions. There is a lack and/or insufficient data on epidemiology, treatment, and outcomes in the EM. The lack of registries impacts the healthcare decisions and the lower incidence from the region might not be reflective of the true disease burden. The treatment outcomes of mUC can be improved by understanding the current disease burden and treatment approach of mUC and identifying the gaps and challenges associated with management. Hence, a literature review was developed to summarize the current disease burden and treatment approach of mUC across EM. The review also highlights the unmet needs for mUC management in EM and suggests a way forward to improve the current situation in order to better serve the patients., Competing Interests: Disclosure All authors are members of the Emerging Markets Genitourinary Cancer Working Group. Additionally, Waleed Al Morsi is a full-time employee of Pfizer., (Copyright © 2024. Published by Elsevier Inc.)

Purpose: To evaluate predictive factors of increasing intravesical recurrence (IVR) rate in patients with upper tract urothelial carcinoma (UTUC) after receiving radical nephroureterectomy (RNUx) with bladder cuff excision (BCE)., Materials and Methods: A total of 2114 patients were included from the updated data of the Taiwan UTUC Collaboration Group. It was divided into two groups: IVR-free and IVR after RNUx, with 1527 and 587 patients, respectively. To determine the factors affecting IVR, TNM stage, the usage of pre-operative ureteroscopy, and pathological outcomes were evaluated. The Kaplan-Meier estimator was used to estimate the rates of prognostic outcomes in overall survival (OS), cancer-specific survival (CSS), disease-free survival (DFS), and bladder recurrence-free survival (BRFS), and the survival curves were compared using the stratified log-rank test., Results: Based on our research, ureter tumor, female, smoking history, age (< 70 years old), multifocal tumor, history of bladder cancer were determined to increase the risk of IVR after univariate analysis. The multivariable analysis revealed that female (BRFS for male: HR 0.566, 95% CI 0.469-0.681, p < 0.001), ureter tumor (BRFS: HR 1.359, 95% CI 1.133-1.631, p = 0.001), multifocal (BRFS: HR 1.200, 95% CI 1.001-1.439, p = 0.049), history of bladder cancer (BRFS: HR 1.480, 95% CI 1.118-1.959, p = 0.006) were the prognostic factors for IVR. Patients who ever received ureterorenoscopy (URS) did not increase the risk of IVR., Conclusion: Patients with ureter tumor and previous bladder UC history are important factors to increase the risk of IVR after RNUx. Pre-operative URS manipulation is not associated with higher risk of IVR and diagnostic URS is feasible especially for insufficient information of image study. More frequent surveillance regimen may be needed for these patients., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: The purpose of this study is to evaluate the rates of pathological complete response (ypT0N0/X) and pathological response (ypT1N0/X or less) in patients with upper tract urothelial cancer who were treated with neo-adjuvant chemotherapy and to examine their impact on oncological outcomes., Methods: This study is a multi-institutional retrospective analysis of patients with high-risk upper tract urothelial cancer who underwent neoadjuvant chemotherapy and radical nephroureterectomy between 2002 and 2021. Logistic regression analyses were used to investigate all clinical parameters for response after neoadjuvant chemotherapy. Cox proportional hazard models were performed to assess the effect of the response on the oncological outcomes., Results: A total of 84 patients with UTUC who received neo-adjuvant chemotherapy were identified. Among them, 44 (52.4%) patients received cisplatin-based chemotherapy, and 22 (26.2%) patients had a carboplatin-based regimen. The pathological complete response rate was 11.6% (n = 10), and the pathological response rate was 42.9% (n = 36). Multifocal tumors or tumors larger than 3 cm significantly reduced the odds of pathological response. In the multivariable Cox proportional hazard model, pathological response was independently associated with better overall survival (HR 0.38, p = 0.024), cancer-specific survival (HR 0.24, p = 0.033), and recurrence-free survival (HR 0.17, p = 0.001), but it was not associated with bladder recurrence-free survival (HR 0.84, p = 0.69)., Conclusion: Pathological response after neo-adjuvant chemotherapy and radical nephroureterectomy is strongly associated with patient survival and recurrence, and it might be a good surrogate for evaluating the efficacy of neo-adjuvant chemotherapy in the future., Competing Interests: Conflicts of interest The authors have no conflicts of interest relevant to this article., (Copyright © 2023 Formosan Medical Association. Published by Elsevier B.V. All rights reserved.)

