Efficacy of immune checkpoint inhibitor therapy for advanced urothelial carcinoma in real-life clinical practice: results of a multicentric, retrospective study.

Clinical trials revealed significant antitumor activity for immune checkpoint inhibitors (ICI) in metastatic urothelial carcinoma (mUC). Due to their strict eligibility criteria, clinical trials include selected patient cohorts, and thus do not necessarily represent real-world population outcomes. In this multicentric, retrospective study, we investigated real-world data to assess the effectiveness of pembrolizumab and atezolizumab and to evaluate the prognostic value of routinely available clinicopathological and laboratory parameters. Clinical and follow-up data from mUC patients who received ICIs (01/2017-12/2021) were evaluated. Overall survival (OS), progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR), and duration of response (DOR) were used as endpoints. Patients' (n = 210, n = 76 atezolizumab and 134 pembrolizumab) median OS and PFS were 13.6 and 5.9 months, respectively. Impaired ECOG-PS, the presence of visceral, liver or bone metastases, and hemoglobin levels were independently associated with poor OS and DCR. Furthermore, Bellmunt risk factors and the enhanced Bellmunt-CRP score were shown to be prognostic for OS, PFS and DCR. In conclusion, ICIs are effective treatments for a broad range of mUC patients. Our results confirmed the prognostic value of numerous risk factors and showed that Bellmunt risk scores can further be improved when adding CRP to the model.

Epigenetic Priming and Development of New Combination Therapy Approaches.

Muscle-invasive urothelial carcinoma of the bladder (MIBC) has been treated with cisplatin-based chemotherapy for over 30 years. With the advent of immune checkpoint inhibitors, antibody drug conjugates and FGFR3 inhibitors new therapeutic options have been approved for patients with urothelial carcinoma (UC) and are still under investigation regarding association between patients' response and recently defined molecular subtypes. Unfortunately, similar to chemotherapy, only a fraction of UC patients responds to these new treatment approaches. Thus, either further new efficacious therapeutic options for treatment of individual subtypes or new approaches to overcome treatment resistance and to increase patients' response to standard of care treatment are needed.Epigenetic modifications of DNA and chromatin are known to mediate cellular plasticity or treatment resistance, and the responsible epigenetic regulators are frequently mutated or aberrantly expressed in UC. Thus, these enzymes provide targets for novel drug combination therapies to "episensitize" toward approved standard therapies by epigenetic priming. In general, these epigenetic regulators comprise writers and erasers like DNA methyltransferases and DNA demethylases (for DNA methylation), histone methyltransferases and histone demethylases (for histone methylation), as well as acetyl transferases and histone deacetylases (for histone and nonhistone acetylation). Such modifications, e.g., acetyl groups, are recognized by further epigenetic reader proteins, e.g., like the bromodomain and extra-terminal domain (BET) family proteins that often interact in multi-protein complexes and finally regulate chromatin conformation and transcriptional activity.Concurringly, epigenetic regulators target a plethora of cellular functions. Their pharmaceutical inhibitors often inhibit enzymatic activity of more than one isoenzyme or may have further noncanonical cytotoxic effects. Thus, analysis of their functions in UC pathogenesis as well as of the antineoplastic capacity of corresponding inhibitors alone or in combination with other approved drugs should follow a multidimensional approach. Here, we present our standard approach to analyze cellular effects of new epigenetic inhibitors on UC cells alone to define their potency and to conclude on putative reasonable combination therapy partners. We further describe our approach to identify efficacious synergistic combination therapies (e.g., with cisplatin or PARP inhibitors) that may have reduced normal toxicity through dose reduction, which can then be further analyzed in animal experiments. This approach may also serve as prototype for the preclinical evaluation of other epigenetic treatment approaches.

BTA stat®, NMP22® BladderChek®, UBC® Rapid Test, and CancerCheck® UBC® rapid VISUAL as urinary marker for bladder cancer: Final results of a German multicenter study.

