Prognostic Marker For Bladder Cancer Research Articles

The SPARC Score: A Multifactorial Outcome Prediction Model for Patients Undergoing Radical Cystectomy for Bladder Cancer

While multiple independent clinicopathological variables are associated with the outcome of radical cystectomy for bladder cancer, limited prediction tools exist to facilitate individualized risk assessment. We developed the SPARC (Survival Prediction After Radical Cystectomy) score, a prediction model for bladder cancer specific survival after radical cystectomy. We evaluated 2,403 patients who underwent radical cystectomy without neoadjuvant therapy at our institution between 1980 and 2008 with pathological re-review of all specimens. Of these patients 1,776 with nonmetastatic urothelial carcinoma were identified for analysis. A multivariate model was developed using stepwise selection to determine variables associated with cancer specific survival. We created a scoring system based on the β coefficients of this model. Median followup after radical cystectomy in patients alive at last followup was 10.5 years (IQR 7.3, 15.3), during which time 610 had died of bladder cancer. In addition to pathological tumor stage, nodal status, multifocality and lymphovascular invasion, the patient specific factors of Charlson comorbidity index, Eastern Cooperative Oncology Group (ECOG) performance status, current smoking, preoperative hydronephrosis and receipt of adjuvant chemotherapy were significantly associated with the risk of bladder cancer death. We used cumulative scores of these variables to stratify patients into risk groups with 95%, 80%, 60%, 38% and 23% 5-year cancer specific survival from the lowest to the highest risk group, respectively (p<0.0001). The concordance index of this model was 0.75. We present a model to individualize the estimation of cancer specific survival after radical cystectomy. Pending external validation, these data may be used for patient counseling, specifically in regard to recommendations for adjuvant therapy and surveillance frequency, as well as for clinical trial development.

Abstract 2111: Characterization of current and novel biomarkers of bladder cancer

Abstract Bladder cancer is one of the most common cancers in industrialized countries. It is reported that 3.51 billion dollars are spent each year on bladder cancer treatment making it the most expensive cancer to treat on a per patient basis. Approximately 80% of bladder cancer patients have at least one reported recurrence thereby prompting meticulous lifetime screening and surveillance by health care professionals. Therefore, careful evaluation of our institution's current fluorescent in situ hybridization surveillance tools in addition to investigation of novel biomarkers is imperative. Our investigation of novel biomarkers focused on apoptosis regulating genes which are often unbalanced in cancerous tissues. Our institution recently adopted BioView, an automated FISH capture/analysis system, to replace the manual protocol. Validation and assessment of the automated system for fluorescent in situ hybridization (FISH) interpretation to detect urothelial cancer was determined using patient cases to evaluate concordance, overall positivity, interpretative time spent, sensitivity, and specificity. To evaluate novel biomarkers, mRNA from patient samples was reversed transcribed to cDNA then utilized for biomarker quantification by RT-PCR. In addition, immunohistochemistry was performed on the most promising gene to further confirm its clinical usefulness. The automated BioView capture/ detection system does prove to be more time efficient and accurate when addressing all parameters described. In evaluation of novel biomarkers, API5, BIRC3, BIRC4, SURVIVIN, and BFAR provided the most promising data with sensitivity and specificity percentages all ranging from 83% to 97%. From this study of bladder cancer markers, we focused on API5 and its role in bladder cancer initiation and progression. API5 is strongly expressed early in tumorigenesis. Our data confirm that the automated FISH capture/detection system improves laboratory efficiency and accuracy. Furthermore, our data indicate that apoptotic regulators are promising biomarkers for clinical use. In addition, API5 is expressed early in tumorigenesis, which makes it an ideal early diagnostic marker. We have noticed increase in the level of API5 protein associated with tumor grade and stage which also suggest that API5 might be a potential prognostic marker for bladder cancer. In conclusion, detection of apoptosis regulating genes is a promising diagnostic and prognostic approach to aid our screening and surveillance tools. 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 2111. doi:1538-7445.AM2012-2111

Abstract 2996: The prognostic value of a cell cycle proliferation gene signature in bladder cancer

