Progression Of Invasive Bladder Cancer Research Articles

The impact of grading scheme on non-muscle invasive bladder cancer progression: potential utility of hybrid grading schemes

Non-muscle invasive bladder cancer (NMIBC) grade is a major determinant of progression risk. The most widely utilised grading systems are the World Health Organization (WHO) 1973 and 2004 schemes. Recent publications suggest the utility of combining both into a four-tier or a hybrid three-tier system, subdividing WHO 2004 high grade into two separate categories while maintaining low grade as a single group. We identified two retrospective cohorts of bladder resections/biopsies of papillary urothelial NMIBC with long term clinical follow-up. The sentinel specimen was assessed for WHO 2004 and 1973 grade, along with pathological stage and carcinoma in situ. Each case was additionally stratified into a hybrid three-tier system (low grade; high grade, grades 2 and 3) and a four-tier system (low grade, grades 1 and 2; high grade, grades 2 and 3). Uni- and multivariable analysis for progression and event free survival (PFS/EFS) were calculated along with the time dependent area under the curve (AUC) for each grading scheme. There were 609 cases (Cohort A, n=343; Cohort B, n=266), including 449 (74%) pTa, 156 pT1 (26%) and four pTx with 338 (56%) low grade (177, grade 1; 161, grade 2) and 271 (44%) high grade (137, grade 2; 134, grade 3). A total of 108 patients progressed (17.7%): 97 high grade, (grade 3, n=59; grade 2, n=38). Multivariable analyses of PFS with the hybrid 3- and 4-tier systems showed higher Harrell's concordance indices (0.851 and 0.853, respectively) than WHO 1973 (0.844) and WHO 2004 (0.846). In both cohorts AUC values were higher (0.77-0.85) for the two hybrid grading systems compared to WHO 1973 or WHO 2004 (0.72-0.82). Similar results were seen on analysis of EFS. The data support the use of a hybrid three-tier or four-tier grading system to improve stratification of NMIBC patients.

Down-regulation of A-FABP predicts non-muscle invasive bladder cancer progression: investigation with a long term clinical follow-up

BackgroundNon-muscle invasive bladder cancers (NMIBC: pTa, pT1) are characterised by a high risk of recurrence and/or progression. Identification of prognostic markers is needed to improve both diagnosis and management of the disease. The aim of this study was to analyse the expression of A-FABP (adipocyte-fatty acid binding protein) and to evaluate its prognostic value in bladder cancer with a long term clinical follow-up.MethodsA-FABP expression was investigated by immunohistochemistry in 236 tumours (114 pTa, 61 pT1, 61 pT2–4). Immunostaining was classified as negative (absent or weak immunostaining and moderate or strong staining on ≤10% of cells) or positive (moderate or strong staining on > 10% of cells). Event-free survival (EFS) and overall survival (OS) were determined with a 87.3 months median follow-up in the overall cohort. Recurrence-free survival (RFS) and progression-free survival (PFS) were established in NMIBC.ResultsLoss of A-FABP was associated with higher mean age, high stage/grade, and the presence of metastatic lymph nodes. It was correlated with shorter median EFS (17.5 vs 62.5 months; p = 0.001) and mean OS (76.7 vs 154.2 months; p = 0.009) and with higher risk of progression in the pTa/pT1 subgroup (HR, 0.36; 95% CI, 0.13–0.96; p = 0.041) and importantly in the pTa tumours (HR, 0.34; 95% CI, 0.10–0.97; p = 0.045).ConclusionThese results demonstrated that loss of A-FABP expression following a long follow-up was predictive of pTa and pTa/pT1 progression. Immunohistochemistry on diagnostic biopsy is easy to use and could be of value to help clinicians to propose appropriate treatment for these tumours.

Abstract 94: DDX31 cooperates with mutant p53 and EGFR to promote the multistep progression of invasive bladder cancer

