Elevated levels of MMP12 sourced from macrophages are associated with poor prognosis in urothelial bladder cancer.

BACKGROUND: Urothelial bladder cancer is most frequently diagnosed at the non-muscle-invasive stage (NMIBC). However, recurrences and interventions for intermediate and high-risk NMIBC patients impact the quality of life. Biomarkers for patient stratification could help to avoid unnecessary interventions whilst indicating aggressive measures when required. METHODS: In this study, immuno-oncology focused, multiplexed proximity extension assays were utilised to analyse plasma (n = 90) and urine (n = 40) samples from 90 newly-diagnosed and treatment-naïve bladder cancer patients. Public single-cell RNA-sequencing and microarray data from patient tumour tissues and murine OH-BBN-induced urothelial carcinomas were also explored to further corroborate the proteomic findings. RESULTS: Plasma from muscle-invasive, urothelial bladder cancer patients displayed higher levels of MMP7 (p = 0.028) and CCL23 (p = 0.03) compared to NMIBC patients, whereas urine displayed higher levels of CD27 (p = 0.044) and CD40 (p = 0.04) in the NMIBC group by two-sided Wilcoxon rank-sum tests. Random forest survival and multivariable regression analyses identified increased MMP12 plasma levels as an independent marker (p < 0.001) associated with shorter overall survival (HR = 1.8, p < 0.001, 95% CI:1.3-2.5); this finding was validated in an independent patient OLINK cohort, but could not be established using a transcriptomic microarray dataset. Single-cell transcriptomics analyses indicated tumour-infiltrating macrophages as a putative source of MMP12. CONCLUSIONS: The measurable levels of tumour-localised, immune-cell-derived MMP12 in blood suggest MMP12 as an important biomarker that could complement histopathology-based risk stratification. As MMP12 stems from infiltrating immune cells rather than the tumor cells themselves, analyses performed on tissue biopsy material risk a biased selection of biomarkers produced by the tumour, while ignoring the surrounding microenvironment.

Nomograms including the UBC® Rapid test to detect primary bladder cancer based on a multicentre dataset.

OBJECTIVES: To evaluate the clinical utility of the urinary bladder cancer antigen test UBC® Rapid for the diagnosis of bladder cancer (BC) and to develop and validate nomograms to identify patients at high risk of primary BC. PATIENTS AND METHODS: Data from 1787 patients from 13 participating centres, who were tested between 2012 and 2020, including 763 patients with BC, were analysed. Urine samples were analysed with the UBC® Rapid test. The nomograms were developed using data from 320 patients and externally validated using data from 274 patients. The diagnostic accuracy of the UBC® Rapid test was evaluated using receiver-operating characteristic curve analysis. Brier scores and calibration curves were chosen for the validation. Biopsy-proven BC was predicted using multivariate logistic regression. RESULTS: The sensitivity, specificity, and area under the curve for the UBC® Rapid test were 46.4%, 75.5% and 0.61 (95% confidence interval [CI] 0.58-0.64) for low-grade (LG) BC, and 70.5%, 75.5% and 0.73 (95% CI 0.70-0.76) for high-grade (HG) BC, respectively. Age, UBC® Rapid test results, smoking status and haematuria were identified as independent predictors of primary BC. After external validation, nomograms based on these predictors resulted in areas under the curve of 0.79 (95% CI 0.72-0.87) and 0.95 (95% CI: 0.92-0.98) for predicting LG-BC and HG-BC, respectively, showing excellent calibration associated with a higher net benefit than the UBC® Rapid test alone for low and medium risk levels in decision curve analysis. The R Shiny app allows the results to be explored interactively and can be accessed at www.blucab-index.net. CONCLUSION: The UBC® Rapid test alone has limited clinical utility for predicting the presence of BC. However, its combined use with BC risk factors including age, smoking status and haematuria provides a fast, highly accurate and non-invasive tool for screening patients for primary LG-BC and especially primary HG-BC.

