Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion.

BACKGROUND: We have previously identified an unsuspected role for GJB3 showing that the deficiency of this connexin protein induces aneuploidy in human and murine cells and accelerates cell transformation as well as tumor formation in xenograft models. The molecular mechanisms by which loss of GJB3 leads to aneuploidy and cancer initiation and progression remain unsolved. METHODS: GJB3 expression levels were determined by RT-qPCR and Western blot. The consequences of GJB3 knockdown on genome instability were assessed by metaphase chromosome counting, multinucleation of cells, by micronuclei formation and by the determination of spindle orientation. Interactions of GJB3 with α-tubulin and F-actin was analyzed by immunoprecipitation and immunocytochemistry. Consequences of GJB3 deficiency on microtubule and actin dynamics were measured by live cell imaging and fluorescence recovery after photobleaching experiments, respectively. Immunohistochemistry was used to determine GJB3 levels on human and murine bladder cancer tissue sections. Bladder cancer in mice was chemically induced by BBN-treatment. RESULTS: We find that GJB3 is highly expressed in the ureter and bladder epithelium, but it is downregulated in invasive bladder cancer cell lines and during tumor progression in both human and mouse bladder cancer. Downregulation of GJB3 expression leads to aneuploidy and genomic instability in karyotypically stable urothelial cells and experimental modulation of GJB3 levels alters the migration and invasive capacity of bladder cancer cell lines. Importantly, GJB3 interacts both with α-tubulin and F-actin. The impairment of these interactions alters the dynamics of these cytoskeletal components and leads to defective spindle orientation. CONCLUSION: We conclude that deregulated microtubule and actin dynamics have an impact on proper chromosome separation and tumor cell invasion and migration. Consequently, these observations indicate a possible role for GJB3 in the onset and spreading of bladder cancer and demonstrate a molecular link between enhanced aneuploidy and invasive capacity cancer cells during tumor cell dissemination.

Improving detection of carcinoma in situ in bladder cancer: urinary cytology vs the Xpert® BC Monitor.

OBJECTIVE: To investigate and compare the performance of urinary cytology and the Xpert BC Monitor test in the detection of bladder cancer in various clinically significant patient cohorts, including patients with carcinoma in situ (CIS), in a prospective multicentre setting, aiming to identify potential applications in clinical practice. PATIENTS AND METHODS: A total of 756 patients scheduled for transurethral resection of bladder tumour (TURBT) were prospectively screened between July 2018 and December 2020 at six German University Centres. Central urinary cytology and Xpert BC Monitor tests were performed prior to TURBT. The diagnostic performance of urinary cytology and the Xpert BC Monitor was evaluated according to sensitivity (SN), specificity (SC), negative predictive value (NPV) and positive predictive value (PPV). Statistical comparison of urinary cytology and the Xpert BC Monitor was conducted using the McNemar test. RESULTS: Of 756 screened patients, 733 (568 male [78%]; median [interquartile range] age 72 [62-79] years) were included. Bladder cancer was present in 482 patients (65.8%) with 258 (53.5%) high-grade tumours. Overall SN, SC, NPV and PPV were 39%, 93%, 44% and 92% for urinary cytology, and 75%, 69%, 59% and 82% for the Xpert BC Monitor. In patients with CIS (concomitant or solitary), SN, SC, NPV and PPV were 59%, 93%, 87% and 50% for urinary cytology, and 90%, 69%, 95% and 50% for the Xpert BC Monitor. The Xpert BC Monitor missed four tumours (NPV = 98%) in patients with solitary CIS, while potentially avoiding 63.3% of TURBTs in inconclusive or negative cystoscopy and a negative Xpert result. CONCLUSION: Positive urinary cytology may indicate bladder cancer and should be taken seriously. The Xpert BC Monitor may represent a useful diagnostic tool for correctly identifying patients with solitary CIS and unsuspicious or inconclusive cystoscopy.

Nivolumab + Ipilimumab as Immunotherapeutic Boost in Metastatic Urothelial Carcinoma: A Nonrandomized Clinical Trial.

