New synergistic combination therapy approaches with HDAC inhibitor quisinostat, cisplatin or PARP inhibitor talazoparib for urothelial carcinoma.

Urothelial carcinoma (UC) urgently requires new therapeutic options. Histone deacetylases (HDAC) are frequently dysregulated in UC and constitute interesting targets for the development of alternative therapy options. Thus, we investigated the effect of the second generation HDAC inhibitor (HDACi) quisinostat in five UC cell lines (UCC) and two normal control cell lines in comparison to romidepsin, a well characterized HDACi which was previously shown to induce cell death and cell cycle arrest. In UCC, quisinostat led to cell cycle alterations, cell death induction and DNA damage, but was well tolerated by normal cells. Combinations of quisinostat with cisplatin or the PARP inhibitor talazoparib led to decrease in cell viability and significant synergistic effect in five UCCs and platinum-resistant sublines allowing dose reduction. Further analyses in UM-UC-3 and J82 at low dose ratio revealed that the mechanisms included cell cycle disturbance, apoptosis induction and DNA damage. These combinations appeared to be well tolerated in normal cells. In conclusion, our results suggest new promising combination regimes for treatment of UC, also in the cisplatin-resistant setting.

Treatment patterns and clinical outcomes in metastatic urothelial carcinoma: a German retrospective real-world analysis.

Aim: This study assessed real-world treatment in patients with metastatic urothelial carcinoma (mUC) in Germany. Materials & methods: Patients diagnosed with mUC from 2015 to 2019 were identified in two claims databases: AOK PLUS and GWQ. Results: 3226 patients with mUC were analyzed; 1286 (39.9%) received systemic treatment within 12 months of diagnosis (platinum-based chemotherapy: 64.2%). Factors associated with receiving treatment were: younger age, male sex, less comorbidity and recent diagnosis. In AOK PLUS and GWQ populations, unadjusted median overall survival (interquartile range) from diagnosis in treated patients was 13.7 (6.8-32.9) and 13.8 (7.1-41.7) months, and in untreated patients was 3.0 (1.2-10.8) and 3.6 (1.2-18.8) months, respectively. Conclusion: A significant proportion of patients with mUC in Germany receive no systemic treatment.

What is this article about? This article reports the results from a study in Germany between 2015 and 2019 that investigated how advanced bladder cancer that has spread to other organs was treated and how long people lived after diagnosis. The study looked at systemic therapies, which means treatments that affect the entire body.What were the results? Only 40% of people diagnosed with advanced bladder cancer received systemic treatment within the first 12 months. Of those who did receive systemic treatment, the majority received combination therapy that included a chemotherapy drug containing platinum (64%). Systemic treatment was more likely to be given to people who were younger, less sick, male, or more recently diagnosed. After 12 months, 56% of treated people were still alive, compared with 26% of people without treatment. On average, people who received systemic treatment lived for about 14 months, while people without systemic treatment lived for only 3 to 4 months.What do the results of the study mean? Many people with advanced bladder cancer in Germany do not receive systemic treatment. People who receive treatment are likely to live longer than those who do not receive treatment.

Exposure of Bladder Cancer Cells to Blue Light (λ = 453 nm) in the Presence of Riboflavin Synergistically Enhances the Cytotoxic Efficiency of Gemcitabine.

