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Since the approval of the COVID-19 vaccines, their safety and efficacy has been widely demonstrated in patients with cancer. However, there remain patients with reservations regarding vaccination. We aimed to assess genitourinary cancer patients' perceptions of the vaccines as well as barriers and influencers of decision-making through the completion of a questionnaire. While vaccine-associated concerns were observed, most patients with genitourinary cancers were willing to receive the vaccine. Moving forward, differing strategies could be considered to enhance patient education on the utility of vaccination in the setting of cancer and beyond.

External beam radiation therapy (EBRT) to oligometastases may improve outcomes in patients with oligometastatic hormone-sensitive prostate cancer (oHSPC). Follow-up on this cohort has been limited to5 years and prospective data on de novo patients with oHSPC are lacking. We reviewed the long-term outcomes of patients with oHSPC treated with EBRT and androgen deprivation therapy on a prospective trial.From 2006 to 2011, patients with oHSPC with 1 to 5 metastases received 36 weeks of androgen deprivation therapy (luteinizing hormone-releasing hormone agonist + bicalutamide) and up to 53 Gy to all visible metastases. When indicated, the primary tumor or prostate bed was treated with EBRT up to 78 or 66 Gy, respectively.Twenty-nine patients were treated: 15 de novo, 14 oligorecurrent, and 21 patients (72.4%) had bone metastases. Median number of metastases per patient was 1 (range, 1-5). EBRT was administered to 52 lesions (38 bone, 12 pelvic lymph nodes [LNs], 2 nonpelvic LNs) up to 53 Gy (range, 47-66). Median follow-up was 9.9 years (years; range, 0.2-14.4). Median overall survival was 9.7 years (95% confidence interval [CI], 5.8-not reached). Median progression-free survival was 1.9 years (95% CI, 1.6-2.2). Patients who presented with prostate cancer-defined de novo metastases had significantly improved (P = .04) median progression-free survival (2.0 years; 95% CI, 1.3-6.0) compared with oligorecurrent patients (1.8 years; 95% CI, 1.0-2.0). Patients who presented with LN-only metastases had numerically improved (P = .13) median PFS (5.8 years; 95% CI, 1.2-not reached) compared with patients with bony metastases (1.8 years; 95% CI, 1.3-2.0). At last follow-up, 17 patients (58.6%) had local control of all EBRT-treated metastases. The metastases that locally progressed had previously been controlled for median 3.5 years (range, 1.7-10.5).Our results compare favorably with other reported studies of patients with oHSPC and provide new insights into their long-term outcomes.

Supplementary Data from Disruption of β-Catenin–Dependent Wnt Signaling in Colon Cancer Cells Remodels the Microenvironment to Promote Tumor Invasion

The recent classification of colon cancer into molecular subtypes revealed that patients with the poorest prognosis harbor tumors with the lowest levels of Wnt signaling. This is contrary to the general understanding that overactive Wnt signaling promotes tumor progression from early initiation stages through to the later stages including invasion and metastasis. Here, we directly test this assumption by reducing the activity of ß-catenin–dependent Wnt signaling in colon cancer cell lines at either an upstream or downstream step in the pathway. We determine that Wnt-reduced cancer cells exhibit a more aggressive disease phenotype, including increased mobility in vitro and disruptive invasion into mucosa and smooth muscle in an orthotopic mouse model. RNA sequencing reveals that interference with Wnt signaling leads to an upregulation of gene programs that favor cell migration and invasion and a downregulation of inflammation signatures in the tumor microenvironment. We identify a set of upregulated genes common among the Wnt perturbations that are predictive of poor patient outcomes in early-invasive colon cancer. Our findings suggest that while targeting Wnt signaling may reduce tumor burden, an inadvertent side effect is the emergence of invasive cancer.Implications:Decreased Wnt signaling in colon tumors leads to a more aggressive disease phenotype due to an upregulation of gene programs favoring cell migration in the tumor and downregulation of inflammation programs in the tumor microenvironment; these impacts must be carefully considered in developing Wnt-targeting therapies.