Abstract An important aspect of personalized medicine involves the identification of high risk patients likely to benefit from neoadjuvant or adjuvant therapy. In the case of bladder cancer, this entails identifying the 20-30% of patients with non-muscle invasive (NMI) tumors that will progress to muscle-invasive (MI) disease, and the approximately 50% of patients diagnosed with MI tumors who do not survive past 5 years. In breast cancer, independently derived prognostic gene signatures are enriched with cell cycle proliferation (CCP) genes. Recently, a CCP score obtained from the average expression level of 31 genes was found to predict recurrence free and disease specific survival (DSS) in prostate cancer. Here, we assess the importance of CCP correlated genes for prognosis in bladder cancer and the prognostic value of the CCP score in 8 publicly available and heterogeneous microarray datasets (N = 762), with samples obtained from both cystectomy and transurethral resection of the bladder (TURBT) and profiled on 7 microarray platforms. Clusters of correlated and predictive genes for stage, grade, and DSS were identified using an iterative subtractive method. A gene set enrichment analysis on the predictive clusters found that CCP was the only consistently enriched class of genes (P &amp;lt; 0.05) for each clinical variable. We then analyzed the extent in which the performance of predictive gene signatures, composed of the top 10-100 genes in step sizes of 10, depended on genes that correlated with the CCP gene signature. When the gene expression data was adjusted for the 31 CCP genes, the predictive ability of the gene signatures was significantly and negatively impaired in most datasets. We next evaluated the CCP score for its prognostic value. Without optimizing the CCP signature for bladder cancer, we found the CCP score to be significantly higher in MI compared to NMI tumors in 3/4 cystectomy datasets (P &amp;lt; 0.05), with AUC ranging from 0.62 to 0.84, and in 2/4 TURBT datasets (P &amp;lt; 0.05) with AUC ranging from 0.60 to 0.75. CCP score was significantly higher in high grade tumors compared to low grade tumors in 3/4 cystectomy datasets (P &amp;lt; 0.05), with AUC ranging from 0.63 to 0.88, and in all 5 TURBT datasets (P &amp;lt; 0.05), with AUC ranging from 0.76 to 0.91. Most notably, although nodal staging was the most consistent predictor of DSS in cystectomy datasets (P &amp;lt; 0.05), CCP score was the only clinical variable marginally predictive of DSS (P &amp;lt; 0.10) in multiple cystectomy datasets when only preoperative variables were considered. In TURBT datasets, CCP score was the only variable predictive of DSS (P &amp;lt; 0.05) in multiple datasets. Finally, CCP score was predictive of NMI to MI progression in low grade tumors (AUC = 0.73, P = 0.020). Altogether, these results indicate that the prognostic value of gene signatures in bladder cancer depend on genes that correlate with CCP and that optimization of the CCP signature may yield clinically relevant prognostic markers in bladder cancer. 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 2996. doi:1538-7445.AM2012-2996

Abstract 1163: HYAL4 is a novel potential prognostic and diagnostic biomarker for bladder cancer

Abstract Introduction and Objectives: The Hyaluronidase (HAase) family of enzymes is responsible for the degradation of hyaluronic acid (HA). There are 6 HAase genes in the human genome, clustered on chromosomes 3p21.3 (HYAL-1, -2, -3) and 7q31.3 (HYAL-4, PH20, HYALP1). Studies have shown that, while PH20 and HYAL-2 are basically testicular and lysosomal HAases, HYAL1 is the tumor-derived HAase, promoting tumor growth, invasion and angiogenesis. Additionally, HYAL1 is a diagnostic and prognostic marker for bladder cancer (BCa). The expression of other HAases has not been investigated in either normal and/or tumor cells and tissues. In this study we examined the expression of all 6 HAases in bladder tissues, cells and urine. Methods: Real time RT-PCR was used to examine the mRNA levels of the 6 HAase genes in BCa cell lines, 59 bladder tissues (normal (NBL) = 22; tumor (TBL) = 37) and 160 urine specimens (BCa = 52; normal = 18; history of BCa = 30; benign conditions = 59). Transcript levels were normalized to β-actin. HYAL1 and HYAL4 were localized in bladder tissues by immunohistochemistry using anti-HYAL1 and anti-HYAL4 antibodies, respectively. The staining was graded for intensity (0 - 3) and area of staining. HYAL4 function was analyzed by siRNA transfection of HT1376 and HT5637 cell lines. Results: HYAL1, HYAL4 and HYALP1 mRNA levels were significantly (6-13-fold) elevated in TBL tissues (12.5 ± 2.7; 29.7 ± 21.9; 26.7 ± 21.9) when compared to NBL tissues (1.2 ± 0.6; 2.2 ± 2.4; 4.2 ± 6.6; P &amp;lt; 0.001). HYAL3 and PH20 were expressed at 100-fold less level. HYAL2 expression was comparable to HYAL1 and HYAL4, but the difference in the expression between NBL and TBL was not significant (P &amp;gt; 0.05). cDNA cloning revealed that HYALP1 is a pseudo gene that encodes a non-functional truncated protein. HYAL1 and HYAL4 staining was significantly elevated in TBL tissues (188 ± 57; 232 ± 91) when compared to NBL tissues (68.5 ± 21.3; 93 ± 46; P &amp;lt; 0.001). The staining for both HAases was exclusively localized in tumor cells. When compared to HYAL3, PH20, HYAL2 mRNA levels, HYAL1, HYAL4 and HYALP1 mRNA levels were significantly elevated in BCa patients’ urine (P &amp;lt; 001). Urinary HYAL4 mRNA levels had higher sensitivity (78.9%) and specificity (86.2%) than HYAL1 mRNA levels (72.3%; 76.2%) to detect BCa. Both HAases detected high-grade BCa with 83% - 86% sensitivity. Down regulation of HYAL4 expression by siRNA decreased proliferation of HT1376 and HT5637 cells (2.3-fold) and induced apoptosis (2.6-fold). Conclusion: This is the first study showing that HYAL4, like HYAL1, is a tumor derived HAase that is exclusively expressed in BCa cells. HYAL4 may be a potentially accurate marker for BCa diagnosis and may promote BCa growth and progression. Support: Grant NCI/NIH R01 CA-72821-09; Bankhead Coley Cancer Research Program Sylvester Comprehensive Cancer Center and CURED 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 1163. doi:1538-7445.AM2012-1163