Non-muscle invasive bladder cancer is a common tumour in men and women. In case of resistance to the standard therapeutic agents, gemcitabine can be used as off-label instillation therapy into the bladder. To reduce potential side effects, continuous efforts are made to optimise the therapeutic potential of drugs, thereby reducing the effective dose and consequently the pharmacological burden of the medication. We recently demonstrated that it is possible to significantly increase the therapeutic efficacy of mitomycin C against a bladder carcinoma cell line by exposure to non-toxic doses of blue light (453 nm). In the present study, we investigated whether the therapeutically supportive effect of blue light can be further enhanced by the additional use of the wavelength-specific photosensitiser riboflavin. We found that the gemcitabine-induced cytotoxicity of bladder cancer cell lines (BFTC-905, SW-1710, RT-112) was significantly enhanced by non-toxic doses of blue light in the presence of riboflavin. Enhanced cytotoxicity correlated with decreased levels of mitochondrial ATP synthesis and increased lipid peroxidation was most likely the result of increased oxidative stress. Due to these properties, blue light in combination with riboflavin could represent an effective therapy option with few side effects and increase the success of local treatment of bladder cancer, whereby the dose of the chemotherapeutic agent used and thus the chemical load could be significantly reduced with similar or improved therapeutic success.

Tailored immunotherapy approach with nivolumab with or without ipilimumab in patients with advanced transitional cell carcinoma after platinum-based chemotherapy (TITAN-TCC): a multicentre, single-arm, phase 2 trial.

BACKGROUND: Nivolumab is used after platinum-based chemotherapy in patients with metastatic urothelial carcinoma. Studies suggest improved outcomes for dual checkpoint inhibition with high ipilimumab doses. We aimed to examine the safety and activity of nivolumab induction and high-dose ipilimumab as an immunotherapeutic boost as a second-line treatment for patients with metastatic urothelial carcinoma. METHODS: TITAN-TCC is a multicentre, single-arm, phase 2 trial done at 19 hospitals and cancer centres in Germany and Austria. Adults aged 18 years or older with histologically confirmed metastatic or surgically unresectable urothelial cancer of the bladder, urethra, ureter, or renal pelvis were eligible. Patients had to have progression during or after first-line platinum-based chemotherapy and up to one more second-line or third-line treatment, a Karnofsky Performance Score of 70 or higher, and measurable disease as per Response Evaluation Criteria in Solid Tumors version 1.1. After four doses of intravenous nivolumab 240 mg induction monotherapy every 2 weeks, patients with a partial or complete response at week 8 continued maintenance nivolumab, whereas those with stable or progressive disease (non-responders) at week 8 received a boost of two or four doses of intravenous nivolumab 1 mg/kg plus ipilimumab 3 mg/kg every 3 weeks. Patients who subsequently had progressive disease during nivolumab maintenance also received a boost, using this schedule. The primary endpoint was the confirmed investigator-assessed objective response rate in the intention-to-treat population and had to exceed 20% for the null hypothesis to be rejected (based on the objective response rate with nivolumab monotherapy in the CheckMate-275 phase 2 trial). This study is registered with ClinicalTrials.gov, NCT03219775, and is ongoing. FINDINGS: Between April 8, 2019, and Feb 15, 2021, 83 patients with metastatic urothelial carcinoma were enrolled and all received nivolumab induction treatment (intention-to-treat population). The median age of enrolled patients was 68 years (IQR 61-76), and 57 (69%) were male and 26 (31%) were female. 50 (60%) patients received at least one boost dose. A confirmed investigator-assessed objective response was recorded in 27 (33%) of 83 patients in the intention-to-treat population, including six (7%) patients who had a complete response. This objective response rate was significantly higher than the prespecified threshold of 20% or less (33% [90% CI 24-42]; p=0·0049). The most common grade 3-4 treatment-related adverse events were immune-mediated enterocolitis (nine [11%] patients) and diarrhoea (five [6%] patients). Two (2%) treatment-related deaths were reported, both due to immune-mediated enterocolitis. INTERPRETATION: Treatment with nivolumab and nivolumab plus ipilimumab boosts in early non-responders and patients who progress late significantly improved objective response rate after previous platinum-based chemotherapy compared with the rate reported with nivolumab in the CheckMate-275 trial. Our study provides evidence for the added value of high-dose ipilimumab 3 mg/kg and suggests a potential role for the combination as a rescue strategy in platinum-pretreated patients with metastatic urothelial carcinoma. FUNDING: Bristol Myers Squibb.