BackgroundRelugolix is the newest form of androgen deprivation therapy (ADT) approved for prostate cancer. However, as an oral drug, several real-world concerns exist, particularly medication compliance, safety with other androgen receptor-targeted agents, and financial burden to patients.MethodsA single institution retrospective chart review was conducted evaluating all patients who were prescribed relugolix for any prostate cancer indication from January 1, 2021 to January 31, 2022. Demographic data, cardiac risk factors, concomitant therapy usage, and PSA/testosterone levels, were abstracted from the chart review. Adverse effects were obtained by examining progress notes. Compliance was assessed by clinic notes as well as prescription fills by specialty pharmacy records. The reasons patients did not fill or discontinued the medication were noted.ResultsHundred and one patients were prescribed relugolix, and 91 patients consented to research. Seventy-one (78%) patients filled the prescription to relugolix, with a median follow-up of 5 months. Prescription fill data were available for 45 (63%) patients, with 94% of days covered. The most commonly reported reason not to fill was cost at 50%. Sixty-six (93%) patients reported never missing a dose. PSA levels were available in 71 (100%) patients with 69 (97%) showing stable or improved PSA. Testosterone levels were available in 61 (86%) of patients, which showed 61 (100%) stable or successful castration. Twenty-four (34%) patients used relugolix in combination. No new major safety signals were seen in combination therapy. Nineteen (27%) patients had switched to another form of ADT. Fifteen of these (79%) felt similar or better on relugolix therapy.ConclusionsCompliance with relugolix seemed acceptable. No major new safety signals were seen, even in combination. Among patients who switched therapy, most tolerated relugolix similarly or better than the previous form of ADT. The cost was a major reason for patients not initiating and for discontinuing therapy.

Introduction To describe the efficacy of infigratinib, a potent, selective fibroblast growth factor receptor (FGFR) 1-3 tyrosine kinase inhibitor, across lines of therapy (LOT) in patients with metastatic urothelial cancer (mUC). Patients and Methods Eligible patients had mUC and prior platinum-based chemotherapy, unless contraindicated, and activating FGFR3 mutation/fusion. Patients received infigratinib 125 mg orally daily (3 weeks on/1 week off) in a single-arm, open-label study. Primary endpoint: investigator-assessed confirmed objective response rate (ORR). Disease control rate (DCR), progression-free survival (PFS), best overall response (BOR) that included unconfirmed responses, and overall survival (OS) were also assessed. Subgroup analysis of efficacy and safety outcomes by LOT was performed. Results Sixty-seven patients were enrolled; 13 (19.4%) received infigratinib as early-line therapy for mUC due to ineligibility to receive platinum-based chemotherapy. Overall, ORR was 25.4% (95% CI 15.5-37.5) and DCR was 64.2% (95% CI 51.5-75.5). ORR was 30.8% (95% CI 9.1-61.4) with early-line infigratinib and 24.1% (95% CI 13.5-37.6) for ≥2 LOT. DCR was 46.2% (95% CI 19.2-74.9) for early-line and 68.5% (95% CI 54.4-80.5) for ≥2 LOT. PFS and OS appeared similar in both groups. Thirteen of 59 patients with a bladder primary tumor received early-line treatment with an ORR of 30.5% (95% CI 9.1-61.4), and 46 received ≥2 LOT with an ORR of 20.3% (95% CI 9.4-33.9); BOR was 38.5% (95% CI: 13.9-68.4%) and 42.6% (95% CI: 29.2-56.8%) in the early-line and salvage settings, respectively. Eight patients with upper tract urothelial carcinoma received salvage therapy (ORR, 50.0%; DCR, 100.0%). No significant differences in toxicities between LOT were observed. Conclusion Infigratinib has notable activity in patients with mUC regardless of LOT. The findings support the evaluation of infigratinib across different settings in mUC.

