center dot The incidence of CNS metastases in patients with mUC either during or following treatment with enfortumab vedotin may be higher than previously reported in similar mUC cohorts. center dot CNS metastases in EV-treated patients with mUC are associated with inferior outcomes and limited systemic treatment options. center dot Poor outcomes associated with CNS metastases highlight the need for greater vigilance regarding CNS progression in the population of mUC patients treated with enfortumab vedotin and a reevaluation of the role of CNS imaging surveillance in this disease state. Clinical Genitourinary Cancer, Vol. 22, No. 2, 315-321 (c) 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )

Histologic variant (HV) subtypes of bladder cancer are clinically aggressive tumors that are more resistant to standard therapy compared to conventional urothelial carcinoma (UC). Little is known about the transcriptional programs that account for the morphological and biological differences in HV tumors. To investigate the tumor biology of HV bladder cancers, we generated a single cell RNA sequencing (scRNA- seq) atlas of nine HV tumors and three UC tumors. Our analyses revealed a tumor cell state specific to HVs that is characterized by expression of MUC16 (CA125) , KRT24 , and WISP2 . This CA125+ cell state bears transcriptional hallmarks of epithelial-mesenchymal transition, is enriched in metastases, is predicted to be highly chemotherapy resistant, and is linked with poor survival, suggesting that this cell state plays an important role in the aggressive biology of HV tumors. Our analyses also provide novel evidence of transcriptional “mimicry” between HVs and histologically similar non-urothelial cell types. Lastly, we identified higher expression of TM4SF1, a cell surface protein associated with cancer metastasis, in HV tumor cells compared to UC tumor cells. Finally, CAR T cells engineered against TM4SF1 protein demonstrated in vitro and in vivo activity against bladder cancer cell lines in a TM4SF1 expression- dependent manner, highlighting its potential as a therapeutic target in bladder cancer.One sentence summary Single cell RNA sequencing of primary bladder cancers identified a CA125+ cell state specific to histologic variants that is associated with aggressive biological features and TM4SF1 as a novel therapeutic target for histologic variant subtypes of bladder cancer which can be targeted by anti- TM4SF1 CAR T cells.### Competing Interest StatementThe authors have declared no competing interest.

602 Background: There are no current standard of care options for patients (pts) with muscle-invasive bladder cancer (MIBC) ineligible for cisplatin-based chemotherapy (cisplatin). This trial investigated the safety and efficacy of escalating doses of neoadjuvant atezolizumab (ATZ) prior to radical cystectomy (RC) (NCT02451423). Methods: This single-arm, single institution, phase II trial investigated treatment with 1 (n=6), 2 (n=6) or 3 (n=11) cycles of ATZ (1200 mg IV every 3 weeks) in pts with MIBC. Key inclusion criteria were urothelial carcinoma of the bladder (T2-T4a,N0-1,M0), cisplatin-ineligibility, and eligibility for RC. High-risk pts (>pT2 or LN+ at RC) could receive adjuvant ATZ for up to 16 total cycles. Primary efficacy endpoint was pathologic complete response (pCR; pT0/Ta/TisN0). Secondary endpoints included rate of pathologic downstaging (≤T1N0), 2-year recurrence-free survival (RFS), overall survival (OS), and biomarker assessments of pre and post-treatment biopsies. Pts had RC between 7/2016 and 6/2021. The censor date for survival outcomes was 9/10/2023, representing the final efficacy analysis. Results: A total of 23 pts received ATZ; 1 pt was excluded from efficacy analyses due to lack of confirmed MIBC (≥T2). Among 22 included pts, median age was 70, 74% were men, 83% Caucasian; reasons for cisplatin-ineligibility were renal impairment (37%), hearing loss (27%) or neuropathy (9%); remainder declined cisplatin (27%). At enrollment, cT2/T3/T4 rates were 77%, 14%, and 9%, while 9% were cLN+. All pts completed intended treatment and had RC in the defined timeframe (>3 weeks from last and <12 weeks from first treatment). pCR at RC was 14% (3/22), occurring in pts receiving 1 and 2 cycles of ATZ. Pathologic downstaging (≤pT1N0) was achieved in 23% (5/22), occurring at all three dose levels. Adjuvant ATZ was given to 8 pts. Another 4 pts received off-study adjuvant therapy with cisplatin (3) or nivolumab (1). After median follow-up of 51.4 months from RC, mRFS and mOS were Not Reached. Two-year RFS and OS were 77% and 90%. In 13 pts with available paired pre and post treatment samples, there was significant increase in T-cell % following ATZ (Wilcoxon signed rank test, p<0.05), driven by increase in % and density of CD8+ T-cells (p<0.05). All T-cell populations were significantly more abundant in tumor than in adjacent normal tissue post-treatment but not pre-treatment (CD3 + ,CD3 + CD8 + , CD3 + CD4 + , p<0.001 for all; CD4 + FOXP3 + , p=0.01). Conclusions: Neoadjuvant ATZ results in durable long-term survival rates in cisplatin-ineligible pts with MIBC. These data are comparable to previously reported trials with immunotherapy based regimens, despite a lower pCR rate. Changes suggestive of T-cell migration into the tumor microenvironment were observed following ATZ treatment. Clinical trial information: NCT02451423 .

