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The Cancer Genome Atlas (TCGA) for bladder cancer was published in 2014 with updated annotation of over 400 patients with muscle-invasive bladder cancer (MIBC) in 2017. This tremendous work established the foundation of the genomic landscape of MIBC. The next steps to utilize information from The Cancer Genome Atlas is to (1) identify the causes of mutation, (2) determine the significant differences and sources of heterogeneity, and (3) apply these tools toward patient care. In this review, we discuss the full spectrum of the genomic landscape of MIBC toward the goal of therapeutic application.

Figure S6 - Figure.S6 shows combination of rapamycin and BCG increase percentage of effector memory γδ T cells and production of cytokine such as IFNγ within γδ T cells.

Figure.S1 shows that irradiated bladder tumor cells can stimulate in vitro proliferation and cytokine production in autologous peripheral T cells from patients.

Reagent information for flow cytometry and ELISPOT assay.

Figure.S4 shows that BCG treatment decreases PD-1 expression on mouse bladder tumor antigen-specific CD8+ T cell infiltrates.

Figure S7 - Figure.S7 shows combination of rapamycin and BCG increase absolute number of human effector memory γδ T cells expanded in vitro and reduce the growth of patient-derived xenograft bladder cancer cells in vivo.

Figure.S2 shows that protective immunity against MB49 tumor in mice can not prevent the growth of B16 melanoma tumor.

Figure S5 - Figure.S5 shows that rapamycin promotes BCG treatment efficacy in mouse bladder cancer in vivo model.

Legends for all 7 supplementary figures

Figure S3 - Figure.S3 shows an increase of γδ T cells in tumor-draining lymph nodes in mouse bearing subcutaneous MB49 tumors and depleted of conventional αβ T cells.

Bacillus Calmette-Guérin (BCG) is the most effective intravesical agent at reducing recurrence for patients with high-grade, non–muscle-invasive bladder cancer. Nevertheless, response to BCG is variable and strategies to boost BCG efficacy have not materialized. Prior work demonstrated a requirement for either conventional αβ or nonconventional γδ T cells in mediating BCG treatment efficacy, yet the importance of T-cell antigen specificity for BCG's treatment effect is unclear. Here, we provide direct evidence to show that BCG increases the number of tumor antigen–specific αβ T cells in patients with bladder cancer and protects mice from subsequent same-tumor challenge, supporting BCG induction of tumor-specific memory and protection. Adoptive T-cell transfers of antigen-specific αβ T cells into immunodeficient mice challenged with syngeneic MB49 bladder tumors showed that both tumor and BCG antigen–specific αβ T cells contributed to BCG efficacy. BCG-specific antitumor immunity, however, also required nonconventional γδ T cells. Prior work shows that the mTOR inhibitor rapamycin induces the proliferation and effector function of γδ T cells. Here, rapamycin increased BCG efficacy against both mouse and human bladder cancer in vivo in a γδ T cell–dependent manner. Thus, γδ T cells augment antitumor adaptive immune effects of BCG and support rapamycin as a promising approach to boost BCG efficacy in the treatment of non–muscle-invasive bladder cancer.

Purpose:To characterize immune cell expression among patients with non–muscle invasive bladder cancer (NMIBC) treated with Bacillus Calmette-Guerin (BCG).Experimental Design:Patients with NMIBC treated with intravesical BCG (2008–2015) were identified, and a tissue microarray was constructed using paired pre- and post-BCG bladder samples. Among patients undergoing BCG, cystoscopic evaluation began 3 months after initiating BCG treatment to determine therapeutic response. IHC was performed for CD8, CD4, FoxP3, PD-L1 (SP-142 and 22C3), and PD-1. A full slide review of PD-L1+ staining tumors was performed to characterize PD-L1 and CD8 colocalization. RNA-seq was performed on cored tumors from available specimens. We compared immune cell populations between BCG responders and nonresponders, and between pretreatment and postreatment tumor samples. Baseline PD-L1 staining in the BCG naïve population was then validated in a separate cohort.Results:The final cohort contained 63 pretreatment NMIBC cases, including 31 BCG responders and 32 BCG nonresponders. No differences in CD4, CD8, or FoxP3 expression were identified between responders and nonresponders. Baseline PD-L1 expression (22C3 and SP-142) was observed in 25% to 28% of nonresponders and 0% to 4% of responders (P < 0.01). PD-L1+ cells in BCG nonresponders colocalized with CD8+ T cells. In addition, BCG therapy did not increase PD-L1 gene expression (RNA-seq) or protein levels (IHC). The number of pretreatment CD4+ T cells was very low among PD-L1+ nonresponders (12%) and high among PD-L1− nonresponders (50%, P < 0.01). In a separate cohort of 57 patients with NMIBC undergoing BCG, baseline PD-L1 (22C3) staining was similar (26%).Conclusions:One mechanism of BCG failure may be adaptive immune resistance. Baseline tumor PD-L1 expression predicts an unfavorable response to BCG and if validated, could be used to guide therapeutic decisions.

PURPOSEOptimal patient selection for neoadjuvant chemotherapy prior to surgical extirpation is limited by the inaccuracy of contemporary clinical staging methods in high-risk upper tract urothelial carcinoma (UTUC). We investigated whether the detection of plasma circulating tumor DNA (ctDNA) can predict muscle-invasive and non-organ confined (MI/NOC) UTUC.PATIENTS AND METHODSPlasma cell-free DNA was prospectively collected from chemotherapy-naïve, high-risk UTUC patients undergoing surgical extirpation and sequenced using a 152-gene panel and low-pass whole-genome sequencing. To test for concordance, whole exome sequencing was performed on matching tumor samples. The performance of ctDNA for predicting MI/NOC UTUC was summarized using area under a receiver-operating curve and the optimal variant count threshold determined using Younden’s J statistic. Kaplan-Meier methods estimated survival, and Mantel-Cox log-rank testing assessed the association between preoperative ctDNA positivity and clinical outcomes.RESULTSOf 30 patients prospectively enrolled, 14 were found to have MI/NOC UTUC. At least one ctDNA variant was detected from 21/30 (70%) patients with 52% concordance with matching tumor samples. Detection of at least two panel-based molecular alterations provided the optimal sensitivity and specificity to predict MI/NOC UTUC. Imposing this threshold in combination with a plasma copy number burden score >6.5 achieved a sensitivity of 79% and specificity of 94% in predicting MI/NOC UTUC. Furthermore, the presence of ctDNA was strongly prognostic for progression-free survival (1-yr PFS 69% vs. 100%, pCONCLUSIONThe detection of plasma ctDNA prior to extirpative surgery was highly predictive of MI/NOC UTUC and strongly prognostic of PFS and OS. Preoperative ctDNA demonstrates promise as a biomarker for selecting patients to undergo neoadjuvant chemotherapy prior to nephroureterectomy.