Stereotactic body radiation therapy (SBRT) has been shown to be safe and effective for delaying systemic treatment change among patients with metastatic renal cell carcinoma (mRCC). In this study, we sought to assess the genomic signatures of patients with mRCC who underwent SBRT for oligoprogression. A total of 30 patients with oligoprogressive disease were identified, the majority of whom had clear cell renal cell carcinoma (83.3%) and were receiving first-line treatment (53.3%). Genomic and transcriptomic sequencing were available in 20 and 16 patients, respectively. Duration of systemic treatment (DOT) was categorized as that prior (DOT[P]) and subsequent (DOT[S]) to radiation treatment. The median DOT(P) and DOT(S) were 15.1 and 18.3 mo, respectively, with a median DOT(S)/DOT(P) ratio of 1.4. Patients who had a DOT(S)/DOT(P) ratio of ≥1 had increased expression in pathways related to cell proliferation and development. In contrast, among patients with a ratio of ≤1, the reactive oxygen species pathway was enriched. This study highlights the potential role of genomics and transcriptomics to refine radiation treatment selection in patients with mRCC. PATIENT SUMMARY: In this study, we looked at mutations and genomic expressions among kidney cancer patients who responded better to stereotactic body radiotherapy. We found that enriched expression of certain pathways might play a role in response to radiotherapy.

PURPOSE Shapley additive explanation (SHAP) values represent a unified approach to interpreting predictions made by complex machine learning (ML) models, with superior consistency and accuracy compared with prior methods. We describe a novel application of SHAP values to the prediction of mortality risk in prostate cancer. METHODS Patients with nonmetastatic, node-negative prostate cancer, diagnosed between 2004 and 2015, were identified using the National Cancer Database. Model features were specified a priori: age, prostate-specific antigen (PSA), Gleason score, percent positive cores (PPC), comorbidity score, and clinical T stage. We trained a gradient-boosted tree model and applied SHAP values to model predictions. Open-source libraries in Python 3.7 were used for all analyses. RESULTS We identified 372,808 patients meeting the inclusion criteria. When analyzing the interaction between PSA and Gleason score, we demonstrated consistency with the literature using the example of low-PSA, high-Gleason prostate cancer, recently identified as a unique entity with a poor prognosis. When analyzing the PPC-Gleason score interaction, we identified a novel finding of stronger interaction effects in patients with Gleason ≥ 8 disease compared with Gleason 6-7 disease, particularly with PPC ≥ 50%. Subsequent confirmatory linear analyses supported this finding: 5-year overall survival in Gleason ≥ 8 patients was 87.7% with PPC < 50% versus 77.2% with PPC ≥ 50% ( P < .001), compared with 89.1% versus 86.0% in Gleason 7 patients ( P < .001), with a significant interaction term between PPC ≥ 50% and Gleason ≥ 8 ( P < .001). CONCLUSION We describe a novel application of SHAP values for modeling and visualizing nonlinear interaction effects in prostate cancer. This ML-based approach is a promising technique with the potential to meaningfully improve risk stratification and staging systems.

Introduction: The treatment paradigm of chronic lymphocytic leukemia (CLL) has changed significantly in the last few years. There are multiple front-line therapy options for the treatment of CLL, including chemoimmunotherapy (CIT), ibrutinib, and most recently venetoclax with obinutuzumab. The role of CIT has declined significantly for patients with CLL and novel agents are now being used more frequently in the front-line setting.Areas covered: Authors reviewed the latest data examining the role of CIT versus ibrutinib and ibrutinib combined with CIT for the treatment of CLL. Data reviewed here include: preliminary results from CLL12, long-term results of CLL8 and MD Anderson Cancer Center (MDACC) data with FCR, 7-year follow-up of PCYC-1102/1103 (phase 2 data) with ibrutinib, results of two-phase 3 randomized trials comparing CIT to ibrutinib, E1912 and A041202, and results of HELIOS and other phase 2 trials evaluating CIT combined with ibrutinib.Expert opinion: Treatment approaches for patients with CLL should be individualized and that there is still a role, albeit diminished, for CIT in the treatment of CLL, predominately in the front-line setting. Clinicians should focus on prognostic factors, patient preference, and evaluate short and long-term effects of CIT versus novel agents. Newly diagnosed patients should be encouraged to enroll in clinical trials.