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.

Prodigiosin Sensitizes Sensitive and Resistant Urothelial Carcinoma Cells to Cisplatin Treatment.

Cisplatin-based treatment is the standard of care therapy for urothelial carcinomas. However, complex cisplatin resistance mechanisms limit the success of this approach. Both apoptosis and autophagy have been shown to contribute to this resistance. Prodigiosin, a secondary metabolite from various bacteria, exerts different biological activities including the modulation of these two cellular stress response pathways. We analyzed the effect of prodigiosin on protein levels of different autophagy- and apoptosis-related proteins in cisplatin-sensitive and -resistant urothelial carcinoma cells (UCCs). Furthermore, we investigated the effect on cell viability of prodigiosin alone or in combination with cisplatin. We made use of four different pairs of cisplatin-sensitive and -resistant UCCs. We found that prodigiosin blocked autophagy in UCCs and re-sensitized cisplatin-resistant cells to apoptotic cell death. Furthermore, we found that prodigiosin is a potent anticancer agent with nanomolar IC50 values in all tested UCCs. In combination studies, we observed that prodigiosin sensitized both cisplatin-sensitive and -resistant urothelial carcinoma cell lines to cisplatin treatment with synergistic effects in most tested cell lines. These effects of prodigiosin are at least partially mediated by altering lysosomal function, since we detected reduced activities of cathepsin B and L. We propose that prodigiosin is a promising candidate for the therapy of cisplatin-resistant urothelial carcinomas, either as a single agent or in combinatory therapeutic approaches.

Predicting immune checkpoint inhibitor response in urothelial carcinoma: another step in personalised medicine?

Prediction of treatment response is a crucial issue in individualised treatment for cancer patients. In this context, Nassar and colleagues in the accompanying study published in the British Journal of Cancer analysed retrospectively a cohort of 62 metastatic urothelial cancer patients treated with immune checkpoint inhibitors and of whom not only clinical but also genomic characteristics were available. Combining molecular and clinical factors in a multivariable analysis they identified lack of visceral metastases, neutrophil-to-lymphocyte ratio (NLR) <5, and high single nucleotide variant (SNV) count (≥10) as independent predictors of treatment response.

Therapeutic implications of PD-L1 expression in bladder cancer with squamous differentiation.

BACKGROUND: Immune checkpoint inhibitors (ICI) are an integral part of bladder cancer therapy, however, the relevance of ICI treatment for mixed and pure squamous cell carcinoma of the bladder remains poorly studied. Therefore, we analysed the expression of programmed death-ligand 1 (PD-L1) in urothelial carcinomas with squamous differentiation (UC/SCC) and pure squamous cell carcinoma (SCC) of the bladder and studied a UC/SCC patient with ICI therapy. METHODS: Tissue microarrays of 45 UC/SCC and 63 SCC samples were immunohistochemically stained with four anti-PD-L1 antibodies (28-8, 22C3, SP142 and SP263). PD-L1 expression was determined for tumour cells (TP-Score), immune cells (IC-Score) and combined (CPS, combined positive score). In addition, we present clinical and histological data of an UC/SCC patient with nivolumab therapy. RESULTS: Overall, positive PD-L1 staining ranged between 4.8 and 61.9% for IC and 0 and 51.2% for TC depending on the used antibody. There were no significant differences between UC/SCC and SCC. According to current FDA guidelines for example for first line therapy of urothelial cancer with pembrolizumab (CPS ≥ 10), a subset of SCC patients up to 20% would be eligible. Finally, our UC/SCC index patient revealed excellent therapy response regarding his lung metastasis. CONCLUSIONS: Our data reveal a PD-L1 expression in squamous differentiated carcinomas comparable with current data shown for urothelial tumours. In accordance with the encouraging clinical data of the index patient we suggest ICI treatment also for mixed and pure SCC of the urinary bladder.

Complication rate after cystectomy following pelvic radiotherapy: an international, multicenter, retrospective series of 682 cases.