Abstract Bladder cancer is the most common malignancy of the urinary tract worldwide. Approximately 70% of cases are diagnosed as non-muscle-invasive bladder cancer (NMIBC), while the remaining 30% of cases are classified as muscle-invasive bladder cancer (MIBC). Evidence based on molecular biology has highlighted that the aggressiveness of MIBC advances through a multistep mechanism due to many genomic alterations. Notably, alterations of TP53 and EGFR pathways frequently occur in bladder cancers and are associated with poor prognosis, respectively. However, the connection between the overexpression of EGFR and the p53 mutation in multistep carcinogenesis and the progression of MIBC remains unknown. Here we report the distinct critical roles of DEAD box polypeptide 31 (DDX31) in the multistep progression of muscle invasive bladder cancer (MIBC) through its sequential interaction with mutant p53 (mutp53) and EGFR. In early MIBC cells, nuclear DDX31 acts as a transcriptional co-activator that binds to mutp53/SP1 and enhances mutp53 transcriptional activation, thereby upregulating the EPB41L4B gene, which plays a critical role in metastatic behavior and promotes the invasion and migration of MIBC. Notably, in advanced MIBC, cytoplasmic DDX31, which is transported from the nucleus, functions as an adaptor scaffold protein that forms a complex with EGFR via its interaction with phosho-nucleolin (NCL), leading to constitutive activation of EGFR-Akt signaling. Significantly, high expression of both cytoplasmic DDX31 and p53 proteins is correlated with a poor prognosis in MIBC patients. More importantly, blocking the DDX31-NCL interaction via a dominant-negative peptide led to downregulation of EGFR-Akt signaling, resulting in a significant anti-tumoral effect of bladder cancer in vivo. Our findings reveal that DDX31 cooperates with mutp53 and EGFR to promote the multistep progression of MIBC and that inhibition of DDX31-NCL formation may lead to potential treatment strategies for advanced MIBC. Citation Format: Toyomasa Katagiri, Kei Daizumoto, Tetsuro Yoshimaru, Yosuke Matsushita, Tomoya Fukawa, Masaya Ono, Hiro-omi Kanayama. DDX31 cooperates with mutant p53 and EGFR to promote the multistep progression of invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 94.

Germline DNA copy number variations as potential prognostic markers for non-muscle invasive bladder cancer progression

Accumulating evidence has suggested that germline DNA copy number variations (CNVs) affect various disorders, including human malignancies. However, the significance of CNVs in non-muscle invasive bladder cancer (NMIBC) remains unclear. The purpose of the present study was to identify the role of CNVs in NMIBC. Array comparative genomic hybridization (CGH) analysis was performed to search for candidate CNVs associated with NMIBC susceptibility. Quantitative polymerase chain reaction was carried out to evaluate CNVs associated with patient outcome in 189 NMIBC cases. In total, 11 CNVs were associated with NMIBC risk in array CGH analysis. Out of the 189 CNVs examined, family with sequence similarity 81 member A (FAM81A) and proprotein convertase subtilisin/kexin type 6 (PCSK6) CNVs exhibited a significant association with recurrence and disease progression in NMIBC. PCSK6 has been reported to regulate proliferation and tumor progression in breast and prostate malignancies. Notably, patients with pT1 stage had significantly lower PCSK6 relative copy number than those with pTa (P=0.0196). In multivariate analyses, PCSK6 copy number was an independent prognostic factor for progression-free survival (P=0.0456; risk ratio, 2.17; 95% confidence interval, 1.02-4.82). These data suggest that PCSK6 CNV is a potential new tumor marker for estimating disease progression in NMIBC.

Positive association of collagen type I with non-muscle invasive bladder cancer progression.

PURPOSENon-muscle invasive bladder cancers (NMIBC) are generally curable, while ~15% progresses into muscle-invasive cancer with poor prognosis. While efforts have been made to identify genetic alternations associated with progression, the extracellular matrix (ECM) microenvironment remains largely unexplored. Type I collagen is a major component of the bladder ECM, and can be altered during cancer progression. We set out to explore the association of type I collagen with NMIBC progression.EXPERIMENTAL DESIGNThe associations of COL1A1 and COL1A2 mRNA levels with progression were evaluated in a multi-center cohort of 189 patients with NMIBCs. Type I collagen protein expression and structure were evaluated in an independent single-center cohort of 80 patients with NMIBCs. Immunohistochemical analysis was performed and state-of-the-art multi-photon microscopy was used to evaluate collagen structure via second harmonic generation imaging. Progression to muscle invasion was the primary outcome. Kaplan-Meier method, Cox regression, and Wilcoxon rank-sum were used for statistical analysis.RESULTSThere is a significant association of high COL1A1 and COL1A2 mRNA expression in patients with poor progression-free survival (P=0.0037 and P=0.011, respectively) and overall survival (P=0.024 and P=0.012, respectively). Additionally, immunohistochemistry analysis of type I collagen protein deposition revealed a significant association with progression (P=0.0145); Second-harmonic generation imaging revealed a significant lower collagen fiber curvature ratio in patients with invasive progression (P = 0.0018).CONCLUSIONSAlterations in the ECM microenvironment, particularly type I collagen, likely contributes to bladder cancer progression. These findings will open avenues to future functional studies to investigate ECM-tumor interaction as a potential therapeutic intervention to treat NMIBCs.