Fewer tumour draining sentinel nodes in patients with progressing muscle invasive bladder cancer, after neoadjuvant chemotherapy and radical cystectomy.

PURPOSE: To examine the relationship between the number of tumour draining sentinel nodes (SNs) and pathoanatomical outcomes, in muscle-invasive bladder cancer (MIBC), in patients undergoing neoadjuvant chemotherapy (NAC) and radical cystectomy (RC). MATERIALS AND METHODS: In an ongoing prospective multicenter study, we included 230 patients with suspected urothelial MIBC from ten Swedish urological centers. All underwent TURb and clinical staging. From the cohort, 116 patients with urothelial MIBC; cT2-cT4aN0M0, underwent radical cystectomy (RC) and lymphadenectomy with SN-detection (SNd). 83 patients received cisplatin-based NAC and 33 were NAC-naïve. The number and locations of detected SNs and non-SNs were recorded for each patient. The NAC treated patients were categorized by pathoanatomical outcomes post-RC into three groups: complete responders (CR), stable disease (SD) and progressive disease (PD). Selected covariates with possible impact on SN-yield were tested in uni -and multivariate analyses for NAC-treated patients only. RESULTS: In NAC treated patients, the mean number of SNs was significantly higher in CR patients (3.3) and SD patients (3.6) compared with PD patients (1.4) (p = 0.034). In a linear multivariate regression model, the number of harvested nodes was the only independent variable that affected the number of SNs (p = 0.0004). CONCLUSIONS: The number of tumor-draining SNs in NAC-treated patients was significantly lower in patients with progressive disease.

Emmprin Expression Predicts Response and Survival following Cisplatin Containing Chemotherapy for Bladder Cancer: A Validation Study.

PURPOSE: Neoadjuvant chemotherapy before cystectomy is recommended. To our knowledge the subset of patients likely to benefit has not been identified. We validate emmprin and survivin as markers of chemotherapy response. MATERIALS AND METHODS: Tumor specimens were obtained before therapy from a total of 250 patients with T1-T4 bladder cancer enrolled in 2 randomized trials comparing neoadjuvant chemotherapy before cystectomy with a surgery only arm. Protein expression was determined by immunohistochemistry. RESULTS: Expression was categorized according to predefined cutoffs reported in the literature. Data were analyzed with the Kaplan-Meier method and Cox models. Patients in the chemotherapy cohort with negative emmprin expression had significantly higher down staging overall survival than those with positive expression (71% vs 38%, p<0.001). The values for cancer specific survival were 76% and 56%, respectively (p<0.027). In the cystectomy only cohort emmprin expression was not associated with overall survival (46% vs 35%, p=0.23) or cancer specific survival (55% vs 51%, p=0.64). Emmprin negative patients had an absolute risk reduction of 25% in overall survival (95% CI 11-40) and a number needed to treat of 4 (95% CI 2.5-9.3). Survivin expression was not useful as a biomarker in this study. Limitations were the retrospective design and heterogeneity coupled with the time difference between the trials. CONCLUSIONS: Patients with emmprin negative tumors have a better response to neoadjuvant chemotherapy before cystectomy than those with positive expression.

5-Year outcome of a randomized prospective study comparing bacillus Calmette-Guérin with epirubicin and interferon-α2b in patients with T1 bladder cancer.