Importance: Studies with nivolumab, an approved therapy for metastatic urothelial carcinoma (mUC) after platinum-based chemotherapy, demonstrate improved outcomes with added high-dose ipilimumab. Objective: To assess efficacy and safety of a tailored approach using nivolumab + ipilimumab as an immunotherapeutic boost for mUC. Design, Setting, and Participants: In this phase 2 nonrandomized trial, patients with mUC composed 2 cohorts. Cohort 1 received first-line or second-/third-line nivolumab with escalating doses of ipilimumab, and cohort 2 received second-/third-line nivolumab with high-dose ipilimumab. Recruitment spanned 26 sites in Germany and Austria from August 8, 2017, to February 18, 2021. All patients had a 70% or higher Karnofsky Performance Score and measurable disease per Response Evaluation Criteria in Solid Tumours, version 1.1. Interventions: All patients initiated 4 doses of 240-mg nivolumab (1× every 2 wk). Week 8 nonresponders received nivolumab + ipilimumab (1× every 3 wk). Cohort 1 received 2 doses of 3-mg/kg nivolumab + 1-mg/kg ipilimumab followed by 2 doses of 1-mg/kg nivolumab + 3-mg/kg ipilimumab if no response. Due to safety concerns, cohort 1 treatment was halted, and first-line cohort 2 treatment was not pursued. Cohort 2 received 2 to 4 doses of 1-mg/kg nivolumab + 3-mg/kg ipilimumab. Responders continued with nivolumab maintenance but could receive nivolumab + ipilimumab for later progression. Main Outcomes and Measures: The primary end point was objective response rate. Results: The study comprised 169 patients (118 [69.8%] men; median [range] age, 68 [37-84] years): 86 in cohort 1 (42 first-line; 44 second-/third-line) and 83 in cohort 2. The median (IQR) follow-up times were 10.4 (4.2-23.5) months (first-line cohort 1), 7.5 (3.1-23.8) months (second-/third-line cohort 1), and 6.2 (3.2-22.7) months (cohort 2). Response rates to nivolumab induction were 12/42 (29%, first-line cohort 1), 10/44 (23%, second-/third-line cohort 1), and 17/83 (20%, cohort 2). Response rates to a tailored approach were 20/42 (48% [90% CI, 34%-61%], first-line cohort 1), 12/44 (27% [90% CI, 17%-40%], second-/third-line cohort 1), and 27/83 (33% [90% CI, 23%-42%], cohort 2). Three-year overall survival rates for first-line cohort 1, second-/third-line cohort 1, and cohort 2 using the Kaplan-Meier method were 32% (95% CI, 17%-49%), 19% (95% CI, 8%-33%), and 34% (95% CI, 23%-44%), respectively. Conclusions and Relevance: In this nonrandomized trial, although first-line cohort 1 treatment improved objective response rates, considerable progression events urge caution with this as a first-line therapy. Second-/third-line cohort 1 treatment did not improve response rates compared with nivolumab monotherapy. However, added high-dose ipilimumab may improve tumor response and survival in patients with mUC. Trial Registration: ClinicalTrials.gov Identifier: NCT03219775.

NECTIN4 Amplification Is Frequent in Solid Tumors and Predicts Enfortumab Vedotin Response in Metastatic Urothelial Cancer.