Bacillus Calmette-Guérin (BCG) is the most effective intravesical agent at reducing recurrence for patients with high-grade, non–muscle-invasive bladder cancer. Nevertheless, response to BCG is variable and strategies to boost BCG efficacy have not materialized. Prior work demonstrated a requirement for either conventional αβ or nonconventional γδ T cells in mediating BCG treatment efficacy, yet the importance of T-cell antigen specificity for BCG's treatment effect is unclear. Here, we provide direct evidence to show that BCG increases the number of tumor antigen–specific αβ T cells in patients with bladder cancer and protects mice from subsequent same-tumor challenge, supporting BCG induction of tumor-specific memory and protection. Adoptive T-cell transfers of antigen-specific αβ T cells into immunodeficient mice challenged with syngeneic MB49 bladder tumors showed that both tumor and BCG antigen–specific αβ T cells contributed to BCG efficacy. BCG-specific antitumor immunity, however, also required nonconventional γδ T cells. Prior work shows that the mTOR inhibitor rapamycin induces the proliferation and effector function of γδ T cells. Here, rapamycin increased BCG efficacy against both mouse and human bladder cancer in vivo in a γδ T cell–dependent manner. Thus, γδ T cells augment antitumor adaptive immune effects of BCG and support rapamycin as a promising approach to boost BCG efficacy in the treatment of non–muscle-invasive bladder cancer.

The long-term survival of patients with advanced urothelial carcinoma (UCa) is limited because of innate resistance to treatment. We identified elevated expression of the histone methyltransferase EZH2 as a hallmark of aggressive UCa and hypothesized that EZH2 inhibition, via a small-molecule catalytic inhibitor, might have antitumor effects in UCa. Here, in a carcinogen-induced mouse bladder cancer model, a reduction in tumor progression and an increase in immune infiltration upon EZH2 inhibition were observed. Treatment of mice with EZH2i causes an increase in MHC class II expression in the urothelium and can activate infiltrating T cells. Unexpectedly, we found that the lack of an intact adaptive immune system completely abolishes the antitumor effects induced by EZH2 catalytic inhibition. These findings show that immune evasion is the only important determinant for the efficacy of EZH2 catalytic inhibition treatment in a UCa model.

Cancer treatment utilizing infusion therapies to enhance the patient’s own immune response against the tumor have shown significant functionality in a small subpopulation of patients. Additionally, advances have been made in the utilization of nanotechnology for the treatment of disease. We have previously reported the potent effects of 3-4 daily intravenous infusions of immune modifying poly(lactic-co-glycolic acid) (PLGA) nanoparticles (IMPs; named ONP-302) for the amelioration of acute inflammatory diseases by targeting myeloid cells. The present studies describe a novel use for ONP-302, employing an altered dosing scheme to reprogram myeloid cells resulting in significant enhancement of tumor immunity. ONP-302 infusion decreased tumor growth via the activation of the cGAS/STING pathway within myeloid cells, and subsequently increased NK cell activation via an IL-15-dependent mechanism. Additionally, ONP-302 treatment increased PD-1/PD-L1 expression in the tumor microenvironment, thereby allowing for functionality of anti-PD-1 for treatment in the B16.F10 melanoma tumor model which is normally unresponsive to monotherapy with anti-PD-1. These findings indicate that ONP-302 allows for tumor control via reprogramming myeloid cells via activation of the STING/IL-15/NK cell mechanism, as well as increasing anti-PD-1 response rates.

BACKGROUND: There is insufficient data to recommend screening for bladder cancer (BC). For future BC screening trials, it is important to understand how and if tumor (T) stage can act as a surrogate outcome marker for overall (OS) and cancer-specific (CSS) survival. OBJECTIVE: To characterize OS and CSS between primary tumor (T) stages in non-metastatic bladder cancer (BC) patients. METHODS: Non-metastatic BC patients were identified in the National Cancer Database (NCDB; 2004-2015) (n = 343,163) and National Cancer Institute Surveillance, Epidemiology, and End Results database (SEER) (n = 130,751). Cox multivariable regression compared relationships between T stage (LGTa, HGTa, Tis, LGT1, HGT1, T2-T4) and OS or CSS for all patients and sub-cohorts. RESULTS: Compared to stage LGTa as a reference, overall (SEER; NCDB) and cancer-specific (SEER) survival significantly declined with increasing T stage. Using SEER, OS ranged from HGTa (HR 1.16, CI 1.13–1.21, p

BACKGROUND: Although bladder cancer is much more common in men than in women, female patients with bladder cancer present with more locally advanced tumors and have worse disease-specific outcomes than male patients, even after controlling for biological differences. There is a paucity of research regarding the optimal approach to caring for female patients with bladder cancer in ways that maximize patient satisfaction, preferences, and values. OBJECTIVE: We sought to explore patient-defined priorities and areas in need of improvement for female patients with bladder cancer from the patient perspective. METHODS: We conducted focus group sessions and semi-structured interviews of women treated for bladder cancer to identify patient priorities and concerns until reaching topic saturation. Transcripts were analyzed thematically. RESULTS: Eight patients with muscle-invasive bladder cancer and six patients with non-muscle-invasive bladder cancer participated in two focus groups and seven interviews total. Three themes emerged as significantly affecting the care experience: physical impacts, mental health and emotional wellbeing, and the patient-provider interaction. Each theme included patient-defined specific recommendations on approaches to optimizing the care experience for women with bladder cancer. CONCLUSIONS: Although most participants were satisfied with the quality of care they received, they identified several opportunities for improvement. These concerns centered around enhancing support for patients’ physical and mental needs and strengthening the patient-provider interaction. Efforts to address these needs and reduce gender disparate outcomes via quality improvement initiatives are ongoing.

