Grade Bladder Cancer Research Articles

MP39-19 L-SELECTIN (CD62L) EXPRESSION IN HIGH GRADE UROTHELIAL CARCINOMA AS A POTENTIAL MARKER OF METASTATIC DISEASE

You have accessJournal of UrologyBladder Cancer: Basic Research IV1 Apr 2014MP39-19 L-SELECTIN (CD62L) EXPRESSION IN HIGH GRADE UROTHELIAL CARCINOMA AS A POTENTIAL MARKER OF METASTATIC DISEASE Dharamainder Choudhary, Poornima Hegde, Shilpa Choudhary, Kevin Claffey, Pramod Srivastava, Carol Pilbeam, and John Taylor Dharamainder ChoudharyDharamainder Choudhary More articles by this author , Poornima HegdePoornima Hegde More articles by this author , Shilpa ChoudharyShilpa Choudhary More articles by this author , Kevin ClaffeyKevin Claffey More articles by this author , Pramod SrivastavaPramod Srivastava More articles by this author , Carol PilbeamCarol Pilbeam More articles by this author , and John TaylorJohn Taylor More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1334AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES L-selectin (CD62L) is a vascular adhesion molecule constitutively expressed on leucocytes with a primary function of directing leucocyte migration and homing of lymphocytes to lymph nodes (LNs). Microarray data from laser captured, micro-dissected human specimens of high grade muscle invasive (MIBC) vs. low grade (LGBC) bladder cancers found CD62L to be the highest differentially expressed gene between the groups. We further characterized the mRNA and protein expression of CD62L in high grade cancer and its potential as a marker for metastatic disease. METHODS MIBC and LGBC fresh frozen, paraffin embedded and serum samples were obtained from the UCHC tumor bank. mRNA was evaluated by quantitative polymerase chain reaction (qPCR) and protein levels by immunohistochemistry (IHC) and enzyme linked immunosorbant assay (ELISA). Flow cytometry (FACS analysis) was used to identify the relative number of cells expressing CD62L in fresh tumor tissue. In silico studies were performed using the Oncomine Database. RESULTS Quantification of CD62L transcripts in high grade metastatic MIBC vs. LGBC frozen specimens(microarray; 3.81 ± 2.94 vs 0.06 ± 0.01 p=0.04 ,confirmed by qPCR 0.14 ± 0.04 vs. 23.31 ± 22.47) and immunohistochemistry on paraffin embedded high grade metastatic MIBC vs. LGBC specimens confirmed the elevated expression of CD62L in MIBC samples. Localization of CD62L was also confirmed in discrete foci of bladder tumor cells in LN specimens of high grade metastatic disease. Upregulated expression of CD62L (9.4-fold, p<0.01) was observed by FACS analysis of freshly isolated tumor cells from high grade cancers vs. LGBC specimens. Circulating CD62L levels were also found to be higher (640 ± 75 vs. 558 ± 34 ng/ml) in serum samples from patients with high grade metastatic vs. high grade non-metastatic MIBC. Furthermore, in silico analysis using the Oncomine microarray database showed an association of CD62L expression with tumor aggressiveness and clinical outcomes. CONCLUSIONS Our preliminary findings are the first report of increased expression of CD62L in MIBC and suggest a role in metastatic spread to LNs. CD62L could act as a potential marker for predicting patients who are at high risk of developing or harboring metastatic disease. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e433 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Dharamainder Choudhary More articles by this author Poornima Hegde More articles by this author Shilpa Choudhary More articles by this author Kevin Claffey More articles by this author Pramod Srivastava More articles by this author Carol Pilbeam More articles by this author John Taylor More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP39-17 MECP2 SILENCING IN HIGH GRADE BLADDER CANCER DERIVED HTB-5 CELL LINE AFFECTS CELLULAR PROLIFERATION AND MIGRATORY ABILITY