PURPOSE: The anti-NECTIN4 antibody-drug conjugate enfortumab vedotin (EV) is approved for patients with metastatic urothelial cancer (mUC). However, durable benefit is only achieved in a small, yet uncharacterized patient subset. NECTIN4 is located on chromosome 1q23.3, and 1q23.3 gains represent frequent copy number variations (CNVs) in urothelial cancer. Here, we aimed to evaluate NECTIN4 amplifications as a genomic biomarker to predict EV response in patients with mUC. MATERIALS AND METHODS: We established a NECTIN4-specific fluorescence in situ hybridization (FISH) assay to assess the predictive value of NECTIN4 CNVs in a multicenter EV-treated mUC patient cohort (mUC-EV, n = 108). CNVs were correlated with membranous NECTIN4 protein expression, EV treatment responses, and outcomes. We also assessed the prognostic value of NECTIN4 CNVs measured in metastatic biopsies of non-EV-treated mUC (mUC-non-EV, n = 103). Furthermore, we queried The Cancer Genome Atlas (TCGA) data sets (10,712 patients across 32 cancer types) for NECTIN4 CNVs. RESULTS: NECTIN4 amplifications are frequent genomic events in muscle-invasive bladder cancer (TCGA bladder cancer data set: approximately 17%) and mUC (approximately 26% in our mUC cohorts). In mUC-EV, NECTIN4 amplification represents a stable genomic alteration during metastatic progression and associates with enhanced membranous NECTIN4 protein expression. Ninety-six percent (27 of 28) of patients with NECTIN4 amplifications demonstrated objective responses to EV compared with 32% (24 of 74) in the nonamplified subgroup (P < .001). In multivariable Cox analysis adjusted for age, sex, and Bellmunt risk factors, NECTIN4 amplifications led to a 92% risk reduction for death (hazard ratio, 0.08 [95% CI, 0.02 to 0.34]; P < .001). In the mUC-non-EV, NECTIN4 amplifications were not associated with outcomes. TCGA Pan-Cancer analysis demonstrated that NECTIN4 amplifications occur frequently in other cancers, for example, in 5%-10% of breast and lung cancers. CONCLUSION: NECTIN4 amplifications are genomic predictors of EV responses and long-term survival in patients with mUC.

An Empirical Survey on the Adaption of Neoadjuvant Chemotherapy in Bladder Cancer.

INTRODUCTION: The aim of the study was to determine the adaption of neoadjuvant chemotherapy (NAC) in patients with muscle-invasive bladder cancer (MIBC) in Germany, Austria, and Switzerland and especially underlying reasons for potential low adherence to guidelines. METHODS: We conducted a non-validated survey among 336 urologic departments in Germany, Austria, and Switzerland. RedCap questionnaires were electronically distributed and included 23 items concerning the general NAC administration standards and guideline compliance in patient counseling regarding the actual treatment. RESULTS: The return rate of the questionnaire was 19.1% (63/336). Although 45 departments (71.4%) claim to perform NAC as the standard of care, only 49% of eligible patients actually receive NAC. An advanced disease stage (≥cT3) and a high tumor volume were mentioned to support the application of NAC, whereas 35% of responders worry about deterioration of patients' preoperative status due to NAC. Furthermore, 26.7% of respondents are concerned about the low extent of survival benefit. CONCLUSION: Application of NAC in eligible MIBC patients in Germany, Austria, and Switzerland remains low. Although the majority of urologic departments discuss NAC and acknowledge the need for intensified treatment in advanced disease stages, not all eligible patients will actually receive NAC before radical cystectomy.

Molecular Urothelial Tumor Cell Subtypes Remain Stable During Metastatic Evolution.

Urothelial cancer (UC) care is moving toward precision oncology. For tumor biology-driven treatment of metastatic UC (mUC), molecular subtypes play a crucial role. However, it is not known whether subtypes change during metastatic evolution. To address this, we analyzed a UC progression cohort (N = 154 patients) with 138 matched primary tumors (PRIM) and synchronous or metachronous distant metastasis (MET) by immunohistochemistry, and mRNA sequencing in a subgroup of 20 matched pairs. Protein-based tumor cell subtypes and histomorphology remained stable during metastatic progression (concordance: 94%, 95% confidence interval [CI] 88-97%). In comparison, transcriptome-based molecular consensus subtypes exhibited higher heterogeneity between PRIM and MET (concordance: 45%, 95% CI 23-69%), with switches particularly occurring between luminal and stroma-rich tumors. Of note, all tumors classified as stroma rich showed luminal tumor cell differentiation. By an in-depth analysis, we found a negative correlation of luminal gene and protein expression with increasing desmoplastic stroma content, suggesting that luminal tumor cell differentiation of "stroma-rich tumors" is superimposed by gene expression signals stemming from the stromal compartment. Immunohistochemistry allows tumor cell subtyping into luminal, basal, or neuroendocrine classes that remain stable during metastatic progression. These findings expand our biological understanding of UC MET and have implications for future subtype-stratified clinical trials in patients with mUC. PATIENT SUMMARY: Urothelial carcinomas (UCs) occur in different appearances, the so-called molecular subtypes. These molecular subtypes will gain importance for the therapy of metastatic UCs in the future. We could demonstrate that the subtype remains stable during metastasis, which is highly relevant for future studies.

