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BACKGROUND: The functional status of immune cells in the tumor microenvironment and tumor characteristics may explain bacillus Calmette-Guérin (BCG) failure in high-risk non-muscle-invasive bladder cancer (NMIBC).OBJECTIVE: To characterize molecular correlates of post-BCG high-grade (HG) recurrence using multiomics analysis.DESIGN, SETTING, AND PARTICIPANTS: Patients with BCG-treated NMIBC (n = 156) were included in the study. Metachronous tumors were analyzed using RNA sequencing (n = 170) and whole-exome sequencing (n = 195). Urine samples were analyzed for immuno-oncology-related proteins (n = 190) and tumor-derived DNA (tdDNA; n = 187).OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary endpoint was post-BCG HG recurrence. Cox regression and Wilcoxon rank-sum, t, and Fisher's exact tests were used for analyses.RESULTS AND LIMITATIONS: BCG induced activation of the immune system regardless of clinical response; however, immunoinhibitory proteins were observed in the urine of patients with post-BCG HG recurrence (CD70, PD1, CD5). Post-BCG HG recurrence was associated with post-BCG T-cell exhaustion (p = 0.002). Pre-BCG tumors from patients with post-BCG T-cell exhaustion had high expression of genes related to cell division and immune function. A high predicted post-BCG exhaustion score for pre-BCG tumors was associated with worse post-BCG HG recurrence-free survival (HGRFS; p = 0.002). This was validated in independent cohorts. Pre-BCG class 2a and 2b tumors (UROMOL2021 scheme) were associated with worse post-BCG HGRFS (p = 0.015). Post-BCG exhaustion was observed in patients with high pre-BCG neoantigen load (p = 0.017) and MUC4 mutations (p = 0.002). Finally, the absence of post-BCG tdDNA clearance identified patients at high risk of recurrence (p = 0.018). The retrospective design and partial overlap for analyses are study limitations.CONCLUSIONS: Post-BCG HG recurrence may be caused by T-cell exhaustion. Tumor subtype and pre-BCG tumor characteristics may identify patients at high risk of post-BCG HG recurrence. Urinary measurements have potential for real-time assessment of treatment response.PATIENT SUMMARY: A dysfunctional immune response to bacillus Calmette-Guérin (BCG) therapy may explain high-grade recurrences of bladder cancer.

Supplementary Figure 1. Mutation rates by cohort. Supplementary Figure 2. ERCC2 mutations. Supplementary Figure 3. ERCC2 mutation status and overall survival. Supplementary Figure 4. ERCC2 mutations occur in highly conserved regions.

Significantly altered genes in clinical subsets. MutSigCV ranked lists for significantly mutated genes in the non-responders only (n = 25), the responders only (n = 25), and the ERCC2WT responders only (n = 16).

All alterations in responders and non-responders. All somatic mutations and short insertion/deletions are listed, with additional annotations regarding genomic coordinates, protein change, allelic fractions, and validation status (if available).

Damaging scores for alterations. This table shows the damaging scores (Methods) for nonsynonymous alterations (missense, nonsense, splice site, frame shift) studied in this cohort. The damaging score was derived as follows: missense mutations were scored using the Polyphen2 score(41) for the amino acid substitution. Nonsense mutations, splice site mutations, and short insertion/deletions were automatically assigned a damaging score of 1. "Unavailable" damaging scores are those that are missense mutations without a PolyPhen2 score.

Treatment information and sequencing metrics for all patients The treatment information and raw sequencing metrics, including mean target coverage, for each case is listed here. In addition, the mutation rate data referred to in the Results and represented in Supplementary Figure 1 is listed here by synonymous rate, non-synonymous rate, and total number of mutations per patient. *Cases that were included in the TCGA effort (UC-0296 and UC-0234). (dd = dose dense, GC = gemcitabine/cisplatin, MVAC = methotrexate, vinblastine, doxorubicin, cisplatin). Unavailable cases for Fluidigm Validation (due to inadequate remaining DNA or technical failure) are also denoted (n = 15).