You have accessJournal of UrologyBladder Cancer: Basic Research IV1 Apr 2014MP39-17 MECP2 SILENCING IN HIGH GRADE BLADDER CANCER DERIVED HTB-5 CELL LINE AFFECTS CELLULAR PROLIFERATION AND MIGRATORY ABILITY Shumaila Bilgrami, Zulfiqar Naqvi, Khalid Shaikh, El Nasir Lalani, and Farhat Abbas Shumaila BilgramiShumaila Bilgrami More articles by this author , Zulfiqar NaqviZulfiqar Naqvi More articles by this author , Khalid ShaikhKhalid Shaikh More articles by this author , El Nasir LalaniEl Nasir Lalani More articles by this author , and Farhat AbbasFarhat Abbas More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1332AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES DNA methylation patterns and chromatin structure is significantly altered in many human neoplasms including bladder cancer. Aberrant promoter hypermethylation of tumor suppressor genes is now a well established mechanism of gene inactivation. Methyl binding proteins are believed to act as interpreters of DNA methylation signal. The methyl binding protein MeCP2 has been shown to bind specifically to the 5mCpGs that are flanked by A/T residues. (Klose R et al., 2005) and mediates transcriptional repression by recruiting histone deacetylases (Adrian Bird at al.,1998) and thus, serving as a bridge between two major regulatory mechanisms of gene expression-DNA methylation and histone deacetylation. METHODS A high grade bladder cancer cell line HTB-5 (ATCC) was transfected with psiRNA-hMeCP2 and psiRNA-LucGL3 (Invivogen), after testing for Mycoplasma contamination. CELL PROLIFERATION ASSAY Cellular proliferation was measured using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide]assay kit (ATCC)according to manufacturer’s protocol. CELL MIGRATION ASSAY Confluent monolayers of HTB-5 cells in 6-well plates were serum starved and incubated in minimum essential medium for 24hrs. A bee line (1mm thick) was created with a 10μl pipette tip. Cell migration was monitored at 24hr interval and photographed. RESULTS The results of proliferation assay showed that cell growth was drastically compromised in the MeCP2 silenced cells as compared to the mock transfected and untransfected HTB-5 cells after 24, 48, 72 and 96 hours of incubation (Fig.1) The migratory potential of MeCP2-silenced cells was evaluated by the cell migration/ wound healing assay. The untransfected and mock transfected HTB-5 cells were able to trespass the trough after 48 hours. On the other hand, MeCP2 silenced cells migrated slower than the control transfected and parental HTB-5 cells (Fig.2). CONCLUSIONS The results of our study demonstrated that MeCP2 silencing in high grade bladder cancer derived HTB-5 cells resulted in reduced proliferation and migratory ability. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e432 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Shumaila Bilgrami More articles by this author Zulfiqar Naqvi More articles by this author Khalid Shaikh More articles by this author El Nasir Lalani More articles by this author Farhat Abbas More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

408 MCJ predicts immunotherapy response in T1 high grade bladder cancer

408 MCJ predicts immunotherapy response in T1 high grade bladder cancer

Sialyl Tn-expressing bladder cancer cells induce a tolerogenic phenotype in innate and adaptive immune cells

Despite the wide acceptance that glycans are centrally implicated in immunity, exactly how they contribute to the tilt immune response remains poorly defined. In this study, we sought to evaluate the impact of the malignant phenotype-associated glycan, sialyl-Tn (STn) in the function of the key orchestrators of the immune response, the dendritic cells (DCs). In high grade bladder cancer tissue, the STn antigen is significantly overexpressed and correlated with the increased expression of ST6GALNAC1 sialyltransferase. Bladder cancer tissue presenting elevated expression of ST6GALNAC1 showed a correlation with increased expression of CD1a, a marker for bladder immature DCs and showed concomitant low levels of Th1-inducing cytokines IL-12 and TNF-α. In vitro, human DCs co-incubated with STn+ bladder cancer cells, had an immature phenotype (MHC-IIlow, CD80low and CD86low) and were unresponsive to further maturation stimuli. When contacting with STn+ cancer cells, DCs expressed significantly less IL-12 and TNF-α. Consistent with a tolerogenic DC profile, T cells that were primed by DCs pulsed with antigens derived from STn+ cancer cells were not activated and showed a FoxP3high IFN-γlow phenotype. Blockade of STn antigens and of STn+ glycoprotein, CD44 and MUC1, in STn+ cancer cells was able to lower the induction of tolerance and DCs become more mature.Overall, our data suggest that STn-expressing cancer cells impair DC maturation and endow DCs with a tolerogenic function, limiting their capacity to trigger protective anti-tumour T cell responses. STn antigens and, in particular, STn+ glycoproteins are potential targets for circumventing tumour-induced tolerogenic mechanisms.