[Urinary incontinence after radical prostatectomy for prostate cancer-data from 17,149 patients from 125 certified centers]. / Harninkontinenz nach radikaler Prostatektomie beim Prostatakarzinom ­ aktuelle Daten von 17.149 Patienten aus 125 zertifizierten Zentren.

BACKGROUND: In addition to erectile dysfunction, urinary incontinence is the most common functional limitation after radical prostatectomy (RPE) for prostate cancer (PCa). The German S3 guideline recommends informing patients about possible effects of the therapy options, including incontinence. However, only little data on continence from routine care in German-speaking countries after RPE are currently available, which makes it difficult to inform patients. OBJECTIVE: The aim of this work is to present data on the frequency and severity of urinary incontinence after RPE from routine care. MATERIALS AND METHODS: Information from the PCO (Prostate Cancer Outcomes) study is used, which was collected between 2016 and 2022 in 125 German Cancer Society (DKG)-certified prostate cancer centers in 17,149 patients using the Expanded Prostate Cancer Index Composite Short Form (EPIC-26). Changes in the "incontinence" score before (T0) and 12 months after RPE (T1) and the proportion of patients who used pads, stratified by age and risk group, are reported. RESULTS: The average score for urinary incontinence (value range: 0-worst possible to 100-best possible) was 93 points at T0 and 73 points 12 months later. At T0, 97% of the patients did not use a pad, compared to 56% at T1. 43% of the patients who did not use a pad before surgery used at least one pad a day 12 months later, while 13% use two or more. The proportion of patients using pads differs by age and risk classification. CONCLUSION: The results provide a comprehensive insight into functional outcome 12 months after RPE and can be taken into account when informing patients.

Radiomics and Clinicopathological Characteristics for Predicting Lymph Node Metastasis in Testicular Cancer.

Accurate prediction of lymph node metastasis (LNM) in patients with testicular cancer is highly relevant for treatment decision-making and prognostic evaluation. Our study aimed to develop and validate clinical radiomics models for individual preoperative prediction of LNM in patients with testicular cancer. We enrolled 91 patients with clinicopathologically confirmed early-stage testicular cancer, with disease confined to the testes. We included five significant clinical risk factors (age, preoperative serum tumour markers AFP and B-HCG, histotype and BMI) to build the clinical model. After segmenting 273 retroperitoneal lymph nodes, we then combined the clinical risk factors and lymph node radiomics features to establish combined predictive models using Random Forest (RF), Light Gradient Boosting Machine (LGBM), Support Vector Machine Classifier (SVC), and K-Nearest Neighbours (KNN). Model performance was assessed by the area under the receiver operating characteristic (ROC) curve (AUC). Finally, the decision curve analysis (DCA) was used to evaluate the clinical usefulness. The Random Forest combined clinical lymph node radiomics model with the highest AUC of 0.95 (±0.03 SD; 95% CI) was considered the candidate model with decision curve analysis, demonstrating its usefulness for preoperative prediction in the clinical setting. Our study has identified reliable and predictive machine learning techniques for predicting lymph node metastasis in early-stage testicular cancer. Identifying the most effective machine learning approaches for predictive analysis based on radiomics integrating clinical risk factors can expand the applicability of radiomics in precision oncology and cancer treatment.

PD-L1 (CD274) promoter hypomethylation predicts immunotherapy response in metastatic urothelial carcinoma.