PURPOSE: Conflicting evidence exists on the complication rates after cystectomy following previous radiation (pRTC) with only a few available series. We aim to assess the complication rate of pRTC for abdominal-pelvic malignancies. METHODS: Patients treated with radical cystectomy following any previous history of RT and with available information on complications for a minimum of 1 year were included. Univariable and multivariable logistic regression models were used to assess the relationship between the variable parameters and the risk of any complication. RESULTS: 682 patients underwent pRTC after a previous RT (80.5% EBRT) for prostate, bladder (BC), gynecological or other cancers in 49.1%, 27.4%, 9.8% and 12.9%, respectively. Overall, 512 (75.1%) had at least one post-surgical complication, classified as Clavien ≥ 3 in 29.6% and Clavien V in 2.9%. At least one surgical complication occurred in 350 (51.3%), including bowel leakage in 6.2% and ureteric stricture in 9.4%. A medical complication was observed in 359 (52.6%) patients, with UTI/pyelonephritis being the most common (19%), followed by renal failure (12%). The majority of patients (86%) received an incontinent urinary diversion. In multivariable analysis adjusted for age, gender and type of RT, patients treated with RT for bladder cancer had a 1.7 times increased relative risk of experiencing any complication after RC compared to those with RT for prostate cancer (p = 0.023). The type of diversion (continent vs non-continent) did not influence the risk of complications. CONCLUSION: pRTC carries a high rate of major complications that dramatically exceeds the rates reported in RT-naïve RCs.

Many Different LINE-1 Retroelements Are Activated in Bladder Cancer.

Human genomes contain about 100,000 LINE-1 (L1) retroelements, of which more than 100 are intact. L1s are normally tightly controlled by epigenetic mechanisms, which often fail in cancer. In bladder urothelial carcinoma (UC), particularly, L1s become DNA-hypomethylated, expressed and contribute to genomic instability and tumor growth. It is, however, unknown which individual L1s are activated. Following RNA-immunoprecipitation with a L1-specific antibody, third generation nanopore sequencing detected transcripts of 90 individual elements in the VM-Cub-1 UC line with high overall L1 expression. In total, 10 L1s accounted for >60% of the reads. Analysis of five specific L1s by RT-qPCR revealed generally increased expression in UC tissues and cell lines over normal controls, but variable expression among tumor cell lines from bladder, prostate and testicular cancer. Chromatin immunoprecipitation demonstrated active histone marks at L1 sequences with increased expression in VM-Cub-1, but not in a different UC cell line with low L1 expression. We conclude that many L1 elements are epigenetically activated in bladder cancer in a varied pattern. Our findings indicate that expression of individual L1s is highly heterogeneous between and among cancer types.

Oncological outcome of patients treated with spot-specific salvage lymphnode dissection (sLND) for positron-emission tomography (PET)-positive prostate cancer (PCa) relapse.