Correlation of mitotic indices, AgNor count, Ki-67 and Bcl-2 with grade and stage in papillary urothelial bladder cancer.

To evaluate the mutual inter-relationship of mitotic indices, argyrophilic nuclear organizer regions (AgNOR) count, Ki-67 and B-cell lymphoma 2 protein (bcl-2) in papillary urothelial bladder cancer (pUBC), and their correlation with grade and stage. To establish the cut-off values of these markers to detect high grade and muscle invasive bladder cancer. Fifty-four patients with primary pUBC who underwent transurethral resection / radical cystectomy were analyzed retrospectively. Cell proliferation was assessed by Ki-67 labelling index, mean AgNOR count, mitotic count, mitotic activity index and mitosis/ volume index. Immunohistochemistry was done to see bcl-2 and Ki-67 expression. Correlation of these indices with tumor grade and stage and amongst themselves was assessed. The receiver operating characteristic (ROC) curves were drawn to establish the cut-off values. We found a strong positive correlation of mitotic indices and Ki-67 with tumor grade (P = .000), stage (P < .05) and bcl-2 (P = .000). AgNOR count correlated positively with the grade (P = .006), mitotic indices and Ki-67 (P = .032) but not with tumor stage and bcl-2. Cytoplasmic bcl-2 immunopositivity was seen in 42.3% of low grade pUBC and 85.7% of high grade pUBC cases (P = .001). bcl-2 positivity was seen in 85% of muscle invasive pUBC as compared to only 52.9% of superficial cases. Ki-67 ≥ 32.5%, ≥ 14 mitoses/10 high power fields (hpf), ≥ 11.20 mitoses/mm2, ≥ 0.75 mitoses/100 tumor cells and AgNOR ≥ 11.55 are 100% specific for high grade bladder carcinoma. Ki-67 ≥ 59% and mitoses ≥ 36.50 per 10 hpf can indicate muscle invasion with 100% specificity. Cut-off values for Ki-67, mitotic indices and AgNOR can confirm high grade bladder carcinoma in equivocal cases. Ki-67 and mitotic count can serve as potential and reliable indicators of muscle invasion.

Expression of HMGA2 in bladder cancer and its association with epithelial‐to‐mesenchymal transition

High mobility group protein2 (HMGA2) and epithelial-to-mesenchymal transition are both related to progress of bladder cancer, however, the relationship between HMGA2, E-cadherin and vimentin in bladder cancer is not yet known. Thus, this study has examined expression of HMGA2, E-cadherin and vimentin in bladder cancer and investigated their relationship. The 5637 bladder cancer cell line and SV-HUC-1 normal uroepithelial cells were used to study expression of HMGA2, E-cadherin and vimentin using RT-PCR and western blotting. Paraffin wax-embedded bladder cancer tissues were used to study protein expression using immunohistochemistry and χ(2) analysis and Kendall's correlation were utilized statistical methods. Overexpression of HMGA2 was associated with down-regulation of E-cadherin and up-regulation of vimentin in the 5637 bladder cancer line. A total of 49 paraffin wax-embedded tissues of transitional cell bladder cancer were used. Positive expression levels of HMGA2 protein and vimentin were 41 and 43% in bladder tissues, respectively. No expression of E-cadherin was found in 43%. Expression of HMGA2, loss of E-cadherin and expression of vimentin are all significantly correlated with bladder cancer grade and stage. Loss of E-cadherin and expression of vimentin both correlated with recurrence of the bladder cancer. Expression of HMGA2 was closely associated with occurrence of epithelial-to-mesenchymal transition. Expression of HMGA2, loss of E-cadherin and expression of vimentin may indicate high degree malignancy of bladder cancer. Loss of E-cadherin expression and positive expression of vimentin may predict recurrence of bladder cancer.