Simple Summary Bladder cancer accounts for a significant burden to global public health. Despite advances in therapeutics with the advent of immunotherapy, only a small subset of patients benefit from immunotherapy. In this review, we examine the evidence that suggests that the TGF-β pathway may present a resistance mechanism to immunotherapy. In addition, we present possible therapies that may overcome the TGF-β resistance pathway in the treatment of bladder cancer. Abstract Bladder cancer accounts for nearly 200,000 deaths worldwide yearly. Urothelial carcinoma (UC) accounts for nearly 90% of cases of bladder cancer. Cisplatin-based chemotherapy has remained the mainstay of treatment in the first-line setting for locally advanced or metastatic UC. More recently, the treatment paradigm in the second-line setting was drastically altered with the approval of several immune checkpoint inhibitors (ICIs). Given that only a small subset of patients respond to ICI, further studies have been undertaken to understand potential resistance mechanisms to ICI. One potential resistance mechanism that has been identified in the setting of metastatic UC is the TGF-β signaling pathway. Several pre-clinical and ongoing clinical trials in multiple advanced tumor types have evaluated several therapies that target the TGF-β pathway. In addition, there are ongoing and planned clinical trials combining TGF-β inhibition with ICI, which may provide a promising therapeutic approach for patients with advanced and metastatic UC.

IMPORTANCE: Preclinical studies suggest that inhibition of single-stranded DNA repair by ataxia telangiectasia and Rad3 (ATR) may enhance the cytotoxicity of cisplatin, gemcitabine, and other chemotherapeutic agents. Cisplatin with gemcitabine remains the standard up-front therapy for treatment in patients with metastatic urothelial cancer. OBJECTIVE: To determine whether the use of the selective ATR inhibitor, berzosertib, could augment the activity of cisplatin with gemcitabine. DESIGN, SETTING, AND PARTICIPANTS: In a phase 2 randomized clinical trial, 87 patients across 23 centers in the National Cancer Institute Experimental Therapeutics Clinical Trials Network were randomized to receive either cisplatin with gemcitabine alone (control arm) or cisplatin with gemcitabine plus berzosertib (experimental arm). Key eligibility criteria included confirmed metastatic urothelial cancer, no prior cytotoxic therapy for metastatic disease, 12 months or more since perioperative therapy, and eligibility for cisplatin receipt based on standard criteria. The study was conducted from January 27, 2017, to December 15, 2020. INTERVENTIONS: In the control arm, cisplatin, 70 mg/m(2), was given on day 1 and gemcitabine, 1000 mg/m(2), was given on days 1 and 8 of a 21-day cycle. In the experimental arm, cisplatin, 60 mg/m(2), was given on day 1; gemcitabine, 875 mg/m(2), on days 1 and 8; and berzosertib, 90 mg/m(2), on days 2 and 9 of a 21-day cycle. MAIN OUTCOMES AND MEASURES: The primary end point of the study was progression-free survival. The analysis was on all patients who started therapy. RESULTS: Of the total of 87 patients randomized, 41 patients received cisplatin with gemcitabine alone and 46 received cisplatin with gemcitabine plus berzosertib. Median age was 67 (range, 32-84) years, and 68 patients (78%) were men. Median progression-free survival was 8.0 months for both arms (Bajorin risk-adjusted hazard ratio, 1.22; 95% CI, 0.72-2.08). Median overall survival was shorter with cisplatin with gemcitabine plus berzosertib compared with cisplatin with gemcitabine alone (14.4 vs 19.8 months; Bajorin risk-adjusted hazard ratio, 1.42; 95% CI, 0.76-2.68). Higher rates of grade 3 vs grade 4 thrombocytopenia (59% vs 39%) and neutropenia (37% vs 27%) were observed with cisplatin with gemcitabine and berzosertib compared with cisplatin with gemcitabine alone; consequently, more dose reductions were needed in the experimental arm. Patients in the experimental arm received a median cisplatin dose of 250 mg/m(2), which was significantly lower than the median dose of 370 mg/m(2) in the control arm (P