OBJECTIVES: To report pre-, postoperative and oncological outcomes in patients treated with spot-specific sLND for patients with exclusive nodal recurrence after PCa primary treatment. MATERIALS AND METHODS: With regard to salvage treatment failure (sTF), 46 consecutive patients, undergoing 52 sLND for nodal recurrence detected by PET/CT scan were stratified in 3 groups (group A: post-sLND PSA nadir < 0.01 ng/ml and in follow-up reaching a value > 0.2 ng/ml, group B: post-sLND PSA nadir > 0.01 ng/ml and in follow-up reaching a value equal to pre-sLND PSA; group C: additional salvage treatment administration). Surgical outcome of patients was analyzed by descriptive statistics (Student's t test for continuous variables, Chi-square and Fisher's test for categorial ones). Time to sTF of each group was analyzed and compared by Kaplan-Meier method and correlations regarding sTF and pre-sLND PSA, time from PCa primary treatment to PET/CT scan, time from PCa primary treatment to sLND and number of positive PET/CT scan spots were assessed. RESULTS: Median PSA at PET/CT scan was 2.9 ng/ml (IQR 1.2-6.1). Open and laparoscopic sLND were performed in 40/52 (77%) and 12/52 (23%), respectively. Median number of removed lymph nodes was 6 (IQR 4-13). Histological report was positive for PCa in 39/52 sLND (75%). Median blood loss was 50 ml (IQR 0-50, range 0-600). Median length of hospital stay was 5 days (IQR 4-6). 4 and 7 patients had low-grade (I/II) and high-grade (≥ III) Clavien-Dindo complications, respectively. Readmission rates at 30 and 90 days were 5/52 (9.6%) and 1/52 (2%), respectively. sTF was observed in 2/7 (group A), 12/12 (group B) and 22/22 patients (group C). Median time to sTF in group B and C was 3.5 (IQR 1.7-13.2) and 4 months (IQR 2.0-10), respectively. CONCLUSION: Even spot-specific PET/CT sLND harbors a measurable (CD > III) morbidity in 1 out of 7 patients. Only patients with positive histological report and a PSA nadir < 0.01 ng/ml after sLND seem to experience a long-term benefit. Patients with a PSA nadir > 0.01 ng/ml have a delay of systemic treatment of up to 4 months. sLND remains an experimental approach and long-term oncological benefit needs an improved selection of patients.

HDAC5 Expression in Urothelial Carcinoma Cell Lines Inhibits Long-Term Proliferation but Can Promote Epithelial-to-Mesenchymal Transition.

Class I histone deacetylases (HDACs) generally promote cell proliferation and tumorigenesis, whereas class IIA HDACs like HDAC4 and HDAC5 may promote or impede cancer development in a tissue-dependent manner. In urothelial carcinoma (UC), HDAC5 is often downregulated. Accordingly, HDAC5 was weakly expressed in UC cell lines suggesting a possible tumor-suppressive function. We therefore characterized the effects of stable HDAC5 expression in four UC cell lines (RT112, VM-Cub-1, SW1710 and UM-UC-3) with different phenotypes reflecting the heterogeneity of UC, by assessing proliferation, clonogenicity and migration ability. Further, we detailed changes in the proteome and transcriptome by immunoblotting, mass spectrometry and RNA sequencing analysis. We observed that HDAC5 overexpression in general decreased cell proliferation, but in one cell line (VM-Cub-1) induced a dramatic change from an epitheloid to a mesenchymal phenotype, i.e., epithelial-mesenchymal transition (EMT). These phenotypical changes were confirmed by comprehensive proteomics and transcriptomics analyses. In contrast to HDAC5, overexpression of HDAC4 exerted only weak effects on cell proliferation and phenotypes. We conclude that overexpression of HDAC5 may generally decrease proliferation in UC, but, intriguingly, may induce EMT on its own in certain circumstances.

Comparison of 2-Year Oncological Outcome and Early Recurrence Patterns in Patients with Urothelial Bladder Carcinoma Treated with Open or Robot-Assisted Radical Cystectomy with an Extracorporeal Urinary Diversion.

BACKGROUND: Data on oncological follow-up after robotic-assisted radical cystectomy (RARC) have been reported only scarcely and individual studies have reported an increase in early recurrences and atypical recurrences. PATIENTS AND METHODS: Clinical data of 89 patients with RARC were compared to 59 patients with open radical cystectomy (ORC) at a single institution. Two-year cancer-specific (2y-CSS) and 2-year overall survival (2y-OS) related to histopathological tumor stage of RARC patients calculated by Kaplan-Meier method were compared to ORC patients using log-rank test. Early clinical recurrence rate (eCR, progression ≤6 months post-cystectomy) and metastatic pattern of both groups were compared by chi-square test. RESULTS: Median follow-up 32 months (RARC) and 47.5 months (ORC), both groups were balanced in baseline characteristics. For RARC pts, -2y-OS and CSS-free survival rates were 80 and 90%, for ORC pts 65 and 71% (all p > 0.05). Margin status was not significantly different. eCR was observed in 10 out of 89 (11%) RARC pts and in 7 out of 59 (12%) ORC pts (p = 0.9). No difference in atypical metastases was seen between groups. CONCLUSION: Two-year oncological outcomes of RARC patients are comparable to ORC patients without differences regarding ePR or metastatic pattern.