Bladder Cancer Exosomes Contain EDIL-3/Del1 and Facilitate Cancer Progression

High grade bladder cancer is an extremely aggressive malignancy associated with high rates of morbidity and mortality. Understanding how exosomes may affect bladder cancer progression could reveal novel therapeutic targets. Exosomes derived from human bladder cancer cell lines and the urine of patients with high grade bladder cancer were assessed for the ability to promote cancer progression in standard assays. Exosomes purified from the high grade bladder cancer cell line TCC-SUP and the nonmalignant urothelial cell line SV-HUC were submitted for mass spectrometry analysis. EDIL-3 was identified andselected for further analysis. Western blot was done to determine EDIL-3 levels inurinary exosomes from patients with high grade bladder cancer. shRNA gene knockdown and recombinant EDIL-3 were applied to study EDIL-3 function. Exosomes isolated from high grade bladder cancer cells and the urine of patients with high grade bladder cancer promoted angiogenesis and migration ofbladder cancer cells and endothelial cells. We silenced EDIL-3 expression and found that shEDIL-3 exosomes did not facilitate angiogenesis, and urothelial and endothelial cell migration. Moreover, exosomes purified from the urine of patients with high grade bladder cancer contained significantly higher EDIL-3 levels than exosomes from the urine of healthy controls. EDIL-3 activated epidermal growth factor receptor signaling while blockade of epidermal growth factor receptor signaling abrogated this EDIL-3 induced bladder cell migration. Exosomes derived from the urine of patients with bladder cancer contains bioactive molecules such as EDIL-3. Identifying these components and their associated oncogenic pathways could lead to novel therapeutic targets and treatment strategies.

Association between the Metabolic Syndrome and High Tumor Grade and Stage of Primary Urothelial Cell Carcinoma of the Bladder

To compare histopathologic findings of patients who underwent transurethral resection of a bladder tumor (TUR-B) between groups with and without the metabolic syndrome. We retrospectively analyzed data of 535 patients who underwent TUR-B in our department between October 2005 and March 2011. All patients had primary urethelial cell carcinoma (UCB). Histologic stage, grade, the presence of hypertension, diabetes mellitus, body mass index (BMI), waist circumference, HDL and trigliseride levels were evaluated. The TNM classification was used, with Ta tumor accepted as lower stage and T1 and T2 tumors as higher stage bladder cancers. Also, the pathological grading adopted by the 2004 World Health Organization grading system were applied. Non-invasive papillary urothelial neoplasms of low malignant potential were regarded as low grade. Among the total of 509 patients analyzed in our study, there were 439 males (86.2%) and 70 females (13.8%). Metabolic syndrome was significantly associated with high histologic grade, and high pathologic stage (p<0.001). The patients with metabolic syndrome were found to have statistically significant higher T stage and grade of bladder cancer. Further studies with more patients are needed to confirm our study.

Stage, grade and pathological characteristics of bladder cancer in the UK: British Association of Urological Surgeons (BAUS) Urological Tumour Registry

To investigate Tumour-Node-Metastasis (TNM) stage and demographics at presentation in a very large, contemporary UK cohort of patients with bladder cancer and compare them with other published series, as little published data exists on the pathological characteristics of bladder cancer at presentation. The British Association of Urological Surgeons (BAUS) Section of Oncology started a new urological tumour registry in 1998. We performed a data analysis of all bladder cancer cases between 1999 and 2008. Tumour TNM stage, grade and histopathological diagnosis were reviewed along with standard epidemiological data. In all, 69,712 bladder cancer registrations were recorded. Complete T, N and M stage and grade was available for 32,240 patients. The male to female ratio of the study population was 3:1 and the overall median (sd, range) age at presentation was 73 (11.6, 6-108) years. Final pathological T staging showed that non-muscle-invasive bladder cancer accounted for 75% of cases with the remaining 25% being muscle-invasive disease. Of these patients, 8% had nodal disease and 4% other metastatic sites at presentation. The tumour grade was G1-2 in 65% and G3 in 35% of cases. Transitional cell carcinoma (TCC) accounted for 92%, squamous cell carcinoma and adenocarcinomas 1.5% each, with 5% other histological variants. Non-muscle-invasive TCC accounted for 75% of bladder cancer cases in the UK. The 1973 World Health Organization classification remains in widespread use amongst pathologists in the UK. Obtaining complete and standardised staging and pathology reporting systems in bladder cancer remains a challenge.