Abstract 4712: Targeting the arginine-nitric oxide pathway to arrest development and progression of invasive bladder cancer

Abstract Background: In United States there will be an estimated 74,690 new diagnoses and 15,580 deaths in 2014 due to bladder cancer. Lack of reliable markers of early disease and limited chemotherapy options have resulted in poor patient outcomes. A better approach is to treat patients with early intervention using targeted therapy. Arginine metabolic pathway has been identified to be deregulated in bladder cancer. Arginine is either derived from the microenvironment or synthesized by cells containing arginosuccinate synthase I (ASS1). Arginine undergoes subsequent conversion to nitric oxide (NO), which which can activate numerous intracellular signaling mediators, including guanylate cyclase (GC). We evaluated dual targeting of this metabolic pathway by arginine deprivation (ADI-PEG 20, Polaris Pharmaceuticals, San Diego, CA) and NO chelation (Cobinamide) as a therapy for bladder cancer. Experimental Design: Expression of arginine-NO pathway components in FFPE tissue sections was assessed by IHC and correlated with histopathology, outcomes and progression to identify pathway activation during early and late disease and stratified based on moderate to strong expression (2+, 3+) compared to absent to weak expression (0, 1+). In addition, a panel of bladder cell lines was screened for arginine-NO pathway components by immunoblotting and representative ASS1 negative and positive cells were used to evaluate the effects of arginine deprivation and/or NO chelation in vitro on colony formation and cell viability by clonogenic and MTT assays. Effects of NO production on bladder cancer survival and motility was assessed utilizing siRNA gene silencing and small molecule inhibitors. Effects of arginine deprivation by ADI-PEG 20 treatment in vivo was assessed by a mouse xenograft model. Results: Analysis of human tumors showed that ASS1 loss and increase in NOS occurred early in the course of bladder neoplastic progression. We identified loss of the ASS1 enzyme in bladder cancer cells and compensatory dependence on extracellular arginine, which can be successfully targeted using arginine removal to induce bladder cancer cell death, both in vitro and in vivo. We also identified increased nitric oxide synthase (NOS; the enzyme that catalyzes NO production) and GC activation in invasive bladder cancer cell lines. We characterized the effects of NO production on bladder cancer cell survival, growth and motility. Disruption of the NO pathway through gene silencing and small molecule inhibitors resulted in decreased cell survival, migration and invasion. Conclusions: Thus, dual targeting of this metabolic pathway using arginine chelation by the arginine degrading enzyme ADI-PEG20 and NO scavenging by a novel compound Cobinamide can be a potential mechanism to prevent development and/or progression of early bladder neoplasia. Citation Format: Divya Sahu, Sounak Gupta, Andrew M. Hau, Paul Elson, John Bomalaski, Gerry R. Boss, Donna E. Hansel. Targeting the arginine-nitric oxide pathway to arrest development and progression of invasive bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4712. doi:10.1158/1538-7445.AM2015-4712

Abstract 4801: Stromal-mediated collagen I signal in promoting bladder cancer progression