INTRODUCTION AND OBJECTIVE: BTA stat®, NMP22® BladderChek®, UBC® Rapid Test, and CancerCheck® UBC® rapid VISUAL are urinary-based rapid tests. This multicenter study is the first study comparing all available rapid tests on a large cohort of bladder cancer patients and healthy controls in one setting. METHODS: In total 732 urine samples (second morning urine) in a real-world assessment have been analyzed. We evaluated clinical samples from 464 patients with histologically confirmed urothelial tumors of the urinary bladder (17 solitary CIS, 189 low-grade, 187 high-grade nonmuscle invasive, 71 high-grade muscle invasive), 77 patients with No Evidence of Disease (NED), and from 191 healthy controls. Urine samples were analyzed by the BTA stat®, NMP22® BladderChek®, UBC® Rapid Test point-of-care (POC) system using the concile Omega 100 POC reader, and CancerCheck® UBC® rapid VISUAL. Sensitivities and specificities were calculated by contingency analyses. RESULTS: All investigated urinary markers detected more pathological concentrations in urine of bladder cancer patients compared to tumor-free patients. The calculated diagnostic sensitivities for BTA stat®, NMP22® BladderChek®, UBC® Rapid Test, CancerCheck® UBC® rapid VISUAL, and cytology were 62.4%, 13.4%, 58.2%, 28.6%, 36.2% for low-grade, 83.4%, 49.5%, 84.5%, 63.1%, 71.2% for high-grade nonmuscle invasive, and 95.8%, 35.2%, 76.1%, 50.7%, 67.7% for high-grade muscle-invasive bladder cancer. The specificity was 67.9%, 95.5%, 79.4%, 94.4%, and 83.7%, respectively. The area under the curve (AUC) after receiver operating characteristics (ROC) analysis for high-grade non-muscle-invasive tumors was 0.757, 0.725, 0.819, 0.787, and 0.774, respectively. CONCLUSIONS: The analysis of more than 700 urine samples offers an objective view on urine-based rapid diagnostics. Elevated pathological concentrations of markers in urine of bladder cancer patients were detected in all investigated tests. The highest sensitivities for high-grade non-muscle-invasive tumors were calculated for BTA stat® and UBC® Rapid Test, whereas NMP22® BladderChek®, and cytology showed the highest specificities. BTA stat® and UBC® Rapid Test have the potential to be used as a clinical valuable urinary protein biomarker for the detection of high-grade non-muscle-invasive bladder cancer patients and could be included in the management of these tumors.

Engineering a single-chain variable fragment of cetuximab for CAR T-cell therapy against head and neck squamous cell carcinomas.

The monoclonal antibody cetuximab recognizes domain III of the epithelial growth factor receptor (EGFR) with high-affinity and is an important element in the treatment of several malignancies that overexpress non-mutated wild-type EGFR. In order to create an EGFR recognizing chimeric antigen receptor (CAR) for cellular immunotherapy of head and neck squamous cell carcinoma (HNSCC), we rationally designed single chain fragments of different lengths based on the cetuximab variable heavy and light chains. We then cloned the different cetuximab fragments into our second generation CAR construct, expressed CARs on primary human T-cells from healthy donors using mono- or biscistronic lentiviral vectors and tested the stability, functionality and specificity of the CARs. Our smallest CAR construct was most efficient with greatly improved vector production and T-cell transduction efficacy. Finally, we demonstrated that the new cetuximab CAR construct expressed on T-cells is highly reactive against EGFR-positive HNSCCs and also malignant cells from other solid cancer entities. In conclusion, we generated an optimized high-affinity EGFR CAR construct for the next steps in cancer immunotherapy, which need to focus on the development of armored CAR T-cells that will be more resistant and effective in the hostile microenvironment present in solid cancers.

Using Circulating Tumor DNA To Guide Adjuvant Therapy in Bladder Cancer: IMvigor010 and IMvigor011.

Recent data from IMvigor010 impressively demonstrate the potential of circulating tumor DNA (ctDNA) as a prognostic and a predictive biomarker in patients with urothelial carcinoma. Although ctDNA status was prospectively assessed, the published data are only exploratory and require further prospective validation. Results from the IMvigor011 trial are therefore eagerly awaited.

Cognitive function in patients undergoing cystectomy for bladder cancer - results from a prospective observational study.