Targeting HMGB1 inhibits bladder cancer cells bioactivity by lentivirus-mediated RNA interference.

High mobility group box 1(HMGB1) has been reported to be associate with tumor clinical stage and pathological grade in bladder cancer (BC). In this study, we investigated the underlying mechanism through lentivirus-mediated HMGB1 knockdown. HMGB1 was strongly inhibited in BC cells stably transfected with shRNA against HMGB1. MTT and colony formation assays demonstrated that the down-regulation of HMGB1 attenuated the growth of BC cells in vitro. The HMGB1 knockdown (KD) group displayed an increased proportion of cells in the G0/G1 phase and higher apoptosis rates of BC cells comparing with the control (CON) group. The transwell assay revealed that the KD group cells had much lower invasive activity. To assess the influence of HMGB1 inhibition on tumorigenicity in BC cells, shRNA-HMGB1 lentivirus were injected into the tumors of xenograft models and the results showed that the tumorigenesis in mice were significantly suppressed by shRNA-HMGB1 lentivirus. Furthermore, the expression level of VEGF-C in KD group was significantly decreased comparing with the CON group. The NF-κB inhibitor PDTC reduced the expression of VEGF-C, while HMGB1, as a NF-κB agonist, enhanced the VEGF-C expression. In conclusion, our results suggested that lentiviruses delivering shRNA against HMGB1 may be a promising tool for BC therapy.

Clinical significance of fibroblast growth factor receptor-3 mutations in bladder cancer: a systematic review and meta-analysis

Mutations in the fibroblast growth factor receptor-3 (FGFR3) gene are frequently found in bladder cancer, but their prognostic value remains controversial. To globally summarize the association between FGFR3 mutations and the grade and stage of bladder cancer, and to analyze the predictive role of FGFR3 mutations with respect to survival, eligible studies were identified and assessed for quality through multiple search strategies. Risk ratio (RR) data were collected from studies comparing the number of FGFR3 mutants among low-grade and early-stage bladder cancer patients to the number among high-grade and late-stage patients. Hazard ratio (HR) data were collected from studies comparing survival in patients with mutant FGFR3 genes to those with wild-type genes. Studies were pooled, and the RRs of grade and stage and the HRs of survival were calculated. Thirty studies were included in the present meta-analysis. FGFR3 mutations were found to be closely associated with low-grade and early-stage bladder cancer, showing pooled RRs = 2.948 [95% confidence interval (CI) = 2.357-3.688] and 2.845 (95%CI = 2.145- 3.773), respectively. Notably, patients with FGFR3 mutations tended to show better disease-, progress-, and recurrence-free survival (HR = 0.561, 95%CI = 0.405-0.779), and better disease-specific survival (HR = 0.363, 95%CI = 0.266-0.496). This study demonstrated that FGFR3 mutations are closely related to low grade, early stage, and better survival among bladder cancer patients.

Evaluation of urinary HURP mRNA as a marker for detection of bladder cancer: relation to bilharziasis

This study was carried out to assess the efficacy of urinary hepatoma up-regulated protein (HURP) RNA in bladder cancer diagnosis and its relation to bilharziasis. Voided urine samples and blood were collected from 344 consecutive participants: 211 patients diagnosed with bladder cancer, 71 patients with benign urological disorders and 62 healthy volunteers. Serologic assessment of schistosomiasis antibody in sera, urine cytology and estimation of HURP RNA by reverse transcription polymerase chain reaction in urothelial cells was carried out in all samples. HURP RNA expression showed a significant difference among the three investigated groups. The best cutoff point for HURP RNA was determined as 0.0132 at 78.67% sensitivity and 94% specificity. The sensitivity of urine cytology was improved when combined with HURP RNA in detection of early stage (77.3%), low grade (85.3%) and bilharzial bladder cancer (78.1%). Detection of urinary HURP RNA is a useful non-invasive test for early detection of bladder cancer and bilharzial bladder cancer and it improves sensitivity of urine cytology up to 91%.