PD-L1 status assessed by immunohistochemistry (IHC) has failed to reliably predict outcomes for patients with metastatic urothelial carcinoma (mUC) on immune checkpoint blockade (ICB). PD-L1 promoter methylation is an epigenetic mechanism that has been shown to regulate PD-L1 mRNA expression in various malignancies. The aim of our present study was to evaluate the predictive potential of PD-L1 promoter methylation status (mPD-L1) in ICB-treated mUC compared to conventional IHC-based PD-L1 assessment. We quantified mPD-L1 in formalin-fixed and paraffin-embedded tissue sections using an established quantitative methylation-specific PCR assay (qMSP) in a well-characterized multicenter ICB-treated cohort comprising N = 107 patients with mUC. Additionally, PD-L1 protein expression in tumor tissues was assessed using regulatory approved IHC protocols. The effect of pharmacological hypomethylation by the DNA methyltransferase inhibitor decitabine in combination with interferon-γ stimulation in urothelial carcinoma cell lines was investigated by IHC and FACS. mPD-L1 hypomethylation predicted objective response rate at the first staging on ICB. Patients with tumors categorized as PD-L1 hypomethylated (lower quartile) showed significantly prolonged progression-free (PFS) and overall survival (OS) after ICB initiation. In contrast, PD-L1 protein expression status neither correlated with response nor survival. In multivariable Cox regression analyses, PD-L1 promoter hypermethylation remained an independent predictor of unfavorable PFS and OS. In urothelial carcinoma cell lines, pharmacological demethylation led to an upregulation of membranous PD-L1 expression and an enhanced inducibility of PD-L1 expression by interferon γ. Hypomethylation of the PD-L1 promoter is a promising predictive biomarker for response to ICB in patients with mUC.

[Opportunities for prostate-specific membrane antigen hybrid imaging in prostate cancer]. / Chancen der PSMA(prostataspezifisches Membranantigen)-Hybridbildgebung beim Prostatakarzinom.

Prostate-specific membrane antigen (PSMA) hybrid imaging is a promising new technique gaining importance in the field of prostate cancer (PCa) diagnosis and treatment planning. By combining PSMA radioligands and computed tomography (CT) or magnetic resonance imaging (MRI), PSMA hybrid imaging opens up new diagnostic opportunities. PSMA-PET/CT (PET: positron-emission tomography) is already well established in high-risk PCa for primary staging and tumor localization when biochemical recurrence occurs. Further potential indications for PSMA-PET/CT include tumor detection in the initial work-up before a rebiopsy with improved accuracy, the identification of target structures for precise local treatment in recurrent PCa (salvage radiotherapy or radio-guided surgery) as well as a prediction of response to PSMA radioligand therapy. This narrative review is based on a recent literature search and aims to highlight the opportunities of PSMA imaging in different disease stages of PCa.

Xpert bladder cancer monitor to predict the need for a second TURB (MoniTURB trial).

To determine whether Xpert bladder cancer monitor, a noninvasive PCR-based biomarker test, can predict the need for 2nd transurethral resection of the bladder (TURB) better than clinical assessment. Patients scheduled for TURB were prospectively screened. After initial TURB, patients were assigned to 2nd TURB or follow-up cystoscopy at 3 months (FU) by clinicians' discretion. Central urine cytology and Xpert monitor tests were performed prior to the 1st TURB and 2nd TURB or FU, respectively. Statistical analysis to compare clinical assessment and Xpert monitor comprised sensitivity (SENS), specificity (SPEC), NPV and PPV. Of 756 screened patients, 171 were included (114 with 2nd TURB, 57 with FU). Residual tumors were detected in 34 patients who underwent 2nd TURB, and recurrent tumors were detected in 2 patients with FU. SENS and SPEC of Xpert monitor were 83.3% and 53.0%, respectively, PPV was 32.6% and NPV was 92.1%. Clinical risk assessment outperformed Xpert monitor. In patients with pTa disease at initial TURB, Xpert monitor revealed a NPV of 96%. Xpert monitor was not superior than clinical assessment in predicting the need for 2nd TURB. It might be an option to omit 2nd TURB for selected patients with pTa disease.

Loss of ORP3 induces aneuploidy and promotes bladder cancer cell invasion through deregulated microtubule and actin dynamics.