Background: Impaired cognitive function of bladder cancer patients plays a role in coping with the kind of urinary diversion and may impact perioperative morbidity. In this study we therefore aimed to assess the prevalence of mild cognitive impairment in patients undergoing radical cystectomy. Secondary objectives included correlation of common cognition tests, assessment of the admitting physician, and perioperative complication rates. Methods: Patients undergoing radical cystectomy for bladder cancer were prospectively screened by neuropsychological tests including cognition tests [DemTect (Dementia Detection test), MMSE (Mini-Mental State Examination), clock drawing test] prior to surgery. Besides, clinical characteristics and perioperative outcomes were documented. Frequency of mild cognitive impairment as assessed by DemTect was correlated with the results of MMSE and clock drawing test, the occurrence of anxiety and depression, the assessment of the admitting physician, and perioperative complication rates as calculated by Spearman rank correlation coefficient. Comparative analysis (parametric and nonparametric) of patient characteristics (nonpathological versus pathological DemTect suggestive of mild cognitive impairment) was performed. Results: A total of 51 patients (80% male, median age 69 years) were analyzed. DemTect was suspicious of mild cognitive impairment in 27% (14/51) of patients, whereas MMSE and clock drawing test showed pathological results only in 10/51 and 6/51 patients, respectively. We found no correlation between mild cognitive impairment and anxiety/depression status. In all, 5/20 patients (25%) with suspicious DemTect results were considered suitable for a continent diversion neobladder by the admitting physician. Suspicious DemTect results were predictive for higher perioperative complication rates (29% versus 5%). Study limitations include small sample size and missing long-term follow-up. Conclusions: Mild cognitive impairment was observed in more than a quarter of radical cystectomy patients prior to surgery. Preoperative assessment should be supplemented by neuropsychological testing such as the DemTect as mild cognitive impairment is often underestimated and associated with significantly higher perioperative complication rates.

[Metastatic urothelial carcinoma-guideline-based therapy and new options]. / Das metastasierte Urothelkarzinom ­ leitlinienorientierte Therapie und neue Optionen.

Due to the approval of immuno-oncological therapies with immune checkpoint inhibitors, the treatment of metastatic urothelial carcinoma has become more complex in all lines of therapy. Thus, in first-line treatment, immunotherapy alone or immune maintenance therapy following platinum-based chemotherapy can be applied in addition to treatment with platinum-based combination therapies alone. In addition to the approval status and guideline recommendation, patient-specific factors such as comorbidities as well as patient preference must always be considered when choosing a therapy. In the following, we summarize the current data on treatment options in the first-line therapy of metastatic urothelial carcinoma and illustrate their practical application using a patient example.

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.

Epigenetic Priming of Bladder Cancer Cells With Decitabine Increases Cytotoxicity of Human EGFR and CD44v6 CAR Engineered T-Cells.

Background: Treatment of B-cell malignancies with CD19-directed chimeric antigen receptor (CAR) T-cells marked a new era in immunotherapy, which yet has to be successfully adopted to solid cancers. Epigenetic inhibitors of DNA methyltransferases (DNMTi) and histone deacetylases (HDACi) can induce broad changes in gene expression of malignant cells, thus making these inhibitors interesting combination partners for immunotherapeutic approaches. Methods: Urothelial carcinoma cell lines (UCC) and benign uroepithelial HBLAK cells pretreated with the DNMTi decitabine or the HDACi romidepsin were co-incubated with CAR T-cells directed against EGFR or CD44v6, and subsequent cytotoxicity assays were performed. Effects on T-cell cytotoxicity and surface antigen expression on UCC were determined by flow cytometry. We also performed next-generation mRNA sequencing of inhibitor-treated UCC and siRNA-mediated knockdown of potential regulators of CAR T-cell killing. Results: Exposure to decitabine but not romidepsin enhanced CAR T-cell cytotoxicity towards all UCC lines, but not towards the benign HBLAK cells. Increased killing could neither be attributed to enhanced target antigen expression (EGFR and CD44v6) nor fully explained by changes in the T-cell ligands PD-L1, PD-L2, ICAM-1, or CD95. Instead, gene expression analysis suggested that regulators of cell survival and apoptosis were differentially induced by the treatment. Decitabine altered the balance between survival and apoptosis factors towards an apoptosis-sensitive state associated with increased CAR T-cell killing, while romidepsin, at least partially, tilted this balance in the opposite direction. Knockdown experiments with siRNA in UCC confirmed BID and BCL2L1/BCLX as two key factors for the altered susceptibility of the UCC. Conclusion: Our data suggest that the combination of decitabine with CAR T-cell therapy is an attractive novel therapeutic approach to enhance tumor-specific killing of bladder cancer. Since BID and BCL2L1 are essential determinants for the susceptibility of a wide variety of malignant cells, their targeting might be additionally suitable for combination with immunotherapies, e.g., CAR T-cells or checkpoint inhibitors in other malignancies.

Optimal pathological response after neoadjuvant chemotherapy for muscle-invasive bladder cancer: results from a global, multicentre collaboration.

OBJECTIVES: To evaluate outcomes of patients achieving a post-treatment pathological stage of

Impact of timing of adjuvant chemotherapy following radical cystectomy for bladder cancer on patient survival.