Low molecular weight cyclin E is associated with p27-resistant, high-grade, high-stage and invasive bladder cancer

Expression of low molecular weight (LMW) isoforms of cyclin E is a strong predictor of poor outcome in patients with breast cancer. The purpose of this study was to examine the expression of full-length and LMW cyclin E in bladder cancer cell lines and patient tumors. We used western blotting, immunoprecipitation and kinase assays to examine the expression and activity of key cell cycle-regulatory proteins in various human bladder cell lines, both tumorigenic and non-tumorigenic. We also analyzed cyclin E expression, kinase activity and immune complex binding partners in 43 tissue samples from grade 2 and 3 transitional cell carcinomas. Cyclin E was overexpressed and LMW isoforms were present only in bladder cancer cells. Overexpression of LMW isoforms of cyclin E and increased cyclin E kinase activity were both significantly associated with tumorigenicity of the bladder cell lines (p = 0.005 and 0.022, respectively). Binding of the cyclin-dependent kinase inhibitors p21 and p27 to LMW cyclin E did not inhibit the kinase activity of cyclin E and cyclin-dependent kinase 2 in primary tumor samples overexpressing LMW cyclin E. Full-length and LMW cyclin E were significantly overexpressed in grade 3 tumors compared with grade 2 tumors (p = 0.004). Finally, LMW cyclin E levels were significantly associated with a non-papillary growth pattern (p = 0.031) and invasiveness (p = 0.021) of the bladder tumors and poor overall survival (p = 0.06). These results suggest that LMW cyclin E can be used as a new prognostic marker for bladder cancer.

Ras Association Domain Family 1A: A Promising Prognostic Marker in Recurrent Nonmuscle Invasive Bladder Cancer

IntroductionAberrant methylation of promoter CpG islands is an important inactivation mechanism of tumor suppressors and tumor-related genes. Ras association domain family 1A (RASSF1A) promoter hypermethylation was shown to be associated with bladder cancer (BC), but its prognostic value remains unclear. The aim of the present study was to investigate the value of RASSF1A methylation as a prognostic marker in BC. Materials and MethodsPrimary BC tissues were obtained from 301 patients and included 186 specimens of nonmuscle invasive bladder cancer (NMIBC) and 115 specimens of muscle invasive bladder cancers (MIBC). RASSF1A hypermethylation was assessed using methylation-specific polymerase chain reaction (MS-PCR). The association between RASSF1A hypermethylation and clinicopathologic features, and the prognostic significance of RASSF1A hypermethylation were evaluated by Kaplan-Meier and multivariate Cox regression analyses. ResultsRASSF1A promoter hypermethylation was detected in 33.6% of BCs and occurred more frequently in MIBC (46.1%) than in NMIBC (25.8%) (P < .001). In NMIBC, RASSF1A methylation was associated with advanced tumor stage (P = .026) and high grade (P < .001). Among patients with recurrent NMIBC, RASSF1A methylation was associated with shorter time to progression by Kaplan-Meier analysis (log-rank test; P = .004) and identified as an independent predictor of cancer progression by multivariate Cox regression analysis (hazard ratio [HR], 8.559; P = .014). ConclusionsOur results suggest that methylated RASSF1A may be a potential prognostic marker in patients with recurrent NMIBC.