Expression of survivin in bladder cancer cell lines using quantitative real-time polymerase chain reaction

ObjectivePrevious studies have reported that survivin expression is significantly associated with various malignancies including bladder cancer. However, the relationship between the expression of survivin and the tumor stage and grade of bladder cancer still require further study. MethodsTo determine whether survivin plays a role in the differentiation of bladder cancer cells, we conducted a preliminary study to examine the expression of survivin in bladder cancer cell lines. ResultsIn this study, we observed that the gene expression fold changes of survivin ranged from 3.2 to 16.7 in various tumor grades (G1–G4) of bladder cancer cell lines, which were higher than that in normal human urothelial cell line. With the worse differentiation of bladder cancer cell lines, the gene expression fold changes of survivin increased significantly (3.2-fold in RT4, 5.8-fold in 5637, 6.6-fold in T24, and 16.7-fold in HT1197). In addition, we observed different genotypes among various cell lines (C/C in HUC4449, C/G in RT4, C/G in 5637, G/G in T24, and C/C in HT1197). The relationship between survivin −31 C/G polymorphism and various bladder cancer cell lines was non-significant. However, the overexpression of survivin may be associated with aggressive features of bladder cancer. ConclusionOur findings suggest that survivin could be a potential therapeutic target through the inhibition of cell proliferation in bladder cancer.

Urine tenascin-C is an independent risk factor for bladder cancer patients

Urine biomarkers offer a non‑invasive method of detecting bladder cancer, monitoring disease progression and predicting disease recurrence and therapeutic treatment efficacy. Tenascin‑C (TN‑C), as a component of the extracellular matrix, is vital in the progression of bladder cancer. However, there is little to report with regard to urine TN‑C and its correlation with bladder cancer grade, stage, recurrence and prognosis. In the present study, 66 samples of voided urine from patients with bladder cancer and 42 samples from volunteers were obtained. The urine TN‑C concentration was determined using an ELISA assay. The correlation between the urine TN‑C concentration and the tumor grade, stage and time from bladder cancer diagnosis to recurrence was analyzed by a rank correlation analysis. Multivariate Cox proportional hazards regression was used for finding the main life‑threatening factors among age, gender, tumor grade, stage, relapse and the urine TN‑C concentration. At the end, the Kaplan‑Meier method was used to evaluate the survival rate affected by urine TN‑C as a single factor. The results indicated that the urine TN‑C concentration in the bladder cancer patients was higher compared with the healthy control volunteers (22.5 times higher). Among all the patients, urine TN‑C concentration had a positive correlation with the bladder cancer grade and stage, with correlation coefficients of 0.905 and 0.308, respectively; however, this correlation was negative between urine TN‑C concentration and the time from bladder cancer diagnosis to recurrence. Moreover, the multivariate Cox proportional hazards model analysis indicated that urine TN‑C, like tumor grade and recurrence, may be an independent risk factor for bladder cancer patient survival. However, it is noteworthy that inflammation may affect the concentration of urine TN‑C. The results of the present study indicate that urine TN‑C may be used as a biomarker for monitoring the recurrence of bladder cancer in patients and for predicting its prognosis. However, inflammation of the urinary tract should be excluded first.

Mixed High and Low Grade Bladder Tumors—Are They Clinically High or Low Grade?

The pathological grade of bladder cancer has an immense impact on patient treatment and prognosis. While most bladder tumors show pure high or low grade patterns, some show a mixed pattern. We explored the incidence and clinical significance of this phenomenon. A total of 642 patients with a mean age of 67.5 years underwent transurethral resection of nonmuscle invasive bladder tumors between June 1998 and December 2008, including 156 and 454 with low and high grade lesions, respectively. In 32 patients (5%) mixed grade tumors were found, defined as low grade tumors with 10% or less of a high grade component. All patients were followed a median of 60 months postoperatively. Mean age, the proportion of men and the proportion of stages Ta/T1 in patients with mixed grade tumors were between those of the high and low grade groups. Five-year recurrence-free survival was similar for high, low and mixed grade tumor types (56.9%, 63.8% and 66.4%, respectively, p=0.252). Five-year progression-free survival was significantly lower in patients with high grade disease (73.9%, p<0.0001) but similar in those with high and mixed grade tumors (99% and 96.9%, respectively, p=0.167). Similarly, disease specific survival was significantly worse in patients with high grade tumors (p<0.0001) but similar in those with high and mixed grade lesions (p=0.679). Mixed grade is found in about 5% of nonmuscle invasive tumors, representing a patient group with unique clinical features. The clinical course of patients with mixed grade tumors parallels that of patients with low grade tumors.