HER2, encoded by the ERBB2 gene, is an important druggable driver of human cancer gaining increasing importance as a therapeutic target in urothelial carcinoma (UC). The genomic underpinnings of HER2 overexpression in ERBB2 nonamplified UC are poorly defined. To address this knowledge gap, we investigated 172 UC tumors from patients treated at the University of California San Francisco, using immunohistochemistry and next-generation sequencing. We found that GATA3 and PPARG copy number gains individually predicted HER2 protein expression independently of ERBB2 amplification. To validate these findings, we interrogated the Memorial Sloan Kettering/The Cancer Genome Atlas (MSK/TCGA) dataset and found that GATA3 and PPARG copy number gains individually predicted ERBB2 mRNA expression independently of ERBB2 amplification. Our findings reveal a potential link between the luminal marker HER2 and the key transcription factors GATA3 and PPARG in UC and highlight the utility of examining GATA3 and PPARG copy number states to identify UC tumors that overexpress HER2 in the absence of ERBB2 amplification. In summary, we found that an increase in copy number of GATA3 and PPARG was independently associated with higher ERBB2 expression in patient samples of UC. This finding provides a potential explanation for HER2 overexpression in UC tumors without ERBB2 amplification and a way to identify these tumors for HER2-targeted therapies.

Improved imaging modalities are needed to accurately stage patients with muscle -invasive bladder cancer (MIBC) and metastatic urothelial carcinoma. Imaging with small -molecule ligands or inhibitors of fibroblast activation protein (FAP) is a promising modality that has demonstrated initial efficacy across a broad range of tumors. We present our experience with the novel FAP-peptide binder 68Ga-FAP-2286 in patients with MIBC. Methods: Patients with histopathologically confirmed bladder cancer who had either localized disease at diagnosis (localized cohort, n = 13) or known metastatic disease (metastatic cohort, n = 8) were imaged with 68Ga-FAP-2286 PET as part of a clinical trial (NCT04621435). The SUVmax of 68Ga-FAP-2286 PET-positive lesions and lesion size were documented. In patients who had available 18F-FDG PET performed within 45 d of 68Ga-FAP-2286 PET (n = 5), uptake on the 2 scans was compared. When there was a discrepancy between imaging modalities on retrospective review, biopsy of suggestive lesions was performed as the standard of care. Results: In the metastatic and localized cohorts, 36 and 18 68Ga-FAP-2286- avid lesions, respectively, were identified across multiple anatomic locations, including lymph nodes, visceral metastases, and bones. Fourteen of 36 lesions in the metastatic cohort and 14 of 18 lesions in the localized cohort were lymph nodes measuring less than 1 cm. Among lesions measuring less than 0.5 cm, 0.5-1 cm, and more than 1 cm, average SUVmax was 5.2 +/- 2.6, 9.6 +/- 3.7, and 13.0 +/- 4.3, respectively, in the metastatic cohort and 10.5 +/- 5.1, 10.8 +/- 5.7, and 9.9 +/- 5.4, respectively, in the localized cohort. Five patients had 18F-FDG PET available for comparison. The average SUVmax for lesions avid on 68Ga-FAP-2286 PET and 18F-FDG PET was 9.9 +/- 3.4 versus 4.2 +/- 1.9, respectively (n = 16 lesions). For 3 patients in the localized cohort, 68Ga-FAP-2286 PET informed clinical management, including identification of both false -positive findings on 18F-FDG PET and false -negative findings on conventional CT. Conclusion: 68Ga- FAP-2286 imaging is highly sensitive in patients with urothelial cancer and is effective in identifying metastatic lesions across a variety of anatomic sites, including subcentimeter lymph nodes that would not have raised suspicion on conventional scans. This novel imaging modality may inform clinical decision -making in patients with MIBC both by refining local nodal staging and by defining metastatic disease that would otherwise be undetectable on conventional imaging.