BACKGROUND: Bladder cancers have high total mutation burdens resulting in genomic diversity and intra- and inter-tumor heterogeneity that may impact the diversity of gene expression, biologic aggressiveness, and potentially response to therapy. To compare bladder cancers among patients, an organizational structure is necessary that describes the tumor at the histologic and molecular level. These “molecular subtypes”, or “expression subtypes” of bladder cancer were originally described in 2010 and continue to evolve secondary to next generation sequencing (NGS) and an increasing public repository of well-annotated cohorts. OBJECTIVE: To review the history and methodology of expression-based subtyping of non-muscle invasive (NMIBC) and muscle invasive bladder cancer (MIBC). METHODS: A literature review was performed of primary papers from PubMed that described subtyping methods and their descriptive feature including search terms of “subtype”, and “bladder cancer”. RESULTS: 21 papers were identified for review. Tumor subtyping developed from N = 2 to N = 6 subtyping schemes with most subtypes comprised of at least luminal and basal tumors. Most NMIBCs are luminal cancers and luminal MIBCs may be associated with less aggressive features, while one study of basal tumors identified a better clinical outcome with systemic chemotherapy. Tumors with a P53-like may have intrinsic resistance to chemotherapy. The heterogeneity of tumors, which is likely derived from stromal components and immune cell infiltration, affect subtype calls. CONCLUSION: Subtyping, while still evolving, is ready for testing in clinical trials. Improved patient selection with tumor subtyping may help with tumor classification and potentially match patient or tumor to therapy.

The presence of detrusor muscle is essential for accurate staging of T1 cancers. Detrusor muscle presence can be a quality indicator of transurethral resection of bladder tumor for nonmuscle invasive bladder cancer. We hypothesized that increasing surgeon awareness of personal and institutional detrusor muscle sampling rates could improve resection quality and long-term oncologic outcomes.A retrospective review of transurethral resections of bladder tumor from 1/2006 to 2/2018 was performed. The presence of detrusor muscle in the pathology report and transurethral resection specimen were extracted from records. Individual surgeon scorecards were created and distributed. Rates of detrusor muscle sampling were compared prior to and 12 months after distribution. Chart review was done to compare 3-year recurrence and progression outcomes before and after distribution of scorecards.The rate of detrusor muscle sampling increased from 36% (1,250/3,488) to 54% (202/373) (p=0.001) in the 12 months after scorecard distribution, ie from 30% (448/1,500) to 55% (91/165) (p0.001) in Ta tumors and from 47% (183/390) to 72% (42/58) (p0.001) in T1 tumors. Pathological reporting of muscle also improved for all samples (73%, 2,530/3,488 to 90%, 334/373, p0.001), Ta (75%, 1,127/1,500 to 94%, 155/165, p0.001) and T1 (93%, 362/390 to 100%, 58/58, p=0.04). On multivariate Cox regression analysis, the surgeon scorecard was associated with decreased 3-year risk of recurrence (HR 0.63, 95% CI 0.40-0.99).Creation and distribution of individual surgeon scorecards improved detrusor muscle sampling on transurethral resection and was associated with decreased risk of disease recurrence. Quality evaluation of transurethral resection of bladder tumor may contribute to improved outcomes of patients with nonmuscle invasive bladder cancer.

506 Background: Despite response to systemic therapy, few metastatic urothelial carcinoma (mUC) patients (pts) achieve durable responses. Mutations in the COMPASS-related proteins KMT2D, KMT2C, and KDM6A are found in 66% of UC patients suggesting histone regulation may be an acquired mechanism of tumor viability. We previously found regression of UC in mice when treated with EZH2 inhibitor, tazemetostat. In vivo administration of tazemtostat enhanced the immune response with direct regulation of cytokines and antigen recognition machinery (MHCI and MHCII). We hypothesized that restoration of epigenetic imbalance, combined with immunotherapy, may improve outcomes in mUC pts. Methods: ETCTN 10183 (NCT03854474) is a pilot study evaluating the efficacy of tazemetostat 800 mg BID + pembrolizumab 200 mg every 3 weeks for cisplatin-refractory (Cohort A) or cisplatin/chemo-ineligible advanced UC (Cohort B) pts, with N=12 pts in each cohort. The primary objective is to identify the recommended phase two dose (RP2D) of tazemetostat in combination with pembrolizumab. Secondary objectives include RECIST-based response rate, safety, and progression-free survival. The maximum planned treatment duration is 2 years. Translational objectives include defining total and COMPASS-related genes tumor mutational burden, tumor subtyping, TCR clonality, T cell infiltration, and PDL-1 expression. Results: In the safety lead-in phase, 6 mUC pts were treated. There were no dose-limiting toxicities (DLTs). The RP2D for tazemetostat was established at 800 mg BID. Here we report results of fully accrued cohort A (N=12); 67% males, 33% females, 83% white, 8.3% Black, and 25% Hispanic/Latino ethnicity. The primary sites of UC were: bladder (n=9, 75%), renal pelvis (n=2, 17%), and ureter (n=1, 8.3%). The median (interquartile range, IQR) time since primary diagnosis was 1.1 years (0.7, 1.9); 11/12 patients had non-nodal metastatic disease; 10/12 had 1 or more visceral/bone metastasis. Median number of treatment cycles was 5 (IQR: 3-11; range 1-35). Median duration on protocol treatment was 12 weeks (IQR: 7-30; range 3-107 weeks). Three pts had treatment related grade 3/4 AEs: grade 4 sepsis (n=1), grade 3 lymphopenia (n=2); anemia, increased alkaline-phosphatase, and HSV oral infection (n=1 each). Best response was partial response (PR) in 3 pts (25%) and 3 (25%) had stable disease. Median PFS was 3.1 months (95%CI: 2.3-NA), and median overall survival was 8.0 months (95% CI: 4.7 – NA). Conclusions: The RP2D dose for tazemetostat is 800 mg + 200 mg Pembrolizumab q 3 weeks. The combination was feasible, well tolerated, and resulted in durable responses in poor-risk chemo-refractory UC patients. Clinical trial information: 03854474 .