Multifaceted Mechanisms of Cisplatin Resistance in Long-Term Treated Urothelial Carcinoma Cell Lines.

Therapeutic efficacy of cisplatin-based treatment of late stage urothelial carcinoma (UC) is limited by chemoresistance. To elucidate underlying mechanisms and to develop new approaches for overcoming resistance, we generated long-term cisplatin treated (LTT) UC cell lines, characterised their cisplatin response, and determined the expression of molecules involved in cisplatin transport and detoxification, DNA repair, and apoptosis. Inhibitors of metallothioneins and Survivin were applied to investigate their ability to sensitise towards cisplatin. Cell growth, proliferation, and clonogenicity were examined after cisplatin treatment by MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, EdU (5-ethynyl-2'-deoxyuridine) incorporation assay, and Giemsa staining, respectively. Cell cycle distribution and apoptosis were quantified by flow cytometry. mRNA and protein expressions were measured by real-time quantitative (qRT)-PCR, western blot, or immunofluorescence staining. LTTs recovered rapidly from cisplatin stress compared to parental cells. In LTTs, to various extents, cisplatin exporters and metallothioneins were induced, cisplatin adduct levels and DNA damage were decreased, whereas expression of DNA repair factors and specific anti-apoptotic factors was elevated. Pharmacological inhibition of Survivin, but not of metallothioneins, sensitised LTTs to cisplatin, in an additive manner. LTTs minimise cisplatin-induced DNA damage and evade apoptosis by increased expression of anti-apoptotic factors. The observed diversity among the four LTTs highlights the complexity of cisplatin resistance mechanisms even within one tumour entity, explaining heterogeneity in patient responses to chemotherapy.

Various Mechanisms Involve the Nuclear Factor (Erythroid-Derived 2)-Like (NRF2) to Achieve Cytoprotection in Long-Term Cisplatin-Treated Urothelial Carcinoma Cell Lines.

Therapeutic efficacy of cisplatin-based chemotherapy for advanced-stage urothelial carcinoma (UC) is limited by drug resistance. The nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway is a major regulator of cytoprotective responses. We investigated its involvement in cisplatin resistance in long-term cisplatin treated UC cell lines (LTTs). Expression of NRF2 pathway components and targets was evaluated by qRT-PCR and western blotting in LTT sublines from four different parental cells. NRF2 transcriptional activity was determined by reporter assays and total glutathione (GSH) was quantified enzymatically. Effects of siRNA-mediated NRF2 knockdown on chemosensitivity were analysed by viability assays, γH2AX immunofluorescence, and flow cytometry. Increased expression of NRF2, its positive regulator p62/SQSTM1, and elevated NRF2 activity was observed in 3/4 LTTs, which correlated with KEAP1 expression. Expression of cytoprotective enzymes and GSH concentration were upregulated in some LTTs. NRF2 knockdown resulted in downregulation of cytoprotective enzymes and resensitised 3/4 LTTs towards cisplatin as demonstrated by reduced IC50 values, increased γH2AX foci formation, and elevated number of apoptotic cells. In conclusion, while LTT lines displayed diversity in NRF2 activation, NRF2 signalling contributed to cisplatin resistance in LTT lines, albeit in diverse ways. Accordingly, inhibition of NRF2 can be used to resensitise UC cells to cisplatin, but responses in patients may likewise be variable.

The long noncoding RNA HOTAIR has tissue and cell type-dependent effects on HOX gene expression and phenotype of urothelial cancer cells.