e17010 Background: Approximately 12% of patients (pts) with metastatic PCa (mPCa) identified by conventional imaging harbor DDRv. The increased use of PSMA PET has identified a group of pts with extra-prostatic PCa which is not apparent on conventional imaging and for whom the frequency of germline DDRv testing remains unknown. Methods: A single-institution retrospective analysis of patients with PCa detected by PSMA PET who had also undergone germline genetic testing was undertaken. Data collected included PSA level at time of PSMA PET imaging, stage/Gleason score at diagnosis (dx), rationale for germline testing, castration status, and whether the metastatic disease identified on PSMA PET represented de novo or recurrent mPCa. Germline sequencing results were evaluated for 16 DNA damage repair genes: ATM, ATR, BAP1, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, GEN1, MSH2, MSH6, NBN, PALB2, PMS2, RAD51C, and RAD51D. The frequency of DDRv was evaluated in groups defined by PSMA imaging results: localized (N0M0), Node-Positive (N+M0), metastatic (M+) PCa. Results: Of 795 PCa pts who underwent germline testing and 2101 PCa pts who underwent PSMA PET imaging, 386 had undergone both, and constitute the study cohort. In the study cohort the distribution of PCa extent identified by PSMA PET was: 81/386 (21.0%) N0M0; 76/386 (19.7%) N+M0; 229/386 (59.3%) M+. There were no statistically significant differences in the prevalence of DDRv with regards to age, Gleason score at dx, PSA at time of imaging, castration status at time of imaging, rationale for germline testing, or if metastatic, whether de novo or recurrent. The distribution of DDRv is shown in the table. There were no statistically significant differences between groups. Conclusions: The overall frequency of pathogenic germline DDRv in PCa pts who had undergone PSMA PET imaging was 4.66%, considerably lower than what has been reported in men with mPCa detected with conventional imaging. The presence or absence of extra-prostatic PCa detected by PSMA PET imaging did not appear to affect the frequency of DDRv. Validation of these findings in other cohorts will be important to guide recommendations for germline genetic testing in the growing group of PCa pts undergoing PSMA PET imaging. [Table: see text]

532 Background: ICIs form the backbone of treatment for mUC. However, only a minority of pts benefit and additional biomarkers of ICI response are needed. Methods: In our institution, we identified mUC pts treated with ICI monotherapy who had next generation sequencing (NGS). Somatic alterations present in ≥10% of pts ( ARID1A, CCND1, CDKN2A, CDKN2B, ERBB2, FGF3, FGF4, FGF19, FGFR3, KDM6A, MDM2, MLL2, PIK3CA, RB1, TERTp, TP53, TSC1), as well as DNA-damage response (DDR) alterations and tumor mutational burden (TMB) were analyzed as biomarkers. These biomarkers were individually evaluated in separate multivariate models while accounting for clinical factors including age, BMI, ECOG PS, primary tumor location, histology, hemoglobin, neutrophil to lymphocyte ratio and albumin. Multivariate cox regression and logistic regression models were used to measure hazard ratios (HR) and odds ratios (OR) for overall survival (OS), progression-free survival (PFS) and observed response rate (ORR). Results: Among 152 mUC ICI-treated pts, 107 had NGS data (FoundationOne, UCSF500, Strata), including 85 with TMB data. For the 107 pts with NGS, median age was 70 yrs, majority were male (69, 64%), Caucasian (70, 65%), had pure urothelial histology (57, 53%), and had first-line ICI (55, 51%). ORR was 35%, median PFS was 3.9 mos (95% CI: 2.6-7.5 mos), and median OS was 17.4 mos (95% CI: 14.1-30.6 mos). Biomarkers associated with improved outcomes to ICI, independent of relevant clinical factors, included alterations in ARID1A and DDR, as well as high TMB (>10 Mut/Mb). Inferior outcomes were seen in pts with CDKN2B, KDM6A, FGF3, FGF4, and FGF19 alterations (Table). Conclusions: In this large retrospective multivariate analysis controlling for clinical factors in ICI-treated mUC pts, we found multiple biomarkers associated with improved or inferior outcomes. These hypothesis-generating findings can inform clinical decision making and trial design for mUC pts treated with ICIs, and should be validated in larger cohorts. [Table: see text]