Abstract 5174: HYAL4: a new player in bladder cancer progression and diagnosis

Abstract BACKGROUND: Hyaluronidase (HAase) family of enzymes degrades hyaluronic acid (HA). In the human genome there are 6 HAase genes clustered on chromosomes 3p21.3 (HYAL-1, -2, -3) and 7q31.3 (HYAL-4, PH20, HYALP1). While PH20 and HYAL-2 are testicular and lysosomal HAases, HYAL1 is a tumor-derived HAase. HYAL1 promotes tumor growth, invasion, and angiogenesis and is a diagnostic and prognostic marker for bladder cancer (BCa). The expression of other HAases has not been investigated in either normal cells or tumor cells and tissues. In this study we examined the expression of all 6 HAases in bladder tissues, cells and urine. METHODS: Using time RT-PCR, we examined the mRNA levels of 6 HAase genes in BCa cell lines, 59 bladder tissues (normal (NBL) = 22; tumor (TBL) = 37) and 160 urine specimens (BCa = 52; normal = 18; history of BCa = 30; benign conditions = 59). The levels were normalized to β-actin. HYAL1 and HYAL4 were localized in bladder tissues by immunohistochemistry using anti-HYAL1 and anti-HYAL4 antibodies, respectively. The staining was graded for intensity (0 – 3) and area of staining. HYAL4 function was analyzed by siRNA transfection of HT1376 and HT5637 cell lines. RESULTS: HYAL1, HYAL4 and HYALP1 mRNA levels were significantly (6-13-fold) elevated in TBL tissues (12.5 ± 2.7; 29.7 ± 21.9; 26.7 ± 21.9) when compared to NBL tissues (1.2 ± 0.6; 2.2 ± 2.4; 4.2 ± 6.6; P 0.05). cDNA cloning revealed that HYALP1 is a pseudo gene that encodes a non-functional truncated protein. HYAL1 and HYAL4 staining was significantly elevated in TBL tissues (188 ± 57; 232 ± 91) when compared to NBL tissues (68.5 ± 21.3; 93 ± 46; P &amp;lt; 0.001). The staining for both HAases was exclusively localized in tumor cells. When compared to HYAL3, PH20, and HYAL2 mRNA levels, HYAL1, HYAL4 and HYALP1 mRNA levels were significantly elevated in BCa patients’ urine (P &amp;lt; 001). Urinary HYAL4 mRNA levels had higher sensitivity (78.9%) and specificity (86.2%) than HYAL1 mRNA levels (72.3%; 76.2%) to detect BCa. Both HAases detected high-grade BCa with 83% – 86% sensitivity. Down regulation of HYAL4 expression by siRNA decreased proliferation of HT1376 and HT5637 cells (2.3-fold) and induced apoptosis (2.6-fold). Conclusion: This is the first study showing that, like HYAL1, HYAL4 is a tumor derived HAase that is exclusively expressed in BCa cells. HYAL4 may be a potentially accurate marker for BCa diagnosis and may promote BCa growth and progression. Support: Grant NCI/NIH R01 CA-72821-09; Bankhead Coley Cancer Research Program Sylvester Comprehensive Cancer Center and CURED Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5174. doi:10.1158/1538-7445.AM2011-5174

Abstract 2259: Identifying causative genes and prognostic markers for bladder cancer

Abstract Upon presentation 70% of the bladder tumors are confined to the epithelial (stage pTa) and stromal layers (stage pT1) of the bladder. These non-muscle invasive tumors (NMI-BC) frequently recur and 10-15% will eventually invade the detrusor muscle and may cause metastases. The management of these tumors is characaterized by the difficult choice between transurethral resection (TURB) or total bladder removal by cystectomy. Our first objective was to identify DNA copy number changes as biomarkers predicting tumor progression in high grade NMI-BC to aid decision making. A set of 18 DNA samples isolated from fresh frozen high grade NMI-BC was hybridized on Illumina SNP arrays. Analysis of alterations was done with Nexus software. Hierarchical clustering of significantly differing SNPs showed that tumors that progressed and those that did not clustered separately. A comparison of the differential alterations between tumor groups resulted in many chromosomal locations encompassing chromosome 1p, 2-6, 8-10, 12p, 13q, 17q, 18p and 22q that were fit for inclusion in a validation assay. A custom Agilent 15K array was designed to include probes for these locations. We collected a retrospective series of 96 high grade NMI-BC paraffin embedded tumors with a median follow-up of 5 years. Half of these samples were from patients who developed a muscle-invasive tumor at a later time. Preliminary results show an interesting region on chromosome 8 with the best independent prognostic value. This region and the genes therein will be studied in more detail. This and other validated regions will be used to design a definitive prognostic assay. Complex losses on chromosome 9 characterize bladder tumors regardless of stage. We hypothesized that the complex rearrangements on this chromosome in BC are the results of attempts of the tumor cell to loose tumor suppressor genes whilst struggling to retain other (onco)genes. Therefore, the second aim was to identify pivotal tumor suppressor and/or oncogenes on chromosome 9 for development of bladder cancer, with the aim to exploit these genes or the pathways in which they function as potential targets for therapy for tumors from all stage categories. A custom Agilent 44K array was designed, containing probes for all genes on chromosome 9 (and 8p). DNA from a series of 144 tumors from different stages was hybridized to the arrays. Analysis of alterations was done with Nexus software. We found many regions of loss as expected but also a region that appeared amplified in most tumors. Several candidate regions were validated of their copy number status with specific MLPA assays. Two potential candidate genes from chromosome 9 were selected for mutational and functional analysis. In one of those genes we found several mutations affecting protein function, suggesting that this gene is a new tumor suppressor gene for bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2259. doi:10.1158/1538-7445.AM2011-2259