266 Background: The frequency of young men with aggressive prostate cancer (PC) at diagnosis is increasing. Clinical, environmental, and genetic drivers of this change have not been well characterized. Methods: This multi-institutional study evaluated two cohorts; Cohort 1 completed enrollment and is reported here. Eligible patients (pts) had early-onset (age ≤ 60 years) PC with metastasis (N+ or M+) at diagnosis, or within 5 years of curative intent local therapy. Data were collected to define clinical, environmental, and genetic profiles, including ctDNA and whole genome and transcriptome sequencing using Tempus xE. Standard descriptive statistics were used. Results: 46 pts were enrolled. Median age at diagnosis was 55 (range 41-60 years); 85% were White, 15% were Black; 4% served in the military and 4% reported biochemical exposure. 85% reported a family history of cancer, 46% had family history of PC. Median PSA at diagnosis was 19 (range 1-534 ng/mL), 56% had a Gleason score of 9-10, and 56% had de novo metastatic PC. 46% had prior local therapy. Genetic data is available for 40 pts. The most frequent clinically significant mutations (≥10% for somatic, ≥2.5% for germline) are summarized. 23 unique germline and over 1,000 unique somatic mutations were identified. Germline mutations associated with hereditary PC were found in 15%, all were associated with DNA damage repair (DDR). Somatic mutations in DDR genes were found in 10%. Co-mutations in TP53 and BRAF were seen in 30%. Interestingly, there were also incidental germline mutations identified that are associated with cardiac ( MYBPC, MYH7) and vascular ( MYH11, ACTA2) conditions, among others. Conclusions: In this cohort study we identified an unexpectedly high frequency of family histories that were positive for cancer (85%), and specifically PC (46%). However, rates of germline mutations associated with hereditary PC were similar to previous studies (15%), suggesting the possibility that other novel hereditary mutations driving increased PC risk may be present. Increased rates of somatic mutations in BRAF (35%) were also seen. The high frequency of BRAF mutations, particularly those that co-occur with TP53 mutations, may be driving more aggressive disease. We also found enrichment of mutations associated with non-cancer hereditary syndromes, including hypertrophic cardiomyopathy. These are not usually included in cancer-focused genetic studies, suggesting broader testing that includes potentially actionable incidental findings should be considered. More work is needed to define characteristics of this high-risk population and optimize management. [Table: see text]

Bacillus Calmette–Guérin (BCG) is the most widely used vaccine worldwide and has been used to prevent tuberculosis for a century. BCG also stimulates an anti-tumour immune response, which urologists have harnessed for the treatment of non-muscle-invasive bladder cancer. A growing body of evidence indicates that BCG offers protection against various non-mycobacterial and viral infections. The non-specific effects of BCG occur via the induction of trained immunity and form the basis for the hypothesis that BCG vaccination could be used to protect against the severity of coronavirus disease 2019 (COVID-19). This Perspective article highlights key milestones in the 100-year history of BCG and projects its potential role in the COVID-19 pandemic., Bacillus Calmette–Guérin (BCG) has been used to prevent tuberculosis for a century and is also a standard approach for the treatment of non-muscle-invasive bladder cancer. However, BCG also has a plethora of non-specific effects that occur via the induction of trained immunity and have raised the hypothesis that BCG vaccination could be used to protect against coronavirus disease 2019 (COVID-19). In this Perspective, the authors describe the history of BCG, discuss the mechanisms of its effects, and consider its potential role during the COVID-19 pandemic.

To assess the reliability of peer-review of TURBT videos as a means to evaluate surgeon skill and its relationship to detrusor sampling.Urologists from an academic health system submitted TURBT videos in 2019. Ten blinded peers evaluated each surgeon's performance using a 10-item scoring instrument to quantify surgeon skill. Normalized composite skill scores for each surgeon were calculated using peer ratings. For surgeons submitting videos, we retrospectively reviewed all TURBT pathology results (2018-2019) to assess surgeon-specific detrusor sampling. A hierarchical logistic regression model was fit to evaluate the association between skill and detrusor sampling, adjusting for patient and surgeon factors.Surgeon skill scores and detrusor sampling rates were determined for 13 surgeons performing 245 TURBTs. Skill scores varied from -6.0 to 5.1 [mean: 0; standard deviation (SD): 2.40]. Muscle was sampled in 72% of cases, varying considerably across surgeons (mean: 64.5%; SD: 30.7%). Among 8 surgeons performing5 TURBTs during the study period, adjusted detrusor sampling rate was associated with sending separate deep specimens (odds ratio [OR]: 1.97; 95% confidence interval [CI]: 1.02-3.81, P = .045) but not skill (OR: 0.81; 95% CI: 0.57-1.17, P = .191).Surgeon skill was not associated with detrusor sampling, suggesting there may be other drivers of variability of detrusor sampling in TURBT.

Purpose: To characterize immune cell expression among patients with non–muscle invasive bladder cancer (NMIBC) treated with Bacillus Calmette-Guerin (BCG). Experimental Design: Patients with NMIBC treated with intravesical BCG (2008–2015) were identified, and a tissue microarray was constructed using paired pre- and post-BCG bladder samples. Among patients undergoing BCG, cystoscopic evaluation began 3 months after initiating BCG treatment to determine therapeutic response. IHC was performed for CD8, CD4, FoxP3, PD-L1 (SP-142 and 22C3), and PD-1. A full slide review of PD-L1+ staining tumors was performed to characterize PD-L1 and CD8 colocalization. RNA-seq was performed on cored tumors from available specimens. We compared immune cell populations between BCG responders and nonresponders, and between pretreatment and postreatment tumor samples. Baseline PD-L1 staining in the BCG naïve population was then validated in a separate cohort. Results: The final cohort contained 63 pretreatment NMIBC cases, including 31 BCG responders and 32 BCG nonresponders. No differences in CD4, CD8, or FoxP3 expression were identified between responders and nonresponders. Baseline PD-L1 expression (22C3 and SP-142) was observed in 25% to 28% of nonresponders and 0% to 4% of responders (P < 0.01). PD-L1+ cells in BCG nonresponders colocalized with CD8+ T cells. In addition, BCG therapy did not increase PD-L1 gene expression (RNA-seq) or protein levels (IHC). The number of pretreatment CD4+ T cells was very low among PD-L1+ nonresponders (12%) and high among PD-L1− nonresponders (50%, P < 0.01). In a separate cohort of 57 patients with NMIBC undergoing BCG, baseline PD-L1 (22C3) staining was similar (26%). Conclusions: One mechanism of BCG failure may be adaptive immune resistance. Baseline tumor PD-L1 expression predicts an unfavorable response to BCG and if validated, could be used to guide therapeutic decisions.