534 Background: ICIs are frequently used as therapy in mUC, but only a minority of patients (pts) respond to treatment. High TMB is associated with improved outcomes to ICIs. However, much is unknown about biomarkers associated with ICI outcomes in pts with high and low TMB respectively. Methods: We retrospectively identified mUC pts with known TMB status and available next generation sequencing (NGS) results treated with ICI monotherapy at our institution. TMB high was defined as ≥ 10 mutations/Mb, with the rest being TMB low. Somatic alterations present in ≥10% pts ( ARID1A, CCND1, CDKN2A, CDKN2B, ERBB2, FGF3, FGF4, FGF19, FGFR3, KDM6A, MDM2, MLL2, PIK3CA, RB1, TERTp, TP53, TSC1), and presence of DNA damage response (DDR) alterations were assessed as biomarkers of interest. Within the TMB-high and TMB-low pt groups we separately assessed patients based on the presence or absence of these somatic alterations, APOBEC mutational signature and high PD-L1 expression. Log rank test was used to determine differences in overall survival (OS) and progression free survival (PFS) among these groups. P-value ≤0.05 was considered significant. Results: Among 107 mUC pts treated with ICI monotherapy between 12/2014 and 3/2022 who had NGS data (UCSF500, FoundationOne, Strata), 85 pts had TMB data, including 47 TMB high pts and 38 TMB low pts. Among 85 pts with known TMB status, median age was 76 yrs, the majority were male (55, 65%), Caucasian (57, 67%), had pure urothelial histology (46, 55%) and were treated with ICIs in frontline setting (47, 55%). Median OS was 17.2 mos and median PFS was 3.42 mos. In TMB high pts, presence of DDR , MLL2, KDM6A, PIK3CA and TERTp alterations were each associated with improved outcomes, while presence of CDKN2B alterations was associated with inferior outcomes (Table). Among TMB low pts, those with RB1 alterations had shorter mOS (11.3 months vs 17.2 months; p=0.04) compared to wild-type pts. Conclusions: In this single-center retrospective analysis of mUC pts, we identified somatic alterations that were predictive of outcomes with ICI treatment in TMB high and TMB low pts respectively. Further exploration of biomarkers in patients stratified by TMB status is warranted in larger cohorts. [Table: see text]

Urothelial carcinoma with squamous differentiation (UCS) is a common variant of bladder cancer for which treatment outcomes with novel agents are largely unknown. In this retrospective analysis comparing 40 patients with UCS and 120 patients with pure UC, we found inferior outcomes for UCS patients treated with immune checkpoint inhibitors or Enfortumab vedotin. This highlights an important unmet clinical need that should be addressed with further studies. Introduction: Urothelial carcinoma with squamous differentiation (UCS) is associated with increased resistance to chemotherapy, but outcomes associated with newer therapies approved in this space over the last 5 to 10 years are less well defined. We investigated clinical outcomes and molecular profiling of patients with UCS treated with an immune checkpoint inhibitor (ICI) and/or Enfortumab vedotin (EV). Patients and Methods: We undertook a retrospective analysis of UC patients treated with ICI and/or EV. Objective response rate (ORR), progression free survival (PFS) and overall survival (OS) were compared between pure UC (pUC) and UCS using X 2 and log-rank tests, respectively. Prevalence of the most commonly detected somatic alterations were also compared between the 2 histologic subgroups. Results: A total of 160 patients (40 UCS, 120 pUC) were identified for this analysis. Among 151 patients treated with ICI (38 UCS, 113 pUC), UCS patients had a shorter mPFS (1.9 vs. 4.8 months, P < 0.01) and mOS (9.2 vs. 20.7 months, P < 0.01) compared to pUC. Among 37 patients treated with EV (12 UCS, 25 pUC), UCS patients had a lower ORR (17% vs. 70%, P < 0.01) and shorter mPFS (3.4 vs. 15.8 months, P < 0.01). UCS samples were enriched for CDKN2A, CDKN2B, PIK3CA, while pUC samples were enriched for ERBB2 alterations. Conclusion: In this single-center retrospective analysis, patients with UCS had a distinct somatic genomic profile relative to patients with pUC. Patients with UCS also had inferior outcomes to ICIs and EV compared to patients with pUC.