Selective alteration enrichment. This table shows the results of applying Fisher's Exact Test to a tabulated set of predicted damaging somatically altered genes in responders compared to non-responders. A Benjamini-Hochberg/FDR q-value is reported for genes with the minimum number of events necessary to be considered for a theoretically significant p-value of {less than or equal to} 0.01 (n = 6). The results are sorted by p-value (lowest to highest).

Aim To evaluate whether surgical trainees undergoing additional simulation, compared to conventional training, are able to achieve proficiency sooner with better patient outcomes. Methods This international, multicentre randomised controlled superiority trial recruited urology trainees (n=94) who had performed ≤10 ureterorenoscopy (URS) cases, as a selected index procedure, with no prior simulation experience. Recruits were randomised to simulation-based training or non-simulation-based training groups, the latter of which is the current standard of training. Training sessions were conducted for the simulation arm, utilising an expert-developed multi-modality training curriculum. The primary outcome was the number of procedures required to achieve proficiency, defined as achieving a score of ≥28 on an OSATS scale, on 3 consecutive operations, without complications. Inpatient surgical complications were also recorded. All participants were followed up for 25 procedures or over 18 months. Results A total of 1140 cases were performed by 65 participants where proficiency was achieved in 21 simulation and 18 conventional participants over a median of 8 and 9 procedures, respectively (HR: 1.41 [95% CI 0.72–2.75]). More participants reached proficiency in the simulation arm in flexible ureterorenoscopy, requiring fewer number of procedures (HR 0.89 [95% CI 0.39–2.02]). Significant differences were observed in overall comparison of OSATS scores between groups (mean difference 1.42 [95% CI 0.91–1.92]; p Conclusions Simulation-based training demonstrated higher overall proficiency and fewer procedures were required to achieve proficiency in the complex form of the index procedure with surgical complications.

To evaluate the perioperative and oncological/functional outcomes of robotic post-chemotherapy retroperitoneal lymph node dissection for testicular cancer.In this retrospective study, we included patients who underwent robotic post-chemotherapy retroperitoneal lymph node dissection at 7 academic centers between 2011 and 2021. Patients' characteristics, perioperative findings, as well as oncological and functional outcomes are reviewed. Relationships with the main outcome (90-day complications) were analyzed using multivariable logistic regression.A total of 90 patients with a median (IQR) age of 30 (25-37) years were included. The main primary histologic type was non-seminomatous germ cell tumor (89%). Seven patients (8%) were electively converted to open. Median estimated blood loss, operative time, and length of hospital stay were 150 ml, 5.6 hours, and 2 days, respectively. Final pathology revealed teratoma in 49 (55%), necrosis/fibrosis in 29 (32%), and viable germ cell tumor in 12 (13%) patients. The 90-day complication rate was 16.7%, most of which were low-grade (Clavien-DindoIII) and managed conservatively. On multivariable analysis, pure seminoma (odds ratio 17.4) and bilateral dissection template (odds ratio 4.2) were independently associated with 90-day complications. No 90-day hospital readmission was recorded. With a median (IQR) follow-up of 16 (4-32) months, 6 (6.7%) patients had disease recurrence and there was 1 cancer-related death.With appropriate patient selection at centers with expertise in testicular cancer and minimally invasive surgery, robotic post-chemotherapy retroperitoneal lymph node dissection appears safe and effective, although longer follow-up is warranted.