BACKGROUND: Trials of adjuvant chemotherapy following radical cystectomy generally require chemotherapy to start within 90 days postoperatively. However, it is unclear, whether the interval between surgery and start of adjuvant therapy (S-AC-interval) impacts the oncological outcome. METHODS: Using the Retrospective International Study of Invasive/Advanced Cancer of the Urothelium (RISC) data base, we identified patients who underwent radical cystectomy for muscle invasive bladder cancer and subsequent adjuvant chemotherapy. Univariate analysis of patient characteristics, surgical factors and tumor characteristics regarding their impact on S-AC-interval was performed using Kruskal-Wallis testing and Fisher's exact test. Analysis of progression-free (PFS) and overall survival (OS) (follow-up time beginning with the start date of adjuvant chemotherapy) was analyzed in relation to S-AC-interval (continuous and dichotomous with a cut-off at 90 days) using Kaplan-Meier method and COX regression analysis. RESULTS: We identified 238 eligible patients (83.5% male, mean age: 63.4 years, 76.1% T3/T4, 66.4% pN+, 14.7% R+, 70.6% urothelial carcinoma, 71% cisplatin-based adjuvant chemotherapy). The majority of patients (n = 207, 87%) started chemotherapy within 90 days after surgery. Median S-AC-interval was 57 days (interquartile range 32.8). S-AC-interval did not have consistent association with any patient/tumor characteristics or surgery related factors (type of surgery, urinary diversion). Survival analysis using continuous S-AC-interval revealed a trend toward an impact of S-AC-interval on OS (hazard ratio 1.004, 95% confidence ratio 0.9997-1.0084, P = 0.071). With regards to PFS, that impact was shown to be statistically significant (hazard ratio 1.004, 95% confidence ratio 1.0003-1.0075, P = 0.032). In multivariate analysis, however, S-AC-interval was negated by tumor and patient related factors (pathological T-stage, N-stage, ECOG performance status). Accounting for eligibility criteria defined in some clinical trials, we extended our analysis dividing S-AC-interval in ≤90 and >90 days. Although we could confirm the trend toward better outcome in patients with a shorter S-AC interval in dichotomous analysis, neither differences in OS nor in PFS reached statistical significance (P = 0.438 and P = 0.056). CONCLUSIONS: In a large multi-institutional experience, 87% of patients who received adjuvant chemotherapy received it within the guideline recommended window of 90 days. While it was not possible to determine whether this is the optimal cut-off, early start of adjuvant chemotherapy seems to be reasonable. Regarding prognosis, tumor-related pathological factors abrogated the importance of the S-AC-interval in our analysis.

Rapid identification and susceptibility testing of Candida spp. from positive blood cultures by combination of direct MALDI-TOF mass spectrometry and direct inoculation of Vitek 2.

Fungaemia is associated with high mortality rates and early appropriate antifungal therapy is essential for patient management. However, classical diagnostic workflow takes up to several days due to the slow growth of yeasts. Therefore, an approach for direct species identification and direct antifungal susceptibility testing (AFST) without prior time-consuming sub-culturing of yeasts from positive blood cultures (BCs) is urgently needed. Yeast cell pellets prepared using Sepsityper kit were used for direct identification by MALDI-TOF mass spectrometry (MS) and for direct inoculation of Vitek 2 AST-YS07 card for AFST. For comparison, MALDI-TOF MS and Vitek 2 testing were performed from yeast subculture. A total of twenty four positive BCs including twelve C. glabrata, nine C. albicans, two C. dubliniensis and one C. krusei isolate were processed. Applying modified thresholds for species identification (score ≥ 1.5 with two identical consecutive propositions), 62.5% of BCs were identified by direct MALDI-TOF MS. AFST results were generated for 72.7% of BCs directly tested by Vitek 2 and for 100% of standardized suspensions from 24 h cultures. Thus, AFST comparison was possible for 70 isolate-antifungal combinations. Essential agreement (minimum inhibitory concentration difference ≤ 1 double dilution step) was 88.6%. Very major errors (VMEs) (false-susceptibility), major errors (false-resistance) and minor errors (false categorization involving intermediate result) amounted to 33.3% (of resistant isolates), 1.9% (of susceptible isolates) and 1.4% providing 90.0% categorical agreement. All VMEs were due to fluconazole or voriconazole. This direct method saved on average 23.5 h for identification and 15.1 h for AFST, compared to routine procedures. However, performance for azole susceptibility testing was suboptimal and testing from subculture remains indispensable to validate the direct finding.