PURPOSE: In a multicenter, prospectively randomized study we evaluated the 5-year outcomes of bacillus Calmette-Guérin alone compared to a combination of epirubicin and interferon-α2b in the treatment of patients with T1 bladder cancer. MATERIALS AND METHODS: Transurethral resection was followed by a second resection and bladder mapping. Stratification was for grade and carcinoma in situ. Followup entailed regular cystoscopy and cytology during the first 5 years. The end points assessed in this analysis were recurrence-free survival, time to treatment failure and progression, cancer specific survival and prognostic factors. RESULTS: The study recruited 250 eligible patients. The 5-year recurrence-free survival rate was 38% in the combination arm and 59% in the bacillus Calmette-Guérin arm (p = 0.001). The corresponding rates for the other end points were not significantly different, as free of progression 78% and 77%, treatment failure 75% and 75%, and cancer specific survival 90% and 92%, respectively. The type of treatment, tumor size and tumor status at second resection were independent variables associated with recurrence. Concomitant carcinoma in situ was not predictive of failure of bacillus Calmette-Guérin therapy. An independent factor for treatment failure was remaining T1 stage at second resection. CONCLUSIONS: Bacillus Calmette-Guérin was more effective than the tested combination therapy. The currently recommended management with second resection and 3-week maintenance bacillus Calmette-Guérin entails a low risk of cancer specific death. More aggressive treatment in patients with infiltrative tumors at second resection might improve these results. In particular, concomitant carcinoma in situ was not a predictive factor for poor outcome after bacillus Calmette-Guérin therapy.

Combination of biomarkers for neoadjuvant systemic chemotherapy before cystectomy in patients with urinary bladder cancer.

Clinical utility of cisplatin based neoadjuvant chemotherapy (NAC) prior to radical cystectomy is limited because of lack of tools that can guide for a better patient selection. We aim to explore if a combination of biomarkers is superior to a single marker. Pretreatment tumor specimens and clinical data from two randomized trials including 250 patients with T2-T4 urothelial bladder cancer, were used. The information on the expressions on tumor tissue of four biomarkers; CCTα, emmprin, survivin, and BCL-2, detected by immunohistochemistry in our previous studies, was used. Cox proportional hazard models, including treatment-by-biomarker interaction terms, were used to assess the predictive value of the biomarkers for efficacy of NAC on overall survival. CCTα provided predictive information about the efficacy of NAC (interaction P=0.009). None of the other biomarkers provided statistically significant information additional to CCTα. The adjusted hazard ratio for NAC treated versus no-NAC was 0.42 (95% CI: 0.27-0.64) for patients with negative CCTα expression, when adding information about emmprin it decreased to 0.33 (95% CI: 0.19-0.56) for patients with both negative CCTα and emmprin. This corresponds to a decrease in number needed to treat from 4 to 3 patients. The combination of CCTα with survivin or BCL-2 yielded similar results. In a group of patients with muscle invasive bladder cancer a combination of two biomarkers might improve the possibility to identify patients most likely to benefit from the use of NAC. Further studies designed to have sufficient power to detect an interaction effect are needed.

Is Bcl-2 a predictive marker of neoadjuvant chemotherapy response in patients with urothelial bladder cancer undergoing radical cystectomy?

Background: Response to neoadjuvant cisplatin treatment in bladder cancer has been linked to expression of Bcl-2 protein by cancer cells. The objective of this study was to test Bcl-2 as a predictive marker of neoadjuvant cisplatin chemotherapy response in a patient cohort from randomized cystectomy trials. Methods: Tumor samples were taken from 247 patients with T2-T4 bladder cancer enrolled in two randomized trials comparing cystectomy with or without neoadjuvant chemotherapy. Tissue microarrays from pre-intervention transurethral resection specimens were assessed for Bcl-2 protein status by immunohistochemistry. Extension of staining above 10% was regarded as positive. Downstaging and survival ratios in relation to Bcl-2 immunoreactivity and neoadjuvant chemotherapy utilization were calculated using the log rank test and multivariate Cox proportional hazards regression analyses. Results: Bcl-2 expression was positive in 38% and negative in 62% of the 236 evaluable patients. Bcl-2 negative patients receiving neoadjuvant chemotherapy had a significant increase in survival (p = 0.009), while Bcl-2 positive patients showed no difference (p = 0.4). However, the interaction variable between neoadjuvant chemotherapy and biomarker status was not significant (p = 0.38). When the prognostic value was assessed in the no-chemotherapy group, 5-year overall survival times were significantly better among Bcl-2 positive patients than among Bcl-2 negative patients (42 months vs 33 months, p = 0.04), but again Bcl-2 status did not remain independent when other factors were adjusted. Also, in a multivariate analysis with all patients, Bcl-2 was not significant. Conclusions: Bcl-2 status is not an independent predictor of neoadjuvant cisplatin chemotherapy response and is not prognostic in muscle-invasive bladder cancer.