Previous studies have suggested that the gut microbiome influences the response to checkpoint inhibitors (CPIs) in patients with cancer. CBM588 is a bifidogenic live bacterial product that we postulated could augment CPI response through modulation of the gut microbiome. In this open-label, single-center study (NCT03829111), 30 treatment-naive patients with metastatic renal cell carcinoma with clear cell and/or sarcomatoid histology and intermediate- or poor-risk disease were randomized 2:1 to receive nivolumab and ipilimumab with or without daily oral CBM588, respectively. Stool metagenomic sequencing was performed at multiple timepoints. The primary endpoint to compare the relative abundance of Bifidobacterium spp. at baseline and at 12 weeks was not met, and no significant differences in Bifidobacterium spp. or Shannon index associated with the addition of CBM588 to nivolumab–ipilimumab were detected. Secondary endpoints included response rate, progression-free survival (PFS) and toxicity. PFS was significantly longer in patients receiving nivolumab–ipilimumab with CBM588 than without (12.7 months versus 2.5 months, hazard ratio 0.15, 95% confidence interval 0.05–0.47, P = 0.001). Although not statistically significant, the response rate was also higher in patients receiving CBM588 (58% versus 20%, P = 0.06). No significant difference in toxicity was observed between the study arms. The data suggest that CBM588 appears to enhance the clinical outcome in patients with metastatic renal cell carcinoma treated with nivolumab–ipilimumab. Larger studies are warranted to confirm this clinical observation and elucidate the mechanism of action and the effects on microbiome and immune compartments.

Background: In a previous randomized phase I trial, addition of CBM-588 to the nivolumab/ipilimumab (N/I) regimen showed improved objective response rate, clinical benefit rate, and progression free survival compared to N/I alone in patients (pts) with metastatic renal cell carcinoma (mRCC; Meza et al., ASCO 2021). Furthermore, genomic alterations such as PBRM1 have been associated with clinical benefit to anti-PD-1 monotherapy in patients with mRCC (Miao et al., Science 2018). The primary aim of this study was to investigate tumor genomic characteristics according to treatment arms. Methods: We retrospectively identified pts with mRCC who received N/I alone or with CBM-588 supplementation along with whole exome and transcriptome sequencing (Ashion Analytics). Responses were measured according to RECIST v1.1. A two-tailed Fischer’s exact test was performed to compare genomic characteristics across arms. Results: In this study, 29 mRCC pts were randomized to receive N/I +/- CBM-588 and 21 (72%) pts (71% in N/I with CBM-588 arm and 29% N/I arm) had available genomic data. Within this cohort, the median age was 66.8 (range 46-90) and 71% of pts were male. Eleven (52.4%) pts had clear-cell histology and 10 (47.6%) pts had sarcomatoid features; 15 pts received N/I with CBM-588 and 6 pts received N/I alone. The most commonly mutated genes in the overall cohort were VHL (61.9%), PBRM1 (42.9%), and SETD2 (33.3%). Alterations in VHL, PBRM1, and SETD2 were seen in 66.7% vs. 73.3% (p=0.115), 50.0% vs. 40.0% (p=0.523) and 33.3% vs. 33.3% (p=0.686), in N/I vs. N/I with CBM-588 arm, respectively. Conclusions: There was no significant difference observed in clinically relevant genomic features across study arms. The clinical benefit from CBM-588 appears to be independent of tumor genomic characteristics. More extensive investigations are needed to characterize the determinants of benefit from CBM-588 supplementation. Citation Format: Daniela V. Castro, Nazli Dizman, Zeynep B. Zengin, Jasnoor Malhotra, Luis A. Meza, Ramya Muddasani, Ameish Govindarajan, Neal S. Chawla, Alex Chehrazi-Raffle, JoAnn Hsu, Paulo G. Bergerot, Cristiane D. Bergerot, Tanya B. Dorff, Yung Lyou, Sumanta K. Pal. Genomic characteristics of nivolumab/ipilimumab with or without CBM-588 supplementation in patients with metastatic renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6293.