Background: Endoscopic management of upper tract urothelial cancer (UTUC) is an important treatment option for low risk UTUC. Although Taiwan is an endemic area for UTUC, endoscopic treatment outcomes in Taiwan are frequently under- reported., Methods: This study retrospectively reviewed the treatment outcomes of endoscopic management for clinically localized UTUC. Patients with biopsy or washing cytology confirmed UTUC who underwent endoscopic or percutaneous management with a curative intent were retrospectively reviewed for eligibility of analysis. Those cases without pre-intervention confirmed UTUC, and metastatic or nodal disease at diagnosis were excluded., Results: In total, 307 patients who underwent endoscopic management were reviewed and 279 cases were eligible for final analysis. With a median follow-up of 44.3 months (inter-quartile range (IQR): 23.4-76.4 months), 117 cases (46.4%) were endoscopic cured after median one session (range:1-8; IQR:1-2) of endoscopic treatment. Those endoscopic cured UTUC was associated with more small-sized tumor, more low-grade biopsied-histology, less concomitant bladder UC and less pre-operative hydronephrosis. In addition, 201(79.7%) cases among 252 cases with confirmed oncological outcome were free of UTUC at the end of follow-up and only 43 (17%) patients had a UTUC related mortality. Salvage RNU offered a better tumor free survival rate (92% vs. 77.5%) than those without salvage RNU in those UTUC refractory to endoscopic management. In multivariable analyses, pre-operative hydronephrosis was the independent risk factor for OS. Multiplicity and concomitant bladder UC were independent risk factors for DFS., Conclusions: We confirmed the consistent safety and efficacy of endoscopic management of clinical localized UTUC in a highly UTUC endemic area like Taiwan. Early salvage RNU is mandatory in those UTUC refractory to endoscopic management in prevention of UTUC related death., Competing Interests: Declaration of competing interest Yung Tai Chen, Hsin-Chih Yeh, Hsiang-Ying Lee, Po-Fan Hsieh, Eric Chieh-lung Chou, Yao-Chou Tsai, Ting-En Tai, Jian-Hua Hong, Chao-Yuan Huang, Yuan-Hong Jiang, Yu-Khun Lee, Jen-Shu Tseng, Chih-Chin Yu, Bing-Juin Chiang, Thomas Y. Hsueh, Chia-Chang Wu, and Chung-You Tsai have no conflicts of interest or financial ties to disclose., (Copyright © 2023 Asian Surgical Association and Taiwan Robotic Surgery Association. Published by Elsevier B.V. All rights reserved.)

Background: Although trimodality therapy resecting tumours followed by chemoradiotherapy is emerged for muscle-invasive bladder cancer (MIBC), chemotherapy produces toxicities. Histone deacetylase inhibitors have been identified as an effective strategy to enhance cancer radiotherapy (RT)., Methods: We examined the role of HDAC6 and specific inhibition of HDAC6 on BC radiosensitivity by performing transcriptomic analysis and mechanism study., Results: HDAC6 knockdown or HDAC6 inhibitor (HDAC6i) tubacin exerted a radiosensitizing effect, including decreased clonogenic survival, increased H3K9ac and α-tubulin acetylation, and accumulated γH2AX, which are similar to the effect of panobinostat, a pan-HDACi, on irradiated BC cells. Transcriptomics of shHDAC6-transduced T24 under irradiation showed that shHDAC6 counteracted RT-induced mRNA expression of CXCL1, SERPINE1, SDC1 and SDC2, which are linked to cell migration, angiogenesis and metastasis. Moreover, tubacin significantly suppressed RT-induced CXCL1 and radiation-enhanced invasion/migration, whereas panobinostat elevated RT-induced CXCL1 expression and invasion/migration abilities. This phenotype was significantly abrogated by anti-CXCL1 antibody, indicating the key regulator of CXCL1 contributing to BC malignancy. Immunohistochemical evaluation of tumours from urothelial carcinoma patients supported the correlation between high CXCL1 expression and reduced survival., Conclusion: Unlike pan-HDACi, the selective HDAC6i can enhance BC radiosensitization and effectively inhibit RT-induced oncogenic CXCL1-Snail-signalling, thus further advancing its therapeutic potential with RT., (© 2023. The Author(s).)