BACKGROUND: Chromatin modifying enzymes, mainly through post translational modifications, regulate chromatin architecture and by extension the underlying transcriptional kinetics in normal and malignant cells. Muscle invasive bladder cancer (MIBC) has a high frequency of alterations in chromatin modifiers, with 76% of tumors exhibiting mutation in at least one chromatin modifying enzyme [1]. Additionally, clonal expansion of cells with inactivating mutations in chromatin modifiers has been identified in the normal urothelium, pointing to a currently unknown role of these proteins in normal bladder homeostasis. OBJECTIVE: To review current knowledge of chromatin modifications and enzymes regulating these processes in Bladder cancer (BCa). METHODS: By reviewing current literature, we summarize our present knowledge of external stimuli that trigger loss of equilibrium in the chromatin accessibility landscape and emerging therapeutic interventions for targeting these processes. RESULTS: Genetic lesions in BCa lead to altered function of chromatin modifying enzymes, resulting in coordinated dysregulation of epigenetic processes with disease progression. CONCLUSION: Mutations in chromatin modifying enzymes are wide-spread in BCa and several promising therapeutic targets for modulating activity of these genes are currently in clinical trials. Further research into understanding how the epigenetic landscape evolves as the disease progresses, could help identify patients who might benefit the most from these targeted therapies.

INTRODUCTION AND OBJECTIVE:Prior work has shown that detrusor muscle sampling (MS) from transurethral resection of bladder tumor (TURBT) is related to oncologic outcomes. It is unknown if surgeon s...

INTRODUCTION AND OBJECTIVE:Men are three times more likely to develop bladder cancer (BC) than women. Mouse models have demonstrated that androgens are necessary for BC tumorigenesis. Herein, we as...

280 Background: Approvals of targeted therapies and immune checkpoint inhibitors hold the promise of improving long-term survival in patients with urothelial carcinoma. Competence gaps that were identified prompted the design of a unique educational series for the urology-oncology team. Optimal management of urothelial carcinoma relies on effective patient-provider communication and decision-making. To provide an integrative learning experience, the patient voice was embedded into the clinical content through shared insights and patient-reported data. Methods: A 4-part CME series was launched live-online in October 2021 in partnership with the Large Urology Group Practice Association and remains on-demand through October 2022 at UroCareLive.com and OMedLive.com. A companion patient program was held in September 2021 in partnership with the Bladder Cancer Advocacy Network and remains on-demand at CancerCoachLive.com. Behavioral assessment of preferences and attitudes toward managing patients were examined throughout the CME series and patient/caregiver program. A planned analysis of the data from these questions will determine patient and clinical impact. Outcomes from the patient program were analyzed and presented during the CME series followed by expert-identified strategies to improve clinical practice. Results: To date, 775 clinicians and 15,193 patients have participated in the educational initiative. Of the patient-reported experiences and preferences: 44% prefer to discuss benefits and risks of treatment options; and while 85% prefer to make decisions with their team, 67% felt overwhelmed and ultimately allowed their team to select therapy. Patient insights revealed challenges related to quality of life, side-effect management, and disease management. Patient-provider disparities were observed across preferences for point-of-care treatment discussions and quality of life challenges. Clinical and patient impact following integration of the patient voice will be analyzed. Conclusions: The initiative contributed to the provision of valuable patient insights and preferences based on real-world experience which were integrated into provider education. Increasing this awareness fostered practical strategies and discussion to improve patient-centered care. Education incorporating the patient voice into provider education can further sensitize clinicians to patient concerns and facilitate point-of-care decision-making.

Testis cancer is the most common solid malignancy in young males. The purpose of this guideline is to provide a useful reference on the effective evidence-based treatment of early stage testicular cancer.The systematic review utilized to inform this guideline was conducted by a methodology team at the Johns Hopkins University Evidence-based Practice Center. The methodology team searched using PubMed®, Embase®, and the Cochrane Central Register of Controlled Trials (CENTRAL) from January 1980 through August 2018. The evidence review team also reviewed relevant systematic reviews and references provided by the panel to identify articles that may have been missed by the database searches.When sufficient evidence existed, the body of evidence was assigned a strength rating of A (high), B (moderate), or C (low). Such evidence-based statements are provided as Strong, Moderate, or Conditional Recommendations. In instances of insufficient evidence, additional guidance is provided as Clinical Principles and Expert Opinions.This guideline attempts to improve a clinician's ability to evaluate and treat patients with testicular cancer, but higher quality evidence in future trials will be essential to improve level of care for these patients.

Analysis of the IMvigor 210 trials involving patients with platinum-refractory or cisplatin-ineligible urothelial carcinoma who were treated with the PD-L1 inhibitor atezolizumab identified a resistance signature as an immune biomarker. Transcriptome profiling of 368 tumor samples from this trial revealed that the "genomically unstable" Lund subtype classification was associated with the best response. We developed and applied a novel single-patient subtype classifier based on The Cancer Genome Atlas 2017 expression-based molecular subtypes. We identified 11 patients with a neuronal subtype, with a 100% response rate in eight confirmed cases (2 complete response, 6 partial response), and 72% overall, including 3/11 patients with an unconfirmed response. The survival probability was extraordinarily high for the neuronal subtype, which represents a high-risk cohort with advanced disease, and may be secondary to low levels of TGFβ expression and high mutation/neoantigen burden. PATIENT SUMMARY: We describe a methodology for genomic classification of an individual patient's bladder cancer tumor and have identified a subtype that is associated with a high response rate to immunotherapy. This is an important step forward in identifying the right treatment for the right patient, which is the goal of personalized precision medicine.