Coordinated epigenetic repression of the miR‐200 family and miR‐205 in invasive bladder cancer

MicroRNAs (miRNA) are small noncoding RNAs commonly deregulated in cancer. The miR-200 family (miR-200a, -200b, -200c, -141 and -429) and miR-205 are frequently silenced in advanced cancer and have been implicated in epithelial to mesenchymal transition (EMT) and tumor invasion by targeting the transcriptional repressors of E-cadherin, ZEB1 and ZEB2. ZEB1 is also known to repress miR-200c-141 transcription in a negative feedback loop, but otherwise little is known about the transcriptional regulation of the miR-200 family and miR-205. Recently, miR-200 silencing was also reported in cancer stem cells, implying that miR-200 deregulation is a key event in multiple levels of tumor biology. However, what prevents miR-200 expression remains largely unanswered. Here we report concerted transcriptional regulation of the miR-200 and miR-205 loci in bladder tumors and bladder cell lines. Using a combination of miRNA expression arrays, qPCR assays and mass spectrometry DNA methylation analyses, we show that the miR-200 and miR-205 loci are specifically silenced and gain promoter hypermethylation and repressive chromatin marks in muscle invasive bladder tumors and undifferentiated bladder cell lines. Moreover, we report that miR-200c expression is significantly correlated with early stage T1 bladder tumor progression, and propose miR-200 and miR-205 silencing and DNA hypermethylation as possible prognostic markers in bladder cancer. In addition, we observe that the mesoderm transcription factor TWIST1 and miR-200 expression are inversely correlated in bladder tumor samples and cell lines. TWIST1 associates directly with the miR-200 and miR-205 promoters, and may act as a repressor of miR-200 and miR-205 expression.

Improved cancer specific‐survival in patients with carcinoma invading bladder muscle expressing cyclo‐oxygenase‐2

Study Type - Prognosis (case series). 4 OBJECTIVE: To determine whether the expression of cyclo-oxygenase (COX)-2 has an influence on survival and on the response to chemotherapy in invasive bladder cancer. A population of 266 patients from a tertiary university centre with carcinoma invading bladder muscle without evidence of metastasis at time of cystectomy was analyzed retrospectively. COX-2 expression was evaluated immunohistochemically with a monoclonal anti-COX-2 antibody. All pertinent clinical and pathological parameters were reviewed and correlated with risk factors influencing outcome, including disease-specific and overall survival, as well as COX-2 expression. Immunoreactivity was categorized as positive if COX-2 staining was present in >5% tumour cells. The expression of COX-2 was not influenced by tumour stage, grade or nodal status, nor any other parameters. The risk factors that influenced disease-specific survival in carcinoma invading bladder muscle on multivariate analysis were lymph node status (hazards ratio, HR = 2.46 for N1, P = 0.001, HR = 2.90 for N2, P < 0.001, HR = 5.19 for N3, P = 0.012), use of neoadjuvant chemotherapy (HR = 3.54; P= 0.004) or adjuvant chemotherapy (HR = 0.57, P = 0.014) and COX-2 expression (HR = 0.64 if >5% cells had positive expression; P = 0.025). Kaplan-Meier analysis showed an increased disease-specific survival (P = 0.0063), as well as longer recurrence-free survival (P = 0.003), in patients with muscle-invasive bladder tumours expressing COX-2 in >5% of the cells. A tendency was also observed in a subgroup with positive nodes treated with adjuvant chemotherapy (P = 0.093). The overexpression of COX-2 is associated with a better recurrence-free and disease-specific survival in a large cohort of 266 patients with carcinoma invading bladder muscle treated by cystectomy. A trend for increased disease-specific survival was also observed for patients with COX-2 overexpression and positive nodes who received adjuvant chemotherapy. Potential of COX-2 as a prognostic marker in bladder cancer should be considered.