Utility of Quantitative MRI Metrics for Assessment of Stage and Grade of Urothelial Carcinoma of the Bladder: Preliminary Results

The purpose of this study was to assess associations between quantitative MRI metrics and pathologic indicators of aggressiveness of urothelial carcinoma of the bladder. In this retrospective biinstitutional study, 37 patients (28 men and nine women; mean age, 73 ± 12 years) who underwent pelvic MRI including diffusion-weighted imaging (b values 0, 400, and 800 s/mm(2)) and T2-weighted imaging before transurethral resection or cystectomy for urothelial carcinoma of the bladder were identified. Tumor diameter (measured on T2-weighted imaging), normalized T2 signal intensity (to muscle; hereafter labeled normalized T2) and apparent diffusion coefficient (ADC) were measured for all tumors. Mann-Whitney test and receiver operating characteristic analyses were used to identify associations between these metrics and histopathologic tumor stage and grade. Thirty-seven tumors were assessed (mean size, 35 ± 23 mm; range 8-88 mm). At histopathologic analysis, 16 of 37 (43%) tumors were stage T2 or greater and 21 of 37 (57%) were stage T1 or lower, whereas 34 of 37 (92%) were high grade and three of 37 (8%) were low grade. High-stage (≥ T2) tumors showed greater tumor diameter, lower normalized T2, and lower ADC (p = 0.005-0.032) than low-stage (≤ T1) tumors. Tumor diameter and ADC were significant independent predictors of stage (p ≤ 0.043), with their combination giving an area-under the-curve (AUC) of 0.804. High-grade tumors showed significantly lower ADC (p = 0.023) but no significant difference in tumor diameter or normalized T2 (p = 0.201-0.559). AUC for differentiating low- and high-grade tumors was higher for ADC (0.902) than for tumor diameter (0.603) or normalized T2 (0.725). A combination of size and quantitative MRI metrics can potentially be used as markers of stage and grade of bladder cancer.

Urinary EpCAM in urothelial bladder cancer patients: characterisation and evaluation of biomarker potential

Background:Epithelial cell adhesion molecule is overexpressed in bladder tumours and released from bladder cancer cells in vitro. We test the hypotheses that urinary EpCAM could act as a biomarker for primary bladder cancer detection and risk stratification.Methods:Epithelial cell adhesion molecule was measured by ELISA in urine from 607 patients with primary bladder tumours and in urine from 53 non-cancer controls. Mann–Whitney tests and ROC analyses were used to determine statistical significance and discrimination between non-cancer controls and different stages and grades of disease. Multivariable modelling and Kaplan–Meier analyses were used to determine prognostic significance. The structure of urinary EpCAM was investigated by western blotting and mass spectrometry.Results:Urinary EpCAM levels increase with stage and grade of bladder cancer. Alongside grade and stage, elevated urinary EpCAM is an independent indicator of poor prognosis with a hazard ratio of 1.76 for bladder cancer-specific mortality. The soluble form of EpCAM in urine is the extracellular domain generated by cleavage between ala243 and gly244. Further studies are required to define the influence of other urinary tract malignancies and benign urological conditions on urinary EpCAM.Conclusion:The extracellular domain of EpCAM is shed into urine by bladder tumours. Urinary EpCAM is a strong indicator of bladder cancer-specific survival, and may be useful within a multi-marker panel for disease detection or as a stand-alone marker to prioritise the investigation and treatment of patients. The mechanisms and effects of EpCAM cleavage in bladder cancer are worthy of further investigation, and may identify novel therapeutic targets.