Purpose: Laparoscopic radical nephroureterectomy (LNU) has gradually become the new standard treatment for localized upper tract urothelial cancer (UTUC). With more blunt dissection and tactile sensation, hand-assisted LNU might shorten the operative time compared with the pure laparoscopic approach. However, whether the use of the hand-assisted or the pure laparoscopic approach has an effect on oncological outcomes remains unclear., Methods: We retrospectively identified 629 patients with non-metastatic UTUC who underwent hand-assisted (n = 515) or pure LNU (n = 114) at 9 hospitals in Taiwan between 2004 and 2019. Overall survival, cancer-specific survival, recurrence-free survival, and bladder recurrence-free survival were compared between these two groups using inverse-probability of treatment weighting (IPTW) derived from the propensity scores for baseline covariate adjustment., Results: The median follow-up period was 32.9 and 28.7 months in the hand-assisted and the pure groups, respectively. IPTW-adjusted Cox proportional hazards models showed that the laparoscopic approach (pure vs. hand-assisted) was not significantly associated with all-cause mortality (HR 0.79, 95% CI 0.49-1.24, p = 0.304), cancer-specific mortality (HR 0.88, 95% CI 0.51-1.51, p = 0.634), or extra-vesical recurrence (HR 0.65, 95% CI 0.41-1.04, p = 0.071). However, the pure laparoscopic approach was significantly associated with lower intra-vescial recurrence (HR 0.64, 95% CI 0.43-0.96, p = 0.029) for patients who underwent LNU. Kaplan-Meier curves also revealed that the pure laparoscopic approach was associated with better bladder recurrence-free survival compared with the hand-assisted laparoscopic approach in both the original cohort and the IPTW-adjusted cohort (log-rank p = 0.042 and 0.027, respectively)., Conclusions: The performance of hand-assisted or pure LNU does not significantly affect the all-cause mortality, cancer-specific mortality, or extra-vesical recurrence for patients with non-metastatic UTUC. However, the hand-assisted laparoscopic approach could increase the risk of intra-vesical recurrence for patients who undergo LNU., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cisplatin-based chemotherapy is the primary treatment for metastatic bladder urothelial carcinoma. However, the response rate is only 40-65%. This study investigated the anti-tumor effect and underlying mechanisms of the combination of cisplatin and the NEDD8-activating enzyme inhibitor MLN4924 in human bladder urothelial carcinoma. The combination of cisplatin and MLN4924 exerted synergistic cytotoxicity on two high-grade bladder urothelial carcinoma cell lines, NTUB1 and T24 (combination index <1). MLN4924 also potentiated the cisplatin-induced apoptosis and activation of caspase-3 and -7, phospho-histone H2A.X and PARP. c-Jun N-terminal kinase (JNK) activation and a down-regulation of B-cell lymphoma-extra large (Bcl-xL) were also observed during cisplatin and MLN4924 treatment. Inhibition of JNK activation partially restored cell viability and Bcl-xL expression. Bcl-xL overexpression also rescued cell viability. MLN4924 significantly potentiated cisplatin-induced tumor suppression in urothelial carcinoma xenograft mice. In summary, MLN4924 synergistically enhanced the anti-tumor effect of cisplatin via an increase in DNA damage, JNK activation and down-regulation of Bcl-xL in urothelial carcinoma cells. These findings provide a new therapeutic strategy for the treatment of bladder cancer.

Clinical reports show males have a higher bladder cancer (BCa) incidence than females. The sexual difference of BCa occurrence suggests that estrogen and its receptors may affect BCa development. Estrogen receptor alpha (ERα) is the classic receptor to convey estrogen signaling, however, the function of ERα in BCa development remains largely unknown. To understand the in vivo role of ERα in BCa development, we generated total and urothelial specific ERα knockout mice (ERαKO) and used the pre- carcinogen BBN to induce BCa. Earlier reports showed that ERα promotes breast and ovarian cancers in females. Surprisingly and of clinical importance, our results showed that ERα inhibits BCa development and loss of the ERα gene results in an earlier onset and higher incidence of BBN-induced in vivo mouse BCa. Supportively, carcinogen induced malignant transformation ability was reduced in ERα expressing urothelial cells as compared to ERα negative cells. Mechanism studies suggest that ERα could control the expression of INPP4B to reduce AKT activity and consequently reduce BCa cell growth. In addition, IHC staining of clinical sample analyses show that INPP4B expression, in correlation with reduced ERα, is significantly reduced in human BCa specimens. Together, this is the first report using the in vivo cre-loxP gene knockout mouse model to characterize ERα roles in BCa development. Our studies provide multiple in vitro cell studies and in vivo animal model data as well as human BCa tissue analyses to prove ERα plays a protective role in BCa initiation and growth at least partly via modulating the INPP4B/Akt pathway.