Introduction: While the prognostic value of immune system biomarkers has been well explored, role of cancer associated macrophages in bladder cancer(BC) is unclear due to lack of consistent results, coupled with tissue nonspecific transcriptomic signature. Methodology: Data acquisition from 3,936 patients of The Cancer Genome Atlas (TCGA) in addition to Gene Expression Omninus (GEO) data sets GSE13507, GSE16945, GSE48277, GSE32894, GSE149582, as well as European Genotype Phenotype EGAS000001004507 was obtained. Tumor associated macrophages M1 and M2 were defined utilizing 188 and 159 gene expression profiles via xCell computational algorithm. To obtain bladder tissue residence macrophage signatures, scRNA seq was performed on tissue collected from surgical resection of 5 HG bladder cancer patients and 2 healthy controls. CellRanger software package with default parameters was utilized giving a total of 8,068 cells. FindAllMarkers was utilized for integration of DEG genes (cancer vs control) among the cluster and identification of BC macrophage specific markers. Endpoint of OS were measured in months from the time of cystectomy to follow up. Results: Surprisingly, presence of M1 infiltration was found to be associated with improved OS in two out of eight cohorts only (GSE32894, GSE70691, p Conclusion: Our study represents the largest TAMs evaluation of BC across 8 cohorts with additional scRNA seq exploration of tissue specific signatures, demonstrating no association with OS in bladder cancer. Citation Format: Laura Bukavina, Spencer Bell, Daniel Geynisman, Ilaha Isali, Daniel Ranti, John Sfakianos, Henkel Valentine, Adam Calaway, Alexander Kutikov, Andres Correa, Robert Uzzo, Lee Ponsky, Philip Abbosh. Macrophage transcriptomic signature validation in scRNA seq and overall survival differences in urothelial carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5141.

Window-of-opportunity trials, during which patients receive short-duration pre-surgical therapies, provide a platform for understanding the therapies’ mechanisms of action, but will require a paradigm shift in trial design, specimen collection and analysis.

BackgroundThe functional status of immune cells within the tumor microenvironment and tumor characteristics may explain Bacillus Calmette-Guérin (BCG)-failure in high-risk non-muscle invasive bladder cancer (NMIBC).ObjectiveTo characterize molecular correlates of BCG-failure using a multiomics approach.Design, Setting, and ParticipantsBCG-treated NMIBC patients (n=156) were included. Metachronous tumors were analyzed using RNA-sequencing (n=170) and whole exome sequencing (n=198). Urine samples were analyzed for immune-oncology related proteins (n=190), and tumor-derived DNA (tdDNA; n=192).Outcome Measurement and Statistical AnalysisPrimary endpoint was BCG-failure. Cox regression, Wilcoxon Rank Sum test, t-test or Fisher’s exact test were used.Results and LimitationsBCG caused activation of the immune system regardless of clinical response; however, immune-inhibitory proteins were observed in the urine of BCG-unresponsive patients post-treatment (CD70, PD1, CD5). BCG-failure was associated with post-BCG T-cell exhaustion (p=0.0021). Pre-BCG tumors from patients with post-BCG T-cell exhaustion were characterized by high expression of cell division and immune-related genes. A high post-BCG exhaustion prediction score in pre-BCG tumors was associated with worse post-BCG high-grade recurrence free survival (HGRFS), reflecting BCG-failure (p=0.0084). Pre-BCG tumors of class 2a and 2b were likewise associated with worse post-BCG HGRFS(p=0.0023). Post-BCG exhaustion was observed in patients with high pre-BCG neoantigen load (p=0.023) and mutations in MUC4 (p=0.0007). Finally, absence of post-BCG tdDNA clearance identified patients at high risk of recurrence (p=0.028). The retrospective design, lack of maintenance BCG, and partial overlap in analyses are limitations to the study.ConclusionsBCG failure may be caused by T-cell exhaustion. Tumor subtype and Pre-BCG tumor characteristics may identify patients at high risk of BCG-failure prior to treatment. Urinary measurements have the potential to be used as a real-time assessment of treatment response.Patient SummaryA dysfunctional immune response to BCG therapy may explain lack of response to the treatment.