Low- and High-Grade Bladder Cancer Determination via Human Serum-Based Metabolomics Approach

To address the shortcomings of urine cytology and cystoscopy for probing and grading urinary bladder cancer (BC), we applied (1)H nuclear magnetic resonance (NMR) spectroscopy as a surrogate method for the identification of BC. This study includes 99 serum samples comprising low-grade (LG; n = 36) and high-grade (HG; n = 31) BC as well as healthy controls (HC; n = 32). (1)H NMR-derived serum data were analyzed using orthogonal partial least-squares discriminant analysis (OPLS-DA). OPLS-DA-derived model validity was confirmed using an internal and external cross-validation. Internal validation was performed using the initial samples (n = 99) data set. External validation was performed on a new batch of suspected BC patients (n = 106) through a double-blind study. Receiver operating characteristic (ROC) curve analysis was also performed. OPLS-DA-derived serum metabolomics (six biomarkers, ROC; 0.99) were able to discriminate 95% of BC cases with 96% sensitivity and 94% specificity when compared to HC. Likewise (three biomarkers, ROC; 0.99), 98% of cases of LG were able to differentiate from HG with 97% sensitivity and 99% specificity. External validation reveals comparable results to the internal validation. (1)H NMR-based serum metabolic screening appears to be a promising and less invasive approach for probing and grading BC in contrast to the highly invasive and painful cystoscopic approach for BC detection.

Activation of cyclic AMP/PKA pathway inhibits bladder cancer cell invasion by targeting MAP4-dependent microtubule dynamics

ObjectiveWith the notorious reputation of the vicious invasion, the bladder cancer is the most common malignant tumor of the urinary system. Inhibiting invasion through microtubule dynamics interruption has emerged as an important treatment of bladder cancer. Here we investigated the role of the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway in human bladder cancer cells invasion. Materials and methodsWith or without the treatment of various cAMP elevators, we assessed invasive and migrated capabilities of T24 and UM-UC-3, two high-grade invasive bladder cancer cell lines, using matrigel transwell inserts assay and scratch wound healing assay. The microtubule (MT) dynamics were examined by immunofluorescence and immunoblotting. Microtubule-Associated Protein 4 (MAP4) was silenced to investigate its role in tumor invasion. We also analyzed gene expression of MAP4 in 34 patients with bladder cancer using immunohistochemical staining assay. The interaction between PKA and MAP4 was examined by co-immunoprecipitation. ResultsWe used cAMP elevators and small interfering RNA of MAP4 here, found that both of them can potently inhibit the invasion and the migration of bladder cancer cells by disrupting microtubule (MT) cytoskeleton. Consistently, the bladder cancer grade is positively correlated with the protein level of MAP4. Furthermore, we found that cAMP/PKA signaling can disrupt MT cytoskeleton by the phosphorylation of MAP4. ConclusionOur results indicated that the cAMP/PKA signaling pathway might inhibit bladder cancer cell invasion by targeting MAP4-dependent microtubule dynamics, which could be exploited for the therapy of invasive bladder cancer.

Application of apparent diffusion coefficient ratio in the diagnosis of bladder cancer grading pre-operation

To explore the value of apparent diffusion coefficient (ADC) ratio in the diagnosis of bladder cancer pre-operation by analyzing its differences among different grades of bladder cancer. A total of 52 cases of bladder cancer were all definitely diagnosed with histological results.Routine examinations of magnetic resonance imaging (MRI) and diffusion-weighted imaging (DWI) were performed preoperatively on each patient. ADC map was constructed in work station and ADC values of tumor and internal obturator muscle were measured (b = 800 s/mm(2)).Ratio of ADC was calculated with internal obturator muscle as reference site. Then the relationship between ADC ratio and bladder cancer grade was analyzed. Mean ratio of ADC of all tumors was 0.98±0.35, G1 (1.12±0.21) and G2 (0.67±0.29), the sensitivity and specificity of ADC ratio was 90.2% and 85.3% respectively with an optimal threshold of 0.96. The ratios of ADC of low-grade group were significantly higher than those of high-grade group while the values of non-muscle-invasive group were significantly higher than those of muscle-invasive group. The ratios of ADC of tumor were inversely associated with the malignancy degree of bladder cancer (r = -0.845, P < 0.05). The ratio of ADC of bladder cancer reflects the lesion tissue properties. And its measurement plays an important role in the diagnosis of bladder cancer grading pre-operation.