Objective: To report a large multi-institutional series of laparoendoscopic single-site (LESS) nephroureterectomy (NU)., Materials and Methods: Data on all cases of LESS-NU performed between 2008 and 2012 at 15 institutions were retrospectively gathered. The main demographic data and perioperative outcomes were analysed., Results: The study included 101 patients whose mean (SD) age was 66.4 (9.9) years and mean (SD) body mass index was 24.8 (4) kg/m², and of whom 29.7% had undergone previous abdominal/pelvic surgery. The mean (SD) operating time was 221.4 (73.7) min, estimated blood loss 231.7 (348.0) mL. A robot-assisted LESS technique was applied in 25.7% of cases. An extra trocar was inserted in 28.7% of cases to complete the procedure. Conversion to open surgery was necessary in three cases (3.0%). There was no bladder cuff excision in 20.8% of cases, and excision was carried out using a variety of techniques in the remaining cases. Six intra-operative complications occurred (5.9%). The mean (SD) length of hospital stay was 6.3 (3.5) days. The overall postoperative complication rate was 10.0%, and most of the complications were low grade (Clavien grades 1 and 2). The mean tumour size was 3.1 (1.9) cm. Pathological staging was pTis in two patients, pTa in 12 patients, pT1 in 42 patients, pT2 in 20 patients, pT3 in 23 patients and pT4 in two patients. Pathological grade was high in 71 and low in 30 patients. At a mean follow-up of 14 months, six patients (5.9%) had died. Disease recurrence (including distant and bladder recurrence) was detected in 22.8% of patients, with a mean time to recurrence of 11.5 months., Conclusions: This study reports the largest multi-institutional experience of LESS-NU to date. Peri-operative outcomes mirror those of published standard laparoscopy series. Despite encouraging early findings, longer follow-up is needed to determine the oncological efficacy of the procedure., (© 2013 BJU International.)

The Polycomb Repressive Complex 2 (PRC2) mediates epigenetic gene silencing by trimethylating histone H3 lysine 27 (H3K27me3) and is known to aberrantly silence tumor suppressor genes in cancer. EZH2, the catalytic subunit of PRC2, enhances tumorigenesis and is commonly overexpressed in several types of cancer. Our microRNA profiling of bladder transitional cell carcinoma (TCC) patient samples revealed that microRNA-101 (miR-101) is down-regulated in TCC, and we showed that miR-101 inhibits cell proliferation and colony formation in TCC cell lines. Furthermore, our results confirm that miR-101 directly represses EZH2 and stable EZH2 knockdowns in TCC cell lines create a similar growth suppressive phenotype. This suggests that abnormal down-regulation of miR-101 could lead to the overexpression of EZH2 frequently seen in cancer. We conclude that miR-101 may be a potent tumor suppressor by altering global chromatin structure through repression of EZH2.

Objective: To evaluate effects of stopping smoking on the outcome of nonmuscle-invasive bladder cancer, as cigarette smoking is a risk factor for bladder cancer and little is known about whether stopping smoking reduces the risk of recurrence or progression., Patients and Methods: Between January 1997 and July 2005, 297 men with primary nonmuscle-invasive bladder cancer were treated with transurethral resection (TUR); their smoking status before and after the diagnosis of bladder cancer was obtained by a post hoc questionnaire and interview. 'Quitters' were those who ceased smoking within a year before and 3 months after the diagnosis. Ex-smokers were those who ceased smoking more than a year before diagnosis. Several pathological and clinical variables were compared, with all statistical comparisons being two-sided., Results: In all, 265 patients completed the questionnaire, including 64 non-smokers, 64 ex-smokers, 59 quitters, and 78 continued smokers. The median follow-up was 38 months. There were no significant differences in the strata of stage, grade, tumour multiplicity, intravesical therapy, or median follow-up duration between the four patient groups. The respective 3-year recurrence-free survival of continued smokers, non-smokers, ex-smokers and quitters was 45%, 57%, 62% and 70%. By multivariate analysis, high-grade, T1-stage, multiple tumours and continued smoking were significant independent predictors for a shorter recurrence-free survival. Quitters had a lower risk of recurrence than did either continued smokers or non-smokers, but had a similar risk to ex-smokers., Conclusion: Stopping smoking might be associated with a lower recurrence rate for patients with nonmuscle-invasive bladder cancer.