Highlights of This Issue

The serine/threonine kinase GSK-β is a positive regulator of human cancer cell survival and proliferation. To explore the role of GSK-β in bladder cancer, Naito and colleagues determined the subcellular localization of GSK-β and evaluated the effect of GSK-3 inhibition. Nuclear accumulation of GSK-β was strongly associated with high-grade tumors, metastasis, and lower survival rates in bladder cancer patients. Further, inhibition of GSK-β suppressed proliferation and survival of bladder cancer cells. These results identify nuclear accumulation of GSK-β as a novel prognostic marker in bladder cancer, and suggest GSK-β as a potential therapeutic target in urothelial carcinoma.Prognostic and chemotherapy predictive biomarkers are needed for melanoma. In this study, Jewell and colleagues performed gene expression profiling of formalin-fixed primary melanoma tumors. They found that DNA repair genes, predominantly involved in double-strand break repair, were over-expressed in tumors from patients with reduced relapsefree survival, and in tumors with higher mitotic rate. Analysis of a subset of patients treated with chemotherapy (predominantly dacarbazine) revealed that DNA repair genes were over-expressed in tumors from patients unresponsive to chemotherapy. These findings highlight the importance of upregulation of DNA repair processes during melanoma progression and indicate a potential role in chemoresistance.Carr and colleagues performed a phase II trial of continuous dosing of sunitinib in patients with advanced thyroid cancer to determine disease response and toxicity. Also, an exploratory analysis of FDG-PET imaging after seven days of therapy was performed to determine if disease response could be predicted. Of 33 patients, 10 had partial responses and 16 had stable disease. Patients with RECIST response and stable disease had a significant decline in average SUVs compared to patients with progressive disease. These results suggest that FDG-PET imaging may help identify patients unlikely to respond to tyrosine kinase inhibitor therapy.Clinically useful predictors of response to specific chemotherapy regimens in breast cancer are needed. Tabchy and colleagues tested a 30- gene predictor of pathologic complete response to neoadjuvant paclitaxel and fluorouracil, doxorubicin, cyclophosphamide (T/FAC) chemotherapy from fine-needle biopsies of breast cancers in a prospective randomized trial. The authors found that the 30-gene predictor could identify patients with greater than average sensitivity to T/FAC chemotherapy but not to FAC treatment. To improve their clinical utility, second generation genomic predictors should be developed separately for different molecular subsets of breast cancers.

Glycogen Synthase Kinase-3β: A Prognostic Marker and a Potential Therapeutic Target in Human Bladder Cancer

Although recent studies have shown glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase, as a positive regulator of pancreatic, colon, and kidney cancer cell survival and proliferation, the role of GSK-3 in bladder cancer remains unknown. Our objectives were to determine the subcellular localization of GSK-3β and to evaluate the effect of GSK-3 inhibition in bladder cancer. We used immunohistochemical staining and nuclear/cytosolic fractionation to determine the expression pattern of GSK-3β in human urothelial carcinomas. To study the effect of GSK-3 inhibition on bladder cancer cell proliferation and survival, we used pharmacologic inhibitors of GSK-3, RNA interference, MTS assay, bromodeoxyuridine incorporation assay, quantitative reverse transcriptase-PCR, and Western blotting. We found aberrant nuclear accumulation of GSK-3β in 62% (43 of 69) and 91% (21 of 23) of noninvasive and invasive human urothelial carcinomas, respectively. GSK-3β nuclear staining was significantly associated with high-grade tumors (P < 0.001), advanced stage of bladder cancer (P < 0.05), metastasis (P < 0.05), and worse cause-specific survival (P < 0.05) in bladder cancer patients. Moreover, we found that pharmacologic inhibition or genetic depletion of GSK-3β resulted in decreased viability of bladder cancer cells. Our results suggest nuclear accumulation of GSK-3β as a novel prognostic marker in bladder cancer, show that GSK-3 contributes to urothelial cancer cell proliferation and survival, and identify GSK-3 as a potential therapeutic target in human bladder cancer.

Survivin as a Prognostic Marker for Urothelial Carcinoma of the Bladder: A Multicenter External Validation Study

The aim of the current study was to externally validate the value of survivin as a prognostic marker for bladder cancer in a large multi-institutional cohort of patients treated with radical cystectomy. The study comprised 726 patients treated with radical cystectomy and bilateral pelvic lymphadenectomy. Survivin staining and scoring were done with automated systems coupled with advanced color detection software. Specimens showing at least 10% reactivity were considered altered. Predictive accuracy was quantified using the concordance index and 200-bootstrap resamples were used to reduce overfit bias. Survivin was an independent predictor of disease recurrence and cancer-specific survival in multivariable analyses that controlled for the effects of standard clinicopathologic features (hazard ratios, approximately 1.6; P values < or = 0.002). In all patients (n = 726), addition of survivin to a model including standard clinicopathologic variables did not improve its predictive accuracy (P = 0.67 for disease recurrence and P = 0.27 for cancer-specific survival). In the subgroup of patients with pT(1-3)N(0)M(0) disease (n = 398), addition of survivin improved the accuracy of standard clinicopathologic features for prediction of disease recurrence and cancer-specific survival (1.3%, P < 0.001 and 1.2%, P < 0.001, respectively). Survivin expression improves our accuracy for prediction of cancer recurrence and survival in pT(1-3)N(0)M(0) patients by a small but statistically significant margin. Our findings support the need for further evaluation of survivin and its signaling pathways as well as survivin-targeted therapies in bladder cancer.