BACKGROUND: Urothelial carcinoma (UC) is the fifth most common cancer in the developed world. Delineation of differentiation subtypes in UC highlighted the importance of aberrant differentiation. Understanding underlying mechanisms may facilitate diagnosis and development of efficient therapy strategies. It is well accepted that epigenetic mechanisms are involved. Long noncoding RNAs (lncRNAs), a new class of epigenetic factors, are thought to mediate molecular differences between cell types to control cellular identity. The present study focuses on the lncRNA HOTAIR, originating from the HOXC locus. Its overexpression induces an aggressive phenotype in many cancers and aberrant expression of homeotic HOX transcription factors, especially HOXD10, that regulate differentiation and tissue homeostasis. The aim of the present study was to determine the functional role of HOTAIR in UC with regard to aggressive phenotype, regulation of aberrant differentiation and altered HOX gene expression. METHODS: We determined RNA expression levels of HOTAIR and HOX genes in UC tissues and cell lines. Knockdown of HOTAIR and ectopic overexpression was performed to determine the effect on reported target genes in UC. Cell lines were stably transfected with HOTAIR to investigate changes in phenotype and HOX gene expression. RESULTS: HOTAIR was overexpressed in approximately half of UC tissues and cell lines. Effects of HOTAIR overexpression differed between cell lines. Whereas VM-CUB1 cells acquired the expected phenotype with increased proliferation, clonogenicity, anchorage independent growth, migratory activity and epithelial-to-mesenchymal transition, 5637 cells grew more slowly displaying induction of senescence and related immune response genes. Other UC lines showed intermediate effects. Expression profiling revealed divergent effects on HOX genes, cell cycle regulators and differentiation according with the phenotypic differences between HOTAIR-overexpressing VM-CUB1 and 5637 cells. CONCLUSIONS: Our data indicate that HOTAIR overexpression may affect differentiation state and aggressiveness of UC cells, but in a cell-type dependent manner. Our functional studies and the comparison of our expression data sets with those from other cancer cell types, which revealed minimal overlaps, indicate that effects of HOTAIR are strongly tissue-dependent and can even differ within one cancer type. Thus, HOTAIR functions and target genes cannot simply be transferred from one cancer type to the other.

Second-Line Treatment of Advanced Urothelial Cancer with Paclitaxel and Everolimus in a German Phase II Trial (AUO Trial AB 35/09).

OBJECTIVE: The efficacy of second-line treatment after failure of platinum-based chemotherapy in patients with advanced urothelial cancer is limited. Based on encouraging preclinical and clinical phase I data, we evaluated the safety and efficacy of the combination of paclitaxel and everolimus in these patients. METHODS: In this trial, patients having failed to respond to prior platinum-based combination treatment of urothelial cancer were treated with paclitaxel (175 mg/m(2) i.v., 3-weekly) and the mTOR-inhibitor everolimus (10 mg p.o., once daily). The patients were treated until tumor progression or until a maximum of 6 cycles was completed. A one-stage design was used to evaluate the objective response rate (ORR) as the primary endpoint. RESULTS: A total of 27 patients (67% male; median age 63 years) were enrolled. The most frequent grade III/IV toxicities were anemia (28%), peripheral neuropathy (28%), and fatigue (24%). No treatment-related deaths were reported. Complete and partial remissions were observed in 0/24 and 3/24 patients eligible for efficacy analysis, respectively (ORR 13%). Progression-free survival was 2.9 months [95% confidence interval (95% CI) 1.9-4.2], and the median overall survival was 5.6 months (95% CI 4.8-10.2). CONCLUSION: The combination of paclitaxel and everolimus has not achieved the expected efficacy in second-line treatment of urothelial cancer and should not be further explored.

Canonical Notch signalling is inactive in urothelial carcinoma.