Among eight human bladder cancer cell lines we examined, only T24 cells were resistant to the growth inhibition effect of genistein, an isoflavone and potent anticancer drug. Since the T24 cell line was the only cell line known to overexpress oncogenic H-Ras(val 12), we investigated the role of H-Ras(val 12) in mediating drug resistance. Herein, we demonstrate that the phenotype of T24 cells could be dramatically reversed and became relatively susceptible to growth inhibition by genistein if the synthesis of H-Ras(val 12) or its downstream effector c-Fos had been suppressed. The inhibition of Ras-mediated signalling with protein kinase inhibitors, such as PD58059 and U0126 which inhibited MEK and ERK, in T24 cells also rendered the identical phenotypic reversion. However, this reversion was not observed when an inhibitor was used to suppress the protein phosphorylation function of PI3 K or PKC. These results suggest that the signal mediated by H-Ras(val 12) is predominantly responsible for the resistance of the cells to the anticancer drug genistein.

Cell division is essential for tumor development and progression. Methylation-mediated silencing caused by aberrant de novo methylation of CpG islands located in the promoter regions of growth regulatory genes occurs frequently in human cancers. We investigated the relationship between cell division and de novo methylation to determine whether de novo methylation can occur in the absence of cell division in cancer cells. We treated T24 bladder carcinoma cells with 5-Aza-2'-deoxycytidine to induce a transient demethylation and then compared the timing and kinetics of remethylation of the p16 gene locus under conditions of either G(0)-G(1) growth arrest induced by serum starvation and confluence or continuous cell proliferation in complete medium. Variable levels of remethylation were detected in CpG poor regions of DNA, as well as repetitive DNA elements in the absence of cell division, yet no remethylation occurred at CpG islands under these conditions. This correlated with continuous expression of p16 protein in these cells. DNA methyltransferase (DNMT)1 and DNMT3b3 proteins were undetectable in 5-Aza-2'-deoxycytidine-treated and untreated nondividing cells, and their mRNA transcripts were down-regulated in these cells. Although DNMT3a mRNA levels were also reduced, they recovered to original levels in nondividing cells after drug treatment. Our results suggest that cell division is required for de novo methylation of CpG islands and that DNMT3a may play a role in methylating CpG poor regions or repetitive DNA elements outside of the S phase of the cell cycle.

We looked for p16/p19 deletion and p16 promoter methylation, as well as loss of 9p21 heterozygosity in pure squamous cell carcinomas (SCC), and in transitional cell carcinomas (TCC) of the bladder with SCC components. Homozygous deletion of p16/p19 was detected in 11 of 21 (52%) cases of pure SCCs and in three of ten (30%) cases of TCC with SCC. Three cases of TCC with SCC had p16/p19 deletion, hypermethylation of the p16 promoter, or LOH on 9p21 only in the SCC components, suggesting that these molecular alterations occurred preferentially in SCC. Interestingly, homozygous deletion of p16/p19 was observed in squamous metaplasia from bladder cancer patients (five of 11, 45%), showing that this change occurred in preneoplastic cells. On the other hand, p16/p19 deletions were not found in squamous metaplasias from non cancerous patients. Hypermethylation of the p16 promoter was observed in two of 14 tumors (14%) and none of seven metaplasias examined. These data suggest that: (a) p16/p19 deletion is associated with early carcinogenesis of SCC of the bladder, and squamous metaplasia of the bladder cancer patient has already sustained genetic changes found in cancer, and (b) genetic mosaicism occurs in cases of TCC with SCC, with the SCC component showing more frequent 9p21 alterations than the TCC component.