We have recently shown that loss of ORP3 leads to aneuploidy induction and promotes tumor formation. However, the specific mechanisms by which ORP3 contributes to ploidy-control and cancer initiation and progression is still unknown. Here, we report that ORP3 is highly expressed in ureter and bladder epithelium while its expression is downregulated in invasive bladder cancer cell lines and during tumor progression, both in human and in mouse bladder cancer. Moreover, we observed an increase in the incidence of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced invasive bladder carcinoma in the tissue-specific Orp3 knockout mice. Experimental data demonstrate that ORP3 protein interacts with γ-tubulin at the centrosomes and with components of actin cytoskeleton. Altering the expression of ORP3 induces aneuploidy and genomic instability in telomerase-immortalized urothelial cells with a stable karyotype and influences the migration and invasive capacity of bladder cancer cell lines. These findings demonstrate a crucial role of ORP3 in ploidy-control and indicate that ORP3 is a bona fide tumor suppressor protein. Of note, the presented data indicate that ORP3 affects both cell invasion and migration as well as genome stability through interactions with cytoskeletal components, providing a molecular link between aneuploidy and cell invasion and migration, two crucial characteristics of metastatic cells.

Supporting SURgery with GEriatric Co-Management and AI (SURGE-Ahead): A study protocol for the development of a digital geriatrician.

INTRODUCTION: Geriatric co-management is known to improve treatment of older adults in various clinical settings, however, widespread application of the concept is limited due to restricted resources. Digitalization may offer options to overcome these shortages by providing structured, relevant information and decision support tools for medical professionals. We present the SURGE-Ahead project (Supporting SURgery with GEriatric co-management and Artificial Intelligence) addressing this challenge. METHODS: A digital application with a dashboard-style user interface will be developed, displaying 1) evidence-based recommendations for geriatric co-management and 2) artificial intelligence-enhanced suggestions for continuity of care (COC) decisions. The development and implementation of the SURGE-Ahead application (SAA) will follow the Medical research council framework for complex medical interventions. In the development phase a minimum geriatric data set (MGDS) will be defined that combines parametrized information from the hospital information system with a concise assessment battery and sensor data. Two literature reviews will be conducted to create an evidence base for co-management and COC suggestions that will be used to display guideline-compliant recommendations. Principles of machine learning will be used for further data processing and COC proposals for the postoperative course. In an observational and AI-development study, data will be collected in three surgical departments of a University Hospital (trauma surgery, general and visceral surgery, urology) for AI-training, feasibility testing of the MGDS and identification of co-management needs. Usability will be tested in a workshop with potential users. During a subsequent project phase, the SAA will be tested and evaluated in clinical routine, allowing its further improvement through an iterative process. DISCUSSION: The outline offers insights into a novel and comprehensive project that combines geriatric co-management with digital support tools to improve inpatient surgical care and continuity of care of older adults. TRIAL REGISTRATION: German clinical trials registry (Deutsches Register für klinische Studien, DRKS00030684), registered on 21st November 2022.

Personalized [177Lu]Lutetium-PSMA Therapy for Patients with Pre-Treated Castration-Resistant Prostate Cancer: A Single Institution Experience from a Comprehensive Cancer Centre.

Castration resistant prostate cancer (CRPC) is characterized by an aggressive biological behavior with a relatively short survival time, especially in progressive tumors pretreated with new hormonal agents and taxane chemotherapy. [177Lu]-Lutetium-PSMA (Lu-PSMA) treatment has proven efficacy in these patients. However, around 30% of the CRPC patients do not benefit from Lu-PSMA treatment, and little is known about predictive factors for treatment success if Lu-PSMA is offered in an individualized approach based on clinical and laboratory features. In this monocentric retrospective study, 86 CRPC patients receiving Lu-PSMA treatment were evaluated. The focus of the study was to describe clinical factors at baseline and during early treatment that are related to overall survival (OS). In addition, PSMA PET/CT-, PSA-response, and safety and tolerability (CTCAE adverse event reporting) were assessed. Efficacy endpoints were calculated using stratified Kaplan-Meier methods and Cox regression models. Mean applied dose was 17.7 GBq (mean 5.3 ± 1.1 GBq per cycle) with an average of 3.6 (range 1-8) therapy cycles. Patients were followed up for a mean of 12.4 months (range 1-39). The median OS was 15 months (95% CI 12.8-17.2). The best overall response rate in patients assessed with PSMA PET/CT and PSA response was 27.9%, and 50.0% had at least stable disease. Nine patients had a ≥grade 3 adverse event with anemia being the most frequent adverse event. Positive predictors for prolonged OS from baseline parameters were pre-treatment hemoglobin level of ≥10 g/dL and a lower PSA values at treatment start, while the presence of visceral or liver metastases were not significantly associated with worse prognoses in this cohort. With careful patient selection, an individualized Lu-PSMA treatment approach is feasible and patients with dose-limiting factors or visceral metastases should be included in prospective trials.