Abstract Bladder cancer is the fifth most common malignancy, which is mostly incurable as invasive disease. It is clinically important to study the mechanisms underlying bladder cancer progression. While the tumor microenvironment is widely established to play an active role in epithelial cancers, its contribution to bladder cancer remains unexplored. The presence of cancer-associated fibroblasts (CAFs), characterized by the co-expression of vimentin, alpha-smooth muscle actin, and tenascin C, has been associated with invasive bladder cancer. Nevertheless, functional contributions of CAFs to bladder cancer progression have not been studied. Here,we report the successful isolation and molecular characterization of bladder CAFs. We further investigate their functional roles on bladder cancer progression, with an emphasis on stromal secreted collagen I in the paracrine activation of discoidin domain receptor (DDR1) signaling in neighboring bladder cancer cells. Using bioinformatics analysis we found that invasive bladder cancer patients with elevated expression of CAF genes have a poorer survival than those with lower CAF gene expression. Subsequently, we isolated and characterized CAFs from patient-derived tissues. Co-transplantation of CAFs and bladder cancer cells as xenograft tumors revealed high collagen I (COL1) deposition in these tumors formed, while molecular analyses uncovered CAFs as the primary source of COL1. Further experiments verified that COL1 as a single extracellular matrix component could phenocopy the tumor phenotype resembling those co-transplanted with CAFs and cancer cells. Further, pre-stimulation with COL1 could also enhance metastatic colonization of bladder cancer cells to lung. Molecular analysis of these COL1 stimulated cancer cells revealed up-regulation of the collagen receptor DDR1, but not integrins. Immunohistochemical analysis confirmed the presence of DDR1+ cancer cells adjacent to CAFs in the primary tumor site, with enhanced and exclusive expression of DDR1 in paired lung metastasis. Oncomine analysis showed that invasive bladder cancer expressed higher mRNA levels of COL1 and DDRs than non-invasive cancer, indicating that collagen I-DDR1 interaction may be a generalized phenomenon during invasive bladder cancer progression. To delineate the molecular mechanism downstream to collagen I-DDR1 we studied the interaction of DDR1 and STAT3, a factor we previously reported to drive invasive bladder cancer progression. Stimulation of bladder cancer cells with COL1 revealed a time kinetic increase in total and activated DDR1 protein associated with STAT3 phosphorylation. Further analysis of lung metastasis confirmed the co-localization of DDR1 and nuclear active STAT3. Collectively, these findings uncovered a role of CAFs in bladder cancer progression via stromal mediated collagen I signaling and warrant further analysis of therapeutic options to target signaling components downstream to collagen I. Citation Format: Antonina V. Kurtova, Jing Xiao, Erica J. Lay, Qianxing Mo, Seth P. Lerner, David R. Rowley, Keith S. Chan. Stromal-mediated collagen I signal in promoting bladder cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4801. doi:10.1158/1538-7445.AM2014-4801

Stat3 Activation in Urothelial Stem Cells Leads to Direct Progression to Invasive Bladder Cancer

Two subtypes of human bladder cancer, noninvasive papillary and muscle-invasive cancer, develop through independent pathologic and molecular pathways. Human invasive bladder cancer frequently develops without prior clinical evidence of a noninvasive tumor stage. However, an animal model that recapitulates this unique clinical progression of invasive bladder cancer has not yet been developed. In this study, we created a novel transgenic mouse model of invasive bladder cancer by targeting an active dimerized form of Stat3 to the basal cells of bladder epithelium. When exposed to the carcinogen nitrosamine, Stat3-transgenic mice developed invasive cancer directly from carcinoma in situ (CIS), bypassing the noninvasive papillary tumor stage. Remarkably, invasive bladder cancer driven by active Stat3 was predominantly composed of stem cells, which were characterized by cytokeratin 14 (CK14) staining and enhanced tumor sphere-forming ability. Active Stat3 was also shown to localize to the nucleus of human invasive bladder cancers that were primarily composed of CK14+ stem cells. Together, our findings show that Stat3-induced stem cell expansion plays a critical role in the unique clinical progression of invasive bladder cancer through the CIS pathway.

Evaluation of epidermal growth factor receptor, transforming growth factor alpha, epidermal growth factor and c-erbB2 in the progression of invasive bladder cancer.

Determination of the risk of invasive bladder tumors progressing is still imprecise due to the heterogeneous biological behavior of this neoplasm. The goals of this study were to evaluate the patterns of expression of the epidermal growth factor (EGF) system in invasive bladder cancer and to assess its prognostic value. This immunohistochemical study was performed using fresh frozen tumor samples and a panel of monoclonal antibodies on a series of 43 invasive bladder cancers treated by cystectomy. EGF was detected in 45% of the tumors and did not correlate with survival from bladder cancer. Transforming growth factor alpha (TGF alpha) was expressed by 60% of the tumors and correlated strongly with death from bladder cancer. Epidermal growth factor receptor (EGF-R) expression was seen in 86% of cases and had no prognostic significance. c-erbB2 was expressed in 50% of cases and was inversely related to a poor prognosis. When EGF and TGF alpha were both expressed, there was little or no expression of c-erbB2. The accumulation of several growth factors and the relevant receptor are necessary for the progression of invasive bladder cancers. They could be used as indicators of tumor aggressiveness.