The most common genetic alteration identified to date in bladder cancer is loss of heterozygosity (LOH) of chromosome 9, suggesting the presence of possible tumor suppressor genes on this chromosome. We attempted to map the location of these genes by analyzing 69 primary transitional cell carcinomas of the bladder with a panel of microsatellite markers for LOH on chromosome 9. Monosomy 9 (defined by LOH of all informative markers analyzed on 9p and 9q) was detected in 26 of 69 (38%) tumors, and 22 of 69 (32%) tumors showed subchromosomal deletions. Twelve tumors (17%) demonstrated partial LOH of chromosome 9 and indicated two distinct regions of LOH. Eight tumors showed distal allelic loss of 9q with a minimal region of common deletion flanked proximally by marker GSN on 9q33. Six tumors showed proximal allelic loss of 9p and 9q with a minimal area of common deletion flanked by markers D9S970 on 9p12 and D9S283 on 9q21. Two tumors showed loss of both the distal region of 9q and the proximal region of 9p and 9q, which were separated by a possible 6-44 cM of retained genetic material. The proximal minimal area of common deletion excluded 9q22.3-q31 to where two putative tumor suppressor genes, the nevoid basal cell carcinoma syndrome and multiple self-healing squamous epithelioma (ESS1) genes, have been mapped. The growth arrest-specific gene (GAS1), a candidate tumor suppressor gene, was included within the proximal minimal region. We evaluated the GAS1 gene for its potential role in bladder cancer using single-strand conformational polymorphism to screen for mutations in GAS1 in 10 bladder cancer cell lines and 14 primary bladder tumors. A polymorphism at codon 88 was noted in one primary bladder tumor, but no other abnormalities were found, suggesting that another potential tumor suppressor gene important to bladder cancer resides in these minimally deleted regions. Because the nevoid basal cell carcinoma syndrome gene has long been speculated to be a putative tumor suppressor gene in bladder cancer and this gene has recently been characterized as the human homologue of the Drosophila patched gene (PTC), 20 primary bladder tumors with chromosome 9q LOH were screened for mutations in PTC using single-strand conformational polymorphism and heteroduplex analysis. No alterations were found in any of the samples analyzed. Furthermore, 4 of 37 noninvasive papillary (Ta) tumors demonstrated loss of all 9q markers with retention of 9p, whereas no Ta tumor showed loss of 9p with retention of all 9q markers, suggesting that LOH of 9q is the earlier event in bladder tumorigenesis. In summary, our results indicate two tumor suppressor loci associated with proximal chromosome 9p to q and distal chromosome 9q that may be important in bladder cancer. GAS1 and PTC do not seem to be frequently mutated in bladder cancer.

An elevated risk of bladder cancer has been reported in the endemic region of 'black foot disease' on the southwest coast of Taiwan and may be related to high arsenic levels in artesian well water. Thirteen urothelial tumors from this endemic region were examined for mutations in exons 5-8 of the p53 gene to identify the effects of possible exogenous factors at the DNA level. DNA was extracted from archival tissue after microdissection of tumors and analyzed by PCR-SSCP (polymerase chain reaction-based single strand conformation polymorphism), followed by direct sequencing. Eight cases (62%) showed mutations and 9 of the 10 point mutations observed were transitions. The type and position of the mutations were not significantly different when compared with the spectra of p53 mutations previously reported for transitional cell carcinomas (TCCs). However, two of the mutations were CGC-->CAC base changes at codon 175, a mutational hotspot for many tumor types but previously unreported in TCCs except in cases associated with inflammatory agents. Three of the tumors examined were found to contain double mutations, a relatively rare mutagenic event in human cancers. Our results suggest that the agents responsible for the high risk of bladder cancer in the black foot disease region may operate through an inflammation-based mechanism which increases the amount of DNA damage per mutagenic event.

Noninvasive transitional cell carcinomas of the bladder can have two distinct morphologies suggesting they contain different genetic alterations. Papillary transitional cell carcinomas (T(a) tumors) are often multifocal and only occasionally progress, whereas flat tumors (carcinomas in situ, CIS), frequently progress to invasive disease. We examined 216 bladder tumors of various stages and histopathologies for two genetic alterations previously described to be of importance in bladder tumorigenesis. Loss of heterozygosity of chromosome 9 was observed in 24 of 70 (34%) T(a) tumors but was present in only 3 of 24 (12%) CIS and dysplasia lesions (P = 0.04). In contrast, only 1 of 36 (3%) T(a) tumors contained a p53 gene mutation compared to 15 of 23 (65%) CIS and dysplasias (P < 0.001), a frequency comparable to that observed in muscle invasive tumors (25 of 49; 51%). The presence of p53 mutations in CIS and dysplasia could explain their propensities to progress since these mutations are known to destabilize the genome. Analysis of several tumor pairs involving a CIS and an invasive cancer provided evidence that the chromosome 9 alteration may in some cases be involved in the progression of CIS to more invasive tumors, in addition to its role in the initiation of T(a) tumors. However, the CIS and secondary tumor were found to contain different genetic alterations in some patients suggesting divergent progression pathways. Bladder carcinogenesis may therefore proceed through two distinct genetic alteration pathways responsible for generating superficial tumors with differing morphologies and pathologies.