Purpose Bladder cancer is initially diagnosed and staged with a transurethral resection of bladder tumor (TURBT). Patient survival is dependent on appropriate sampling of layers of the bladder, but pathology reports are dictated as free text, making large-scale data extraction for quality improvement challenging. We sought to automate extraction of stage, grade, and quality information from TURBT pathology reports using natural language processing (NLP). Methods Patients undergoing TURBT were retrospectively identified using the Northwestern Enterprise Data Warehouse. An NLP algorithm was then created to extract information from free-text pathology reports and was iteratively improved using a training set of manually reviewed TURBTs. NLP accuracy was then validated using another set of manually reviewed TURBTs, and reliability was calculated using Cohen’s κ. Results Of 3,042 TURBTs identified from 2006 to 2016, 39% were classified as benign, 35% as Ta, 11% as T1, 4% as T2, and 10% as isolated carcinoma in situ. Of 500 randomly selected manually reviewed TURBTs, NLP correctly staged 88% of specimens (κ = 0.82; 95% CI, 0.78 to 0.86). Of 272 manually reviewed T1 tumors, NLP correctly categorized grade in 100% of tumors (κ = 1), correctly categorized if muscularis propria was reported by the pathologist in 98% of tumors (κ = 0.81; 95% CI, 0.62 to 0.99), and correctly categorized if muscularis propria was present or absent in the resection specimen in 82% of tumors (κ = 0.62; 95% CI, 0.55 to 0.73). Discrepancy analysis revealed pathologist notes and deeper resection specimens as frequent reasons for NLP misclassifications. Conclusion We developed an NLP algorithm that demonstrates a high degree of reliability in extracting stage, grade, and presence of muscularis propria from TURBT pathology reports. Future iterations can continue to improve performance, but automated extraction of oncologic information is promising in improving quality and assisting physicians in delivery of care.

e14552 Background: Recent advances in the field of cancer immunology have highlighted the importance of the immune system for eliminating tumors. Numerous studies have shown that tumor-infiltrating immune cells such as antigen-presenting cells (APCs), T cells, and natural killer (NK) cells play critical roles in tumor control. However, the inflammatory anti-tumor immune response is counteracted by the induction of immune regulatory mechanisms within the tumor microenvironment (TME). These findings have led to the development of immune-targeted therapies, which are aimed at activating anti-tumor immune signaling pathways and enhancing anti-tumor immune function. While immunotherapies, have revolutionized the treatment of several solid tumors and leukemias, at best response rates remain low at 25%-30%, and a portion of patients eventually develop resistance to therapy leading to disease progression and mortality. Methods: We have previously reported the potent effects of 3-4 daily intravenous infusions of immune modifying poly(lactic- co-glycolic acid) (PLGA) nanoparticles, ONP-302, free from drugs or other bioactive agents, for the amelioration of acute inflammatory diseases by targeting myeloid cells. The present studies describe a novel use for ONP-302 nanoparticles, employing an altered dosing scheme to reprogram myeloid cells resulting in significant enhancement of tumor immunity. The efficacy of ONP-302 nanoparticles at inducing an anti-tumor immune response was evaluated using syngeneic mouse tumor models. Results: ONP-302 infusion decreased tumor growth via the activation of the cGAS/STING pathway within myeloid cells, and subsequently increased NK cell activation via an IL-15-dependent mechanism. Additionally, ONP-302 treatment increased PD-1/PD-L1 expression in the tumor microenvironment, thereby allowing for functionality of anti-PD-1 for treatment in the B16.F10 melanoma tumor model which is normally unresponsive to monotherapy with anti-PD-1. Conclusions: These findings indicate that ONP-302 allows for tumor control via reprogramming myeloid cells via activation of the STING/IL-15/NK cell mechanism, as well as increasing anti-PD-1 response rates.

TPS4617 Background: Patients [pts] withlymph node positive (LN+), non-metastatic bladder cancer (BC) have a better prognosis than those with metastatic (M1) disease. However, this population is under-represented in advanced bladder trials and ineligible for bladder-sparing trials. Therefore, there have been no larger prospective trials establishing the standard of care in LN+ BC. Given the promise of immunotherapy in advanced BC and potential synergy between immunotherapy and radiation, INSPIRE was designed to determine the role of concurrent and adjuvant durvalumab (durva) in this patient population when treated with induction chemotherapy (IC) followed by concurrent chemoRT. Methods: This is a randomized phase II study that is enrolling BC pts with stage III [N1-3 M0], pure or mixed urothelial cancer. Pts must have received ≥3 cycles of IC [either before or after registration, prior to randomization] without progression. LN+ is defined as radiologically LN ≥1.0 cm in short axis, with or without biopsy prior to IC. As long as pts do not progress on induction chemotherapy, they will be randomized to chemoRT+/- durva using 5 stratification factors (Simon Pocock minimization method) a) IC prior vs. post registration b) cisplatin vs non-cisplatin regimen during RT c) LN size d) response to IC e) extent of TURBT. Pts on the chemoRT+durva arm will get chemotherapy per physician choice + IMRT + 3 x doses of Q3wk durva for 6.5-8 wks, whereas those on the control arm will get chemoRT alone. The primary end point is clinical complete response [CR], defined as no radiologically measurable disease in the LNs and negative cystoscopy and bladder biopsy 8-10 weeks post-chemoRT +/- durva. Pts on the chemoRT + durva arm who have a CR or clinical benefit ( > T0 and ≤T2 in bladder per cystoscopy, biopsy + CR/PR/SD in LN by imaging) will get adjuvant Q4wk durva for 9 doses, while those on the chemoRT arm will undergo observation. Secondary end points include OS, PFS, bladder-intact event-free survival, rate of toxicity and salvage cystectomy. This study is designed to detect an improvement of 25% in clinical CR between both arms (37.5% to 62.5%). A total accrual of 114 pts (in order to enroll 92 evaluable pts) will provide 81% power to detect this difference using a Fisher’s exact test (assuming 10% drop out + anticipating that 20% chemotherapy-naïve pts will progress post IC). We are banking blood and primary tumor tissue pre- and post-chemoRT in both groups. The study was activated in August 2020 and accrual is ongoing. We expanded eligibility to include N3 in 9/2021. INSPIRE is the first prospective study designed for only LN+ BC and will define both short-term and long-term outcomes for bladder sparing in this patient population and has the potential to define a new treatment strategy for stage III BC. Clinical trial information: NCT04216290.