Take Home Message Pelvic organ-preserving robot-assisted radical cystectomy with orthotopic neobladder or ileal conduit allows return to sexual activity after surgery while not compromising oncological outcome. This approach should be considered in sexually active female undergoing cystectomy., Background For females undergoing cystectomy and urinary diversion, decreases in sexual and urinary functions can have a significant impact on quality of life. Pelvic organ–preserving (POP) radical cystectomy (RC) has been proposed as an approach to improve postoperative functional outcomes. Objective To evaluate postoperative functional outcomes of a robotic approach for female POP RC with intracorporeal urinary diversion. Design, setting, and participants This was a multicenter retrospective study evaluating sexual, urinary, and oncological outcomes for sexually active females undergoing POP robot-assisted RC for ≤T2 bladder cancer. Exclusion criteria included multifocal, trigonal, or locally advanced tumors. Surgical procedure We describe a step-by-step technique for POP robot-assisted RC with intracorporeal urinary diversion. Measurements The primary outcome of the study was evaluation of sexual and urinary functions following surgery. Oncological outcomes were evaluated as a secondary endpoint. Results and limitations Our study included 23 females who underwent POP robot-assisted RC between 2008 and 2020 with intracorporeal neobladder (87%) or ileal conduit (13%) reconstruction. The median follow-up was 20 mo. A postoperative sexual function questionnaire was completed by 15 patients (65%). Of those, 13 (87%) resumed sexual activity at a median of 6 mo after surgery. Of the patients with a neobladder, 14 (70%) achieved daytime continence and 16 (80%) achieved nighttime continence. Cancer-specific and overall survival were both 91%. The results are limited by their retrospective nature. Conclusions POP robot-assisted RC with orthotopic neobladder allows a majority of female patients to return to sexual activity after surgery. This approach should be considered for selected sexually active women. Patient summary We evaluated 23 women with bladder cancer who underwent surgical removal of the bladder with preservation of their reproductive organs. Following this surgery, a majority of patients resumed sexual activity. For selected patients, this technique can be performed without compromising cancer control.

In the last two decades, surgical techniques for intracorporeal urinary diversion have been developed with the aim of reducing surgical morbidity. Although increasing constantly, the numbers of urologists offering intracorporeal neobladder reconstruction remain limited due to the complex nature of the procedure. In this article, we aim to provide a detailed description of the surgical technique we currently use at our institution. This technique was initially developed and perfected at the Karolinska Institutet in Sweden starting in 2003. It is a reproducible surgical approach with standardized and well-defined surgical steps. We give a detailed description of the surgical steps and provide tips and tricks to address specific situations and to increase efficiency. We also review the indications, the preoperative considerations, equipment necessary, postoperative considerations, and clinical outcomes for this procedure. Finally, we provide an accompanying didactic surgical video. We believe that this standardized approach can be learned and reproduced safely by motivated robotic surgeons.

Background Although PD-1/PD-L1 immune checkpoint blockade (ICB) therapy has achieved durable clinical responses in a subset of bladder cancer patients (15–25%), the majority do not respond. This has led to a need to identify clinically predictive biomarkers. Urine is an accessible material that may reflect cellular and/or genetic signatures related to ICB response. It has been shown that bladder cancer patient urine contains immune cells in addition to tumor cells. To study the concordance between urinary immune cells and the tumor immune microenvironment (TIME), we un-biasedly characterized bladder cancer patient urine and compared it to tumor and peripheral blood mononuclear cells (PBMC). Methods Matched tumor, urine, and PBMC from 8 bladder cancer patients were dissociated for single cell RNA sequencing (scRNAseq) and Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) using 10X Genomics. Results scRNAseq of bladder cancer patient urine revealed CD4+ and CD8+ T, T regulatory (Treg), natural killer (NK), and B cells as well as neutrophils, dendritic cells, monocytes, and macrophages. The composition and transcriptional profiles of these cells were more similar to the tumor immune cells than to PBMC. Urine immune cells expressed hypoxia, anergy, and pro-inflammatory gene signatures that were more similar to tumor immune cells than PBMC. Conclusions Our work represents the first scRNAseq and CITE-seq of cancer patient urine. Our study shows several immune cells shed in bladder cancer patient urine and suggests they look phenotypically similar to the TIME. This has implications for future clinical applications as urine can be sampled non-invasively in scenarios when tumor resection may not be feasible. Supported by the NIH grant: R01 CA249175-01.

INTRODUCTION AND OBJECTIVES:The aim of this study is to compare the long-term renal function outcome after robotic partial nephrectomy in completely endophytic tumors, partially endophytic tumors, ...