BackgroundEnfortumab vedotin (EV) is an antibody-drug conjugate approved for patients with treatment-refractory advanced urothelial carcinoma (aUC), however data on biomarkers of response is lacking.MethodsWe retrospectively identified all aUC patients at our institution who received EV monotherapy and had next-generation sequencing (NGS) data available. Patients were considered responders if they had a complete response or partial response on restaging scans during treatment. Observed response rate (ORR) was evaluated by local investigator and compared between responders and non-responders using Chi-squared test. A univariable analysis was conducted using the Cox proportional hazard test to assess for associations between baseline characteristics and most common somatic alterations (in ≥10% of patients) with patient survival outcomes [progression-free survival (PFS) and overall survival (OS)]. Somatic alterations were then individually evaluated in separate multivariate models while accounting for patient and clinical characteristics using Cox regression models.ResultsAmong 29 patients treated with EV monotherapy, 27 had available NGS data. Median age was 70, 24 (83%) were men, 19 (62%) were Caucasian, 15 (52%) had pure urothelial histology and 22 (76%) had primary tumor in the bladder. ORR was 41%, and PFS and OS for the overall cohort were 5.1 months and 10.2 months. Responders were enriched among patients with TP53, KDM6A and MDM2 alterations. Patients with these alterations, as well as those with composite TP53/MDM2 alterations (alterations in either TP53 or MDM2), also had increased ORR with EV treatment compared to patients without these alterations. In the univariable analysis, baseline albumin level ≥ 3.0g/dL and presence of composite TP53/MDM2 alterations were associated with a prolonged OS. Baseline ECOG 0/1, TP53 alterations and TP53/MDM2 alterations were associated with a prolonged PFS. In the multivariable analysis, TP53 and TP53/MDM2 alterations were genomic markers predictive of improved PFS after accounting for the relevant clinical characteristics.ConclusionIn this single-center retrospective analysis of aUC patients treated with EV, presence of TP53 or MDM2 somatic alterations, lower ECOG PS scores (ECOG 0 or 1) and higher albumin levels (≥3 g/dL) were associated with improved outcomes with EV treatment. Prospective and external validation of these findings in larger cohorts is warranted.

You have accessJournal of UrologyCME1 Apr 2023PD09-12 THE USE OF 68Ga-FAP-2286 PET IMAGING IN PATIENTS WITH LOCALIZED BLADDER CANCER Domenique Escobar, Vadim Koshkin, Brad Kline, Jonathan Chou, Maxwell Meng, Terence Friedlander, Vipul Kumar, Thomas Hope, and Sima Porten Domenique EscobarDomenique Escobar More articles by this author , Vadim KoshkinVadim Koshkin More articles by this author , Brad KlineBrad Kline More articles by this author , Jonathan ChouJonathan Chou More articles by this author , Maxwell MengMaxwell Meng More articles by this author , Terence FriedlanderTerence Friedlander More articles by this author , Vipul KumarVipul Kumar More articles by this author , Thomas HopeThomas Hope More articles by this author , and Sima PortenSima Porten More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003240.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Bladder cancer is a common and often aggressive malignancy. Computed tomography (CT), magnetic resonance imaging (MRI) and fluorodeoxyglucose-positron emission tomography (FDG-PET) are used for staging but have limitations in identifying nodal or metastatic disease. Fibroblast activation protein (FAP) is a cell surface protein that is highly expressed on cancer-associated fibroblasts present in many cancers, including bladder cancer. We present the results of a pilot study assessing the diagnostic performance of 68Ga-FAP-2286 (FAP)-PET imaging in a cohort of patients with bladder cancer. METHODS: Patients with solid tumors underwent FAP-PET as part of a clinical trial (NCT04621435), including 16 patients with bladder cancer. We report on ten patients with clinically localized disease treated with curative intent at the time of imaging. Standard imaging with CT, MRI, FDG-PET, or bone scan was performed within 8 weeks of FAP-PET. The maximum standardized uptake value (SUV) and size of FAP-PET-positive lesions (defined as any focus of activity with at least 1.5 times higher SUV compared with mediastinal blood pool) were documented. RESULTS: Patient demographics and clinical stage are summarized in Table 1. Concordance of 12 FAP-PET scans was 33% with standard imaging and 43% with FDG-PET. Of 8 patients with pathologic or radiographic follow up, FAP-PET was concordant with true disease status in 100%. Two patients had FAP-PET before and after neoadjuvant chemotherapy; one had resolution of lymph node uptake and one had decreased but persistent uptake on (pathology from pelvic lymph node dissection positive for urothelial carcinoma). We highlight two cases where FAP-PET altered treatment decisions (Figure 1). CONCLUSIONS: FAP-PET has the potential to change treatment pathways and improve patient care by better identifying metastatic sites or falsely positive lymph nodes in cancers considered clinically localized and by improving response assessment to systemic therapy. Source of Funding: IIT from Clovis © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e245 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Domenique Escobar More articles by this author Vadim Koshkin More articles by this author Brad Kline More articles by this author Jonathan Chou More articles by this author Maxwell Meng More articles by this author Terence Friedlander More articles by this author Vipul Kumar More articles by this author Thomas Hope More articles by this author Sima Porten More articles by this author Expand All Advertisement PDF downloadLoading ...