BACKGROUND: Notch signalling regulates cell fate in most tissues, promoting precursor cell proliferation in some, but differentiation in others. Accordingly, downregulation or overactivity variously contributes to cancer development. So far, little is known about Notch pathway activity and function in the normal urothelium and in urothelial carcinoma (UC). We have therefore investigated expression of Notch pathway components in UC tissues and cell lines and studied the function of one receptor, NOTCH1, in detail. METHODS: Expression of canonical Notch pathway components were studied in UC and normal bladder tissues by immunohistochemistry and quantitative RT-PCR and in UC cell lines and normal cultured urothelial cells by qRT-PCR, immunocytochemistry and Western blotting. Pathway activity was measured by reporter gene assays. Its influence on cell proliferation was investigated by γ-secretase inhibition. Effects of NOTCH1 restoration were followed by measuring cell cycle distribution, proliferation, clonogenicity and nuclear morphology. RESULTS: NOTCH1 and its ligand, DLL1, were expressed at plasma membranes and in the cytoplasm of cells in the upper normal urothelium layer, but became downregulated in UC tissues, especially in high-stage tumours. In addition, the proteins were often delocalized intracellularly. According differences were observed in UC cell lines compared to normal urothelial cells. Canonical Notch pathway activity in reporter assays was repressed in UC cell lines compared to normal cells and a mammary carcinoma cell line, but was induced by transfected NOTCH1. Inhibitors of Notch signalling acting at the γ-secretase step did not affect UC cell proliferation at concentrations efficacious against a cell line with known Notch activity. Surprisingly, overexpression of NOTCH1 into UC cell lines did not significantly affect short-term cell proliferation, but induced nuclear abnormalities and diminished clonogenicity. CONCLUSION: Our data indicate that canonical Notch signalling is suppressed in urothelial carcinoma mainly through downregulation of NOTCH1. These findings can be explained by proposing that canonical Notch signalling may promote differentiation in the urothelium, like in many squamous epithelia, and its suppression may therefore be advantageous for tumour progression. As an important corollary, inhibition of canonical Notch signalling is unlikely to be efficacious and might be counter-productive in the treatment of urothelial carcinoma.

A nomogram including baseline prognostic factors to estimate the activity of second-line therapy for advanced urothelial carcinoma.

OBJECTIVE: To study the impact of the prognostic factors liver metastasis (LM), anaemia (haemoglobin [Hb] <10 g/dL), Eastern Cooperative Oncology Group performance status (ECOG-PS) ≥1 and time from previous chemotherapy (TFPC) on the activity of second-line therapy for advanced urothelial carcinoma (UC). PATIENTS AND METHODS: Twelve phase II trials evaluating second-line chemotherapy and/or biological characteristics (n = 748) in patients with progressive disease were pooled. Progression-free survival (PFS) was defined as tumour progression or death from any cause. The PFS rate at 6 months (PFS6) was defined from the date of registration and calculated using the Kaplan-Meier method. Response rate (RR) was defined using Response Evaluation Criteria in Solid Tumours (RECIST) 1.0. A nomogram predicting PFS6 was constructed using the rms software package in R (http://www.r-project.org). RESULTS: Data regarding progression, anaemia, LM, ECOG-PS and TFPC were available from 570 patients in nine phase II trials. The overall median PFS was 2.7 months, PFS6 was 22.2% (95% confidence interval 18.8-25.9) and the RR was 17.5% (95% CI: 14.5-20.9%). For every unit increase in risk group, the hazard of progression in 6 months increased by 41% and the odds of response decreased by 48%. A nomogram was constructed to predict PFS6 on an individual patient level. The model was internally validated and was shown to have acceptable calibration performance. CONCLUSIONS: The RR and PFS6 vary as a function of baseline prognostic factors in patients receiving second-line therapy for advanced UC. A nomogram incorporating prognostic factors facilitates the evaluation of outcomes across phase II trials enrolling heterogeneous populations and helps select suitable agents for phase III testing.