e17049 Background: Although prostate cancer (PC) is heterogeneous, the rate of patients diagnosed with aggressive metastatic disease at a young age has been increasing. Prior studies defining genetic abnormalities in high-risk PC have not focused on this unique population of patients, thus the clinical and molecular features of these lethal PC phenotypes are not well described. Methods: This multi-institutional study evaluated two cohorts. Cohort 1, reported here, included early-onset (age ≤ 60) PC that was metastatic (N+ or M+) at diagnosis or PC that metastasized within 5 years post curative intent/local therapy. Cohort 2 included men with metastatic hormone sensitive PC who rapidly progressed (≤ 14 months) after systemic therapy. Data was collected to define clinical and genomic profiles, including sequencing of somatic & germline DNA, circulating tumor DNA and tumor RNA. Standard descriptive statistics were used. Results: 44 patients were enrolled. Median age at diagnosis was 55 years (range 41-60); 84% were White and 14% were Black. Median prostate specific antigen at diagnosis was 20 (range 1-534 ng/mL). 54% reported a family history of PC, while breast and colorectal cancer were reported in 35% and 14%, respectively. 4.5% reported a history of biochemical agent exposure. 58.5% of patients had De Novo distant metastatic disease (56% of these were low-volume) and 59.5% had a Gleason score of 9-10. 59% had received prior local therapy. Germline and somatic genetic data are available for 36 patients (4 are pending). The most common somatic mutation was in TP53 (n=15), followed by BRAF (n=14), AR (n=7), ERBB3 & MYC (n=5 each), CDKN2B, HRAS, MUC4, OBSCN & SPOP (n=4 each). Additional unique mutations in over 1,000 genes were also identified. Germline mutations were detected in BRCA2, ATM, ATP7B & FBN1 (n=3 each), RB1, CDH1, MYBPC3, MYH11 & MYH7 (n=2 each). 11 other unique germline mutations were also identified. Germline mutations were identified in genes previously implicated in hereditary PC ( BRCA2, ATM, PALB2, BRIP1 & CHEK2), with an overall germline incidence of 25%. There were also incidental germline mutations in genes related to hereditary cardiac conditions ( MYBPC, MYH11, MYH7), as well as other hereditary cancers ( RB1 and CDH1). Conclusions: This study evaluated specific criteria to define risk factors associated with the development of aggressive PC at a young age. Nearly 90% of these patients had a family history of cancer, with over 50% reporting a family history of PC. Somatic mutations were identified in genes such as TP53 that are frequently associated with aggressive disease. Additionally, there was enrichment for germline mutations associated with PC that exceeded what has previously been reported and enrichment of mutations not commonly included in PC genetic risk panels. Thus, more work is needed to define characteristics of this high-risk population and optimize management.

4506 Background: Neoadjuvant chemotherapy is the standard of care in muscle-invasive bladder cancer patients. However, treatment is intense, the overall benefit is small, and there is no established marker to identify patients who benefit most. The aim of the study is to characterize cell-free DNA (cfDNA) methylation from patients receiving neoadjuvant chemotherapy in SWOG S1314, a prospective cooperative group trial, and to correlate the methylation signatures with pathologic response. Methods: Blood samples were collected prospectively from 73 patients before and during standard neoadjuvant chemotherapy. At radical cystectomy, pathologic response was documented. Plasma cfDNA was profiled using Infinium MethylationEPIC BeadChip array. Differential methylation between pathologic responders (≤pT1N0M0) and non-responders was analyzed, and a Random Forest model was used to generate a classifier predictive of treatment response. Results: Using pre-chemotherapy plasma cfDNA, we developed a methylation-based response score (mR-score) predictive of pathologic response. The mR-score also could be calculated using plasma samples collected after the first cycle of neoadjuvant chemotherapy, resulting in a similar predictive ability. Furthermore, we used cfDNA methylation data to calculate the circulating bladder DNA fraction, which had a modest but independent predictive ability for treatment response. When we combined the mR-score and circulating bladder DNA fraction, we successfully predicted pathologic response outcomes in 79% of patients based on their plasma collected before chemotherapy and after 1 cycle of chemotherapy. Conclusions: Our study provides proof of concept that cfDNA methylation may be used to predict treatment response in bladder cancer patients receiving neoadjuvant chemotherapy. Clinical trial information: NCT02177695.

Among veterans, prostate cancer is the most common malignancy and has a higher incidence compared to the rest of the nation. No study has compared the effectiveness of Robotic-assisted-laparoscopic radical prostatectomy (RALP) vs. open radical prostatectomy (ORP) in the Veteran's Affairs (VA) hospital setting during the adoption of RALP.Institutional Review Board approval was obtained. Retrospective review was completed on Veterans with prostate cancer who underwent ORP or RALP from March 2011 to January 2017 during the introduction of RALP at one VA hospital. Perioperative and functional outcomes between ORP and RALP were compared as well as between the initial 50 and final 53 RALPs.Among 91 ORPs and 153 RALPs, RALP had significant reductions in blood transfusions [2(1.3%) vs. 44(40%), p0.001], length of stay [2 days(1-2) vs. 3 days(2-4), p0.001], Clavien grade2 complications [1(0.7%) vs. 20 (22.0%), p0.001], urine leak [2(1.3%) vs. 11 (12.1%), p0.001], and ICU readmissions [0(0%) vs. 3(3.3%), p0.001]. There were no significant differences in positive margin status or functional outcomes. Compared to the first 50 cases, the last 53 RALPs demonstrated a shorter operative time (349 vs. 292 min, p0.001), lower EBL (300 vs. 150 mL, p0.001), more frequent 1-day length of stay (34% vs. 60%, p = 0.02), and fewer composite adverse events (82% vs. 51%, p = 0.004). Operative time for the final 53 RALPs (292 minutes) was shorter than that of ORP (325 minutes, p = 0.013).During the introduction of RALP at one VA hospital, RALP was associated with several improved parameters compared to ORP and similar operative times were noted after the first 100 cases of RALPs. RALP is safe to introduce in a VA setting without compromising outcomes.