Somatic instability at microsatellite repeats was detected in 6 of 200 transitional cell carcinomas of the bladder. Instabilities were apparent as changes in (GT)n repeat lengths on human chromosome 9 for four tumors and as alterations in a (CAG)n repeat in the androgen receptor gene on the X chromosome for three tumors. Single locus alterations were detected in three tumors, while three other tumors revealed changes in two or more loci. In one tumor we found microsatellite instability in all five loci analyzed on chromosome 9. The alterations detected were either minor 2-base pair changes or larger (> 2 base pairs) alterations in repeat length. All six tumors were low stage (Ta-T1), suggesting that these alterations can occur early in bladder tumorigenesis.

The tumors of 20 patients with multifocal primary transitional cell carcinoma of the bladder or lymph node metastases were examined for molecular genetic defects which we have previously found to be present in > 50% of invasive tumors. These included loss of heterozygosity (LOH) of chromosome 9, which occurs in superficial as well as invasive bladder tumors, and LOH of chromosome 17p and p53 mutations, which are commonly found only in invasive tumors. Analysis of multiple or recurrent primary tumors in 7 patients for these markers was generally consistent with recently published data that the tumors are monoclonal in origin and that p53 mutations occur as a late event in the generation of invasive bladder cancers. Comparison of the primary tumors and metastases to regional lymph nodes in 14 patients demonstrated a complete concordance between the molecular genetic defects present, showing that LOH of chromosomes 9 and 17p and p53 mutations occurred in the primary tumors before metastasis. Because of the importance of chromosome 9 in bladder cancer, we mapped the location of a putative tumor suppressor gene by restriction fragment length polymorphism analysis of 123 cases obtained in this and earlier studies. Most of the tumors showed LOH for more than one marker on chromosome 9. Results of mapping of 4 tumors with partial deletion of chromosome 9 suggests that the tumor suppressor gene is located between 9p12 and 9q34.1.

Forty-three transitional cell carcinomas of the bladder of differing grades and stages were examined for reduction to homozygosity for chromosomes 9q, 11p, and 17p. Allelic loss of chromosome 9q was seen in 24 of 38 informative grades II, III, and IV tumors providing further evidence for a bladder tumor suppressor gene on this chromosome. In contrast to the grade-independent involvement of chromosome 9q, allelic losses of chromosomes 11p and 17p were seen only in grade III and IV tumors. The results with chromosome 17p were particularly striking and showed that 0 of 10 grade II versus 20 of 31 grade III and IV tumors had allelic losses for this chromosome harboring the p53 tumor suppressor gene often mutated in other human cancers. The data suggest that cumulative genetic damage is sustained in transitional cell carcinomas and that one of the underlying molecular mechanisms distinguishing low grade from high grade tumors involves chromosome 17p.

Twenty-five human bladder tumors were examined for loss of heterozygosity of markers on chromosomes 6p, 9q, 11p, 14q, and 17p. These studies show that all of the markers were reduced to homozygosity in at least some of the tumors. They also confirmed earlier studies by Fearon et al. [Nature (Lond.), 318: 377-380, 1985] that approximately 40% of bladder tumors were reduced to homozygosity for markers on chromosome 11p. However, the greatest frequency of allelic loss was seen for chromosomes 9q (67% of informative cases) and 17p (63% of informative cases) with both chromosomes being lost concordantly in 10 out of 20 informative tumors. Allelic loss of chromosome 9q has not been previously observed with other human cancers; however, deletions of 17p have been reported in breast, lung, and colorectal carcinomas. The data raise the interesting possibility that allelic losses of specific chromosomes might be a feature of cancer in a particular differentiated cell type whereas loss of other chromosomes harboring more generally acting tumor suppressor genes might be a common feature of human cancers.