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.

TBX3 is dynamically expressed in pancreatic organogenesis and fine-tunes regeneration.

BACKGROUND: The reactivation of genetic programs from early development is a common mechanism for injury-induced organ regeneration. T-box 3 (TBX3) is a member of the T-box family of transcription factors previously shown to regulate pluripotency and subsequent lineage commitment in a number of tissues, including limb and lung. TBX3 is also involved in lung and heart organogenesis. Here, we provide a comprehensive and thorough characterization of TBX3 and its role during pancreatic organogenesis and regeneration. RESULTS: We interrogated the level and cell specificity of TBX3 in the developing and adult pancreas at mRNA and protein levels at multiple developmental stages in mouse and human pancreas. We employed conditional mutagenesis to determine its role in murine pancreatic development and in regeneration after the induction of acute pancreatitis. We found that Tbx3 is dynamically expressed in the pancreatic mesenchyme and epithelium. While Tbx3 is expressed in the developing pancreas, its absence is likely compensated by other factors after ablation from either the mesenchymal or epithelial compartments. In an adult model of acute pancreatitis, we found that a lack of Tbx3 resulted in increased proliferation and fibrosis as well as an enhanced inflammatory gene programs, indicating that Tbx3 has a role in tissue homeostasis and regeneration. CONCLUSIONS: TBX3 demonstrates dynamic expression patterns in the pancreas. Although TBX3 is dispensable for proper pancreatic development, its absence leads to altered organ regeneration after induction of acute pancreatitis.

Increasing Biomarker Guidance in the Treatment of Urothelial Carcinoma: Systematic Review of International Clinical Trials.

PURPOSE: Precision oncology requires biomarker testing from tumor tissue for clinical decision-making and selection of targeted therapies. We systematically evaluated the role of tissue biomarker testing within interventional clinical trials for locally advanced and metastatic urothelial carcinoma (UC). METHODS: A systematic search within the publicly available ClinicalTrials.gov database was performed for the period 1995 to January 2020. We searched for all interventional studies on systemic treatments for advanced UC. Two investigators independently screened the records and extracted the data for statistical analyses. RESULTS: We included 356 studies out of 827 initial records in the final analysis. The overall number of interventional trials in UC patients significantly increased during the past 25 years. Forty-three studies (12.1%) required specific biomarker testing as a prerequisite for inclusion. Of the remaining 313 trials, explorative biomarkers of interest were studied in 83 studies (23.3%). In trials with obligate biomarker testing as a precondition for study inclusion, only 3 studies (7%) required an actual fresh pretreatment biopsy, while the majority of studies did not state any tissue requirements (55.8%) or accepted archival tissue samples (37.3%). Among studies without biomarker prerequisites, freshly obtained tissue samples were required in 16.3% of studies evaluating immune checkpoint inhibition and 5.7% evaluating targeted therapy. The collection of archival tissue was allowed in 67.4% and 20% of studies evaluating immune checkpoint inhibitors and targeted therapies, respectively. CONCLUSION: There has been an increase in the number of studies using biomarker-guided interventions for the treatment of advanced UC over the past 25 years. Studies investigating druggable targets in actual UC biopsies immediately before treatment are still rare. Standardized criteria for tissue-based biomarker testing may further accelerate personalized treatment of patients with advanced UC.