Bladder cancer is the fourth most common cancer in men and fifth most common overall. The use of next-generation sequencing (NGS) approaches is crucial to precisely characterize the molecular defects of tumors, and this information could be combined with other clinical data, such as tumor histology and TNM staging, with the goal of precise tumor classification. In many settings, targeted NGS is evaluated in patients with first- and second-line metastatic cancer. Yet, in the decade to come we anticipate increased application of precision oncology at all stages of bladder cancer with the aim of customizing cancer treatment. Here, we review the genomic and transcriptomic features associated with risk stratification in bladder cancer and summarize the current efforts for precision oncology in localized urothelial carcinomas.

The molecular landscape in non-muscle-invasive bladder cancer (NMIBC) is characterized by large biological heterogeneity with variable clinical outcomes. Here, we perform an integrative multi-omics analysis of patients diagnosed with NMIBC (n = 834). Transcriptomic analysis identifies four classes (1, 2a, 2b and 3) reflecting tumor biology and disease aggressiveness. Both transcriptome-based subtyping and the level of chromosomal instability provide independent prognostic value beyond established prognostic clinicopathological parameters. High chromosomal instability, p53-pathway disruption and APOBEC-related mutations are significantly associated with transcriptomic class 2a and poor outcome. RNA-derived immune cell infiltration is associated with chromosomally unstable tumors and enriched in class 2b. Spatial proteomics analysis confirms the higher infiltration of class 2b tumors and demonstrates an association between higher immune cell infiltration and lower recurrence rates. Finally, the independent prognostic value of the transcriptomic classes is documented in 1228 validation samples using a single sample classification tool. The classifier provides a framework for biomarker discovery and for optimizing treatment and surveillance in next-generation clinical trials., Multiple molecular profiling methods are required to study urothelial non-muscle-invasive bladder cancer (NMIBC) due to its heterogeneity. Here the authors integrate multi-omics data of 834 NMIBC patients, identifying a molecular subgroup associated with multiple alterations and worse outcomes.

Despite the frequency of bladder cancer as the fourth most common cancer in men in the USA, there is insufficient data to recommend screening of the general population to detect bladder cancer prior to symptoms. Yet, several small trials of patients at increased risk for bladder cancer have identified cancers at an earlier stage, potentially improving survival. Most patients with bladder cancer will have gross or microscopic hematuria, with other common symptoms including frequency and urgency. An evaluation for bladder cancer includes cystoscopy and imaging of the kidneys and ureters. In the future, a risk-stratified approach for screening and evaluation may improve the specificity of screening and potentially improve survival of patients with bladder cancer.

Checkpoint immunotherapy (CPI) has increased survival for some patients with advanced-stage bladder cancer (BCa). However, most patients do not respond. Here, we characterized the tumor and immune microenvironment in pre- and post-treatment tumors from the PURE01 neoadjuvant pembrolizumab immunotherapy trial, using a consolidative approach that combined transcriptional and genetic profiling with digital spatial profiling. We identify five distinctive genetic and transcriptomic programs and validate these in an independent neoadjuvant CPI trial to identify the features of response or resistance to CPI. By modeling the regulatory network, we identify the histone demethylase KDM5B as a repressor of tumor immune signaling pathways in one resistant subtype (S1, Luminal-excluded) and demonstrate that inhibition of KDM5B enhances immunogenicity in FGFR3-mutated BCa cells. Our study identifies signatures associated with response to CPI that can be used to molecularly stratify patients and suggests therapeutic alternatives for subtypes with poor response to neoadjuvant immunotherapy.

TPS594 Background: Patients [pts] withlymph node positive (LN+), non-metastatic bladder cancer (BC) have a better prognosis than those with metastatic (M1) disease. However, this population is under-represented in advanced bladder trials and ineligible for bladder-sparing trials. Therefore, there have been no larger prospective trials establishing the standard of care in LN+ BC. Given the promise of immunotherapy in advanced BC and potential synergy between immunotherapy and radiation, INSPIRE was designed to determine the role of concurrent and adjuvant durvalumab (durva) in this patient population when treated with induction chemotherapy (IC) followed by concurrent chemoRT. Methods: This is a randomized phase II study that is enrolling BC pts with stage III [N1-3 M0], pure or mixed urothelial cancer. Pts must have received ≥3 cycles of IC [either before or after registration, prior to randomization] without progression. LN+ is defined as radiologically LN ≥1.0 cm in short axis, with or without biopsy prior to IC. As long as pts do not progress on induction chemotherapy, they will be randomized to chemoRT+/- durva using 5 stratification factors (Simon Pocock minimization method) a) IC prior vs. post registration b) cisplatin vs non-cisplatin regimen during RT c) LN size d) response to IC e) extent of TURBT. Pts on the chemoRT+durva arm will get chemotherapy per physician choice + IMRT + 3 x doses of Q3wk durva for 6.5-8 wks, whereas those on the control arm will get chemoRT alone. The primary end point is clinical complete response [CR], defined as no radiologically measurable disease in the LNs and negative cystoscopy and bladder biopsy 8-10 weeks post-chemoRT +/- durva. Pts on the chemoRT + durva arm who have a CR or clinical benefit (>T0 and ≤T2 in bladder per cystoscopy, biopsy + CR/PR/SD in LN by imaging) will get adjuvant Q4wk durva for 9 doses, while those on the chemoRT arm will undergo observation. Secondary end points include OS, PFS, bladder-intact event-free survival, rate of toxicity and salvage cystectomy. This study is designed to detect an absolute improvement of 25% in clinical CR between both arms (37.5% to 62.5%). The accrual goal is 114, assuming 10% drop out + anticipating that 20% chemotherapy-naïve pts will progress post IC, and 92 evaluable pts that will provide 81% power to detect this difference using a Fisher’s exact test. We are banking blood and primary tumor tissue pre- and post-chemoRT in both groups. The study was activated in August 2020 and accrual is ongoing. We expanded eligibility to include N3 in 9/2021. INSPIRE is the first prospective study designed for only LN+ BC and will define both short-term and long-term outcomes for bladder sparing in this patient population and has the potential to define a new treatment strategy for stage III BC. Clinical trial information: NCT04216290.