Natural Killer cell (NK) effector functions are diminished during cancer but the phenotype and mediators of this process are unclear. In contrast, dysfunctional T cells express markers of “exhaustion” (e.g. CTLA-4 and PD-1), and inhibition of signaling through these molecules has improved clinical outcome in multiple tumors. Our data from 100+ patients with bladder cancer (BC) suggests that NK undergo an analogous process of exhaustion marked by up-regulation of Tim-3 and TIGIT in both the periphery and tumor. Tim-3 and TIGIT are induced on both immature and mature NK subsets suggesting implications for normal NK maturation. The magnitude of Tim-3 expression in both tissues is tuned to the invasiveness of the primary tumor, while TIGIT is not, making Tim-3 function as a rheostat for primary tumor invasiveness. Both molecules are expressed at equivalent frequencies in both tissues independent of the magnitude of overall expression but define NK with different effector functions. Peripheral NK from BC patients are functionally comparable to NK from healthy donors in their ability to produce IFNγ and degranulate in response to target cells, while tumor NK are refractory to both stimuli. NK from tumors are not terminally exhausted as they make IFNγ, TNFa, and degranulate after “resting” ex vivo prior to stimulation. Blockade of Tim-3, but not TIGIT, enhances effector function in peripheral NK from BC patients, but is ineffective for NK in tumor tissue implicating tumor-specific factors in mediating NK dysfunction. Tim-3 blockade was most efficient in NK from BC patients after activation with IL-15 suggesting specific inflammatory cues affect responsiveness to checkpoint blockade. Ongoing work will develop strategies to reverse NK exhaustion.

Background: Urothelial bladder cancer kills over 16,000 people annually. Approximately 30% of affected patients have cancer cells invading the muscularis propria at the time of diagnosis. Standard management for muscle-invasive bladder cancer (MIBC) patients involves radical cystectomy and pelvic lymph node dissection. Approximately 50% of these patients will develop fatal metastatic recurrence. In an attempt to eradicate micrometastatic disease, neoadjuvant chemotherapy (NAC) was integrated into treatment. Even though studies show that this improves these patients’ prognosis, population-based studies have demonstrated that NAC is still underutilized. The difficulty of predicting an individual patient’s outcome with cystectomy alone and the potential added benefit with NAC was cited as a common reason for this. Objective: The aim of this study is to develop a web-based app for MIBC patients treated with cystectomy, with or without NAC, designed to improve prediction and enhance communication of these patients’ prognosis. Methods: This study included patients from the National Cancer Database (2003 through 2011) who were diagnosed with MIBC and were subsequently treated with cystectomy. Patient, tumor, and facility-level predictors were incorporated in the outcome prediction model and a state-transition model was synthesized to calculate the 5-year death risk with and without NAC. Internal and external cross-validations were performed to validate the predictions. Using U.S. Life Tables, bladder cancer-specific and other cause mortality were distinguished from all cause mortality rates. The effect of NAC was integrated using a literature-derived hazard ratio (HR). Finally, a web-based tool was developed using the state transition model and usability testing was performed. Results: A total of 9,824 patients who had MIBC and underwent cystectomy met the eligibility criteria and were included in the prediction model (Figure 1). Factors such as race, advanced age, higher clinical T stage, and higher comorbidity index were associated with shorter survival. On the other hand, factors like private insurance, higher income, and undergoing cystectomy at a higher volume facility were associated with longer survival. The prediction model was well-calibrated across geographical regions. Individualized survival estimates of each patient can be generated using the web-based app (BladderCancerRisk.org) by feeding in the predictor variables and a user-defined HR associated with the effect of NAC. The output of the tool is displayed using infographics (Figures 2 and 3). A cohort consisting of 13 clinicians field-tested the usability of the tool. Conclusions: A web-based user-friendly app was developed for patients with MIBC treated with cystectomy, with or without NAC, which individualizes outcome prediction and communication in these patients, and may also facilitate physician-patient shared decision-making. This app can be easily accessed or prescribed by the physicians using the Rx Universe platform (a digital platform that enables physicians to directly “prescribe” evidence-based mobile health applications to patients). [iproc 2017;3(1):e43]