4510 Background: The negative association between ICI response and abx therapy is well defined (Derosa et al Cancer Discov 2021). Paradoxically, a retrospective assessment of the live bacterial product (LBP) CBM588 in patients (pts) with advanced lung cancer showed improved outcome with ICIs when the combination of CBM588 and abx (as compared to CBM588 alone) was employed (Tomita et al Cancer Immunol Res 2020). We postulated that the microbial resistome (genes encoding antimicrobial resistance) could shift in a manner with CBM588 therapy that facilitated ICI response. Methods: Pts with newly diagnosed mRCC with clear cell and/or sarcomatoid histology and intermediate/high risk disease per IMDC criteria were randomized to nivolumab/ipilimumab (nivo/ipi) or nivo/ipi/CBM588 in a 1:2 ratio. Stool samples were collected at baseline and week 12. Whole-metagenome sequencing was performed to analyze stool microbiome composition. Abx resistance genes (RGs) were inferred using publicly available database (McArthur et al. Antimicrob Agents Chemother 2013), and groups of abx RGs for various classes of abx were characterized. Wilcoxon signed-rank test was used for comparison of abx RG abundance between baseline and week 12 in each treatment arm and in responders (R) and non-responders (NR). Results: The study enrolled 30 pts, with the final analysis including 29 eligible pts (median age: 66 years, M:F 21:8, nivo/ipi: 19 pts, nivo/ipi/CBM588:10 pts). Objective response was 20% and 58% in nivo/ipi and nivo/ipi/CBM588 arms, respectively. The overall abundance of abx RGs remained unchanged between baseline and week 12 in pts receiving nivo/ipi alone. In contrast, a decrease in abx RGs was observed in pts receiving nivo/ipi with CBM588 arm from baseline to week 12 (p = 0.042 in Rs; p = 0.078 in NRs). More specifically, nivo/ipi/CBM588 treatment led to a significant reduction in fosfomycin RGs and nitroimidazole (e.g., metronidazole) RGs in both pts with R (p = 0.019 and 0.042, respectively) and NR (p = 0.031 and p = 0.031, respectively). A multitude of other clinically relevant abx RGs were downregulated in pts receiving CBM588, including those mediating resistance to glycopeptide (e.g., vancomycin) and lincosamide (e.g., clindamycin) abx. Conclusions: In the first interrogation of the resistome in mRCC, we demonstrate that CBM588 decreases abx RGs associated with multiple commonly used classes of abx. Abx clear commensals and increase pathogenic (abx resistant) bacteria in the gut. Based on our data, we formulate the hypothesis that combining abx with CBM588 may decrease potentially pathobionts and favor butyrogenic species, thereby improving CPI response. Clinical studies using CBM588 with abx priming may be warranted. Clinical trial information: NCT03829111.

TPS4606 Background: Combination therapy with the immune checkpoint inhibitor (ICI) nivolumab (nivo) and the tyrosine kinase inhibitor cabozantinib (cabo) is a new standard of care for first line treatment of patients with clear cell mRCC. However, despite the improved clinical benefit obtained with this regimen, a subgroup of patients still presents with progressive disease as best response (Choueiri et al NEJM 2021). There is now evidence supporting the role of the gut microbiome in mediating ICI activity (Routy et al Science 2018) and