Choline-phosphate cytidylyltransferase-α as a possible predictor of survival and response to cisplatin neoadjuvant chemotherapy in urothelial cancer of the bladder.

OBJECTIVES: The aim of this study was to test choline-phosphate cytidylyltransferase-α (CCT-α) protein as a biomarker for neoadjuvant cisplatin chemotherapy response in a bladder tumor setting. MATERIALS AND METHODS: A total of 238 patients with T2-T4 bladder cancer enrolled into two prior randomized trials comparing neoadjuvant cisplatin-based chemotherapy (NAC) plus cystectomy with cystectomy only (no-NAC) were used as discovery and validation cohorts. Protein expression was determined with immunohistochemistry and assessed with Histo (H)-scoring. RESULTS: In the discovery cohort, comprising 61 patients, the survival ratio after NAC treatment for CCT-α-negative patients was significantly increased (p = 0.001) while there was no survival advantage in the CCT-α-positive patient group. Similarly, in the validation cohort with 177 patients, NAC treatment improved survival only in the CCT-α-negative group (p = 0.006). Although there was a tendency for a good NAC response with negative CCT-α status, the interaction variable between biomarker and treatment was not significant (p = 0.24). In the cystectomy-only group, patients with positive CCT-α expression had a better survival than CCT-α-negative patients. This prognostic effect of CCT-α expression remained significant after adjusting for well-known prognostic factors in a multivariate analysis. In a pooled database of both patient data sets, multivariate analyses showed CCT-α status as an independent factor for overall survival (p = 0.018; hazard ratio = 1.80, 95% confidence interval 1.11-2.93). CONCLUSION: CCT-α status was not predictive of outcome of NAC response; however, in the control group with cystectomy only it was found to have prognostic value.

Validation of the ezrin, CK20, and Ki-67 as potential predictive markers for BCG instillation therapy of non-muscle-invasive bladder cancer.

OBJECTIVE: The aim of our study was to try to validate 3 promising predictive biomarkers in a database based on prospective trials comparing bacillus Calmette-Guerin (BCG) with mitomycin-C and a combination of epirubicin and interferon, respectively. BACKGROUND: The most common form of bladder cancer is non-muscle-invasive tumors treated initially with transurethral resection. Unfortunately more than half recur and some also progress. Consequently, an attempt to prevent poor outcome is frequently made by intravesical instillations either by chemo- or immunotherapy. The response to such treatment is unpredictable, which is why markers predicting outcome would be valuable. PATIENTS AND METHODS: Immunohistochemical expression of ezrin, CK20, and Ki-67 was evaluated in a tumor tissue microarray based on 2 nordic multicenter trials comparing treatment with BCG vs. other intravesical adjuvant therapies. Kaplan-Meier analysis, log-rank test, and Cox regression were used to evaluate the effect of the biomarkers on recurrence-, progression-, and treatment failure-free survival. RESULTS: Of the 294 available patients immunoreactivity could be assessed in 285 patients for ezrin (97%), 285 patients for CK20 (97%), and 294 patient׳s for Ki-67 (100%). The 3 biomarkers did not predict time to any of the endpoints. Multifocality was the only predictive factor for time to recurrence (P = 0.029) and progression (P = 0.031). Ezrin was, however, predictive for treatment failure (P = 0.029) in a subgroup (BCG treated in one of the trials). In a multivariate analysis among BCG treated, none of the variables correlated to recurrence and only multifocality correlated to progression. Limitations in our study are the retrospective design and those inherent to immunohistochemistry. CONCLUSIONS: The negative results from this validation study question the ability of the tested biomarkers to predict therapy effect.