Expression of Bmi-1 is a prognostic marker in bladder cancer

BackgroundThe molecular mechanisms of the development and progression of bladder cancer are poorly understood. The objective of this study was to analyze the expression of Bmi-1 protein and its clinical significance in human bladder cancer.MethodsWe examined the expression of Bmi-1 mRNA and Bmi-1 protein by RT-PCR and Western blot, respectively in 14 paired bladder cancers and the adjacent normal tissues. The expression of Bmi-1 protein in 137 specimens of bladder cancer and 30 specimens of adjacent normal bladder tissue was determined by immunohistochemistry. Statistical analyses were applied to test the relationship between expression of Bmi-1, and clinicopathologic features and prognosis.ResultsExpression of Bmi-1 mRNA and protein was higher in bladder cancers than in the adjacent normal tissues in 14 paired samples (P < 0.01). By immunohistochemical examination, five of 30 adjacent normal bladder specimens (16.7%) versus 75 of 137 bladder cancers (54.3%) showed Bmi-1 protein expression (P < 0.05). Bmi-1 protein expression was intense in 20.6%, 54.3%, and 78.8% of tumors of histopathological stages G1, G2, and G3, respectively (P < 0.05). Expression of Bmi-1 protein was greater in invasive bladder cancers than in superficial bladder cancers (81.5% versus 32.5%, P < 0.05). In invasive bladder cancers, the expression of Bmi-1 protein in progression-free cancers was similar to that of cancers that have progressed (80.0% versus 82.4%, P > 0.5). In superficial bladder cancers, the expression of Bmi-1 protein in recurrent cases was higher than in recurrence-free cases (62.5% versus 13.7%, P < 0.05). Bmi-1 expression was positively correlated with tumor classification and TNM stage (P < 0.05), but not with tumor number (P > 0.05). Five-year survival in the group with higher Bmi-1 expression was 50.8%, while it was 78.5% in the group with lower Bmi-1 expression (P < 0.05). Patients with higher Bmi-1 expression had shorter survival time, whereas patients with lower Bmi-1 expression had longer survival time (P < 0.05).ConclusionExpression of Bmi-1 was greater in bladder cancers than in the adjacent normal tissues. The examination of Bmi-1 protein expression is potentially valuable in prognostic evaluation of bladder cancer.

Forkhead box O-class 1 and Forkhead box G1 as Prognostic Markers for Bladder Cancer

Forkhead box O-class 1 (FOXO1) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. Its functions are modulated by forkhead box G1 (FOXG1), which acts as a transcriptional repressor with oncogenic potential. Real-time PCR and immunohistochemical staining were performed in 174 primary bladder cancer specimens and 21 normal bladder mucosae to evaluate these genes. FOXO1 and FOXG1 mRNA expression in cancer tissues were higher than in normal mucosae (each P<0.001). FOXO1 mRNA levels were significantly higher in samples of non-progressed patients (P<0.001), but FOXG1 were enhanced in those of progressed patients (P=0.019). On univariate analysis, FOXO1 mRNA expression was significantly associated with grade, stage, recurrence, progression and survival (each P<0.05). On multivariate analysis, increased FOXO1 mRNA expression was associated with both reduced disease progression (odds ratio [OR], 0.367; 95% confidence interval [CI], 0.163-0.826, P=0.015) and enhanced disease-free survival (OR, 3.262; 95% CI, 1.361-7.820, P=0.008). At a median follow-up of 33 months (range 2 to 156), the patients with a high FOXO1 mRNA expression had a significantly prolonged survival (P=0.001). Immunohistochemical findings of FOXO1 were generally concordant with mRNA expression levels. In conclusion, FOXO1 may be a promising marker for predicting progression in human bladder cancers.

EBAG9 is a tumor‐promoting and prognostic factor for bladder cancer

Upregulation of EBAG9 expression has been observed in several malignant tumors such as advanced breast and prostate cancers, indicating that EBAG9 may contribute to tumor proliferation. In the present study, we assess the role of EBAG9 in bladder cancer. We generated human bladder cancer EJ cells stably expressing FLAG-tagged EBAG9 (EJ-EBAG9) or empty vector (EJ-vector), and investigated whether EBAG9 overexpression modulates cell growth and migration in vitro as well as the in vivo tumor formation of EJ transfectants in xenograft models of BALB/c nude mice. EBAG9 overexpression promoted EJ cell migration, while the effect of EBAG9 to cultured cell growth was rather minimal. Tumorigenic experiments in nude mice showed that the size of EJ-EBAG9-derived tumors was significantly larger than EJ-vector-derived tumors. Loss-of-function study for EBAG9 using small interfering RNA (siRNA) in xenografts with parental EJ cells showed that the intra-tumoral injection of EBAG9 siRNA markedly reduced the EJ tumor formation compared with control siRNA. Furthermore, immunohistochemical study for EBAG9 expression was performed in 60 pathological bladder cancer specimens. Intense and diffuse cytoplasmic immunostaining was observed in 45% of the bladder cancer cases. Positive EBAG9 immunoreactivity was closely correlated with poor prognosis of the patients (p = 0.0001) and it was an independent prognostic predictor for disease-specific survival in multivariate analysis (p = 0.003). Our results indicate that EBAG9 would be a crucial regulator of tumor progression and a potential prognostic marker for bladder cancer.

Pax-5 protein expression in bladder cancer: a preliminary study that shows no correlation to grade, stage or clinical outcome

Pax (paired box) genes comprise a gene family crucial for cell differentiation that encodes a set of transcription factors. Recently, Pax-5 mRNA expression was suggested as a prognostic marker in bladder cancer (BC). However, a functional role of Pax-5 in BC is questionable because the protein expression was not determined in these studies. Therefore, we evaluated Pax-5 protein expression in an unselected, consecutive series of BC. We immunohistochemically investigated Pax-5 protein expression in 100 archival bladder tumours and 22 normal urothelial samples using tissue microarray (TMA) technology and a monoclonal antibody against Pax-5. Staining intensity and percentage of positively stained cells were determined and correlated to histopathological characteristics of the tumours and clinical follow-up data. All 22 samples of histopathologically normal urothelium were negative for Pax-5 protein expression. Overall, 70 of 100 tumours gave interpretable results. Only seven of 70 (10%) cases showed a positive nuclear Pax-5 staining but without significant correlation to clinicopathological characteristics. Interestingly, we could observe Pax-5 positive lymphocytes located within the tumour or closely adjacent in the underlying stroma in 24 of 70 (34%) cases in our series. Pax-5 protein expression is infrequent in BC. Absence of correlation to clinicopathological characteristics suggests a minor functional role of Pax-5 in BC. Pax-5 positive lymphocytes within reactive infiltrates adjacent to the tumour warrant further studies evaluating biological, immunological and clinical relevance.

Hydronephrosis as a Prognostic Marker in Bladder Cancer in a Cystectomy-Only Series

Hydronephrosis as a Prognostic Marker in Bladder Cancer in a Cystectomy-Only Series

Tissue Inhibitor of Metalloproteinases-3 Promoter Methylation is an Independent Prognostic Factor for Bladder Cancer

TIMP-3 (tissue inhibitor of metalloproteinases-3) is 1 of 4 members of a family of proteins that were originally classified according to their ability to inhibit matrix metalloproteinases. We analyzed TIMP-3 methylation in 175 urine sediment DNA samples from patients with bladder cancer with well characterized clinicopathological parameters, including patient outcome. We examined urine sediment DNA for aberrant methylation of 9 genes, including TIMP-3, by quantitative fluorogenic real-time polymerase chain reaction. Using an optimal cutoff value by TaqMan(R) quantitation we found that the risk of death was statistically significantly higher in patients with higher TIMP-3 and ARF methylation (HR 1.99, 95% CI 1.12 to 3.27, p = 0.01 and HR 1.66, 95% CI 1.00 to 2.76, p = 0.05, respectively) than in patients without/lower TIMP3 and ARF methylation in urine. A significant correlation was also seen between the risk of death and stage 3 tumor (HR 2.73, 95% CI 1.58 to 4.72, p = 0.003) and metastasis (HR 3.32, 95% CI 1.98 to 5.57, p = 0.0001). Multivariate analysis subsequently revealed that TIMP-3 methylation was an independent prognostic factor for bladder cancer survival with stage and metastasis (p = 0.001 and 0.02, respectively). These results suggest that TIMP-3 promoter methylation could be a clinically applicable marker for bladder cancer progression.

Serum tissue inhibitor of metalloproteinase-2 (TIMP-2) and matrix metalloproteinase-2 in complex with the inhibitor (MMP-2:TIMP-2) as prognostic markers in bladder cancer

Objectives: To examine whether the circulating TIMP-2 or MMP-2:TIMP-2 complex associate with disease-free or cause-specific survival in bladder cancer patients. Design and methods: Levels of circulating TIMP-2 and MMP-2:TIMP-2 complex from 50 patients and 44 healthy volunteers were measured by ELISA and compared with the clinical data. Results: In cancer patients the levels of TIMP-2 and MMP-2:TIMP-2 complex were significantly lower than in healthy volunteers ( p = < 0.0001). Low TIMP-2 levels and low MMP-2:TIMP-2 complex levels correlated significantly with poor prognosis ( p = 0.0032, p = 0.0022, respectively). The 5-year cause-specific survival rate was 67% and 60% in patients with a high serum level for TIMP-2 and MMP-2:TIMP-2 versus 18% and 20% in those with low levels of TIMP-2 and MMP-2:TIMP-2 complex. Conclusions: The results indicate that TIMP-2 and MMP-2:TIMP-2 complex associate with favorable clinical course and could be used as a novel prognostic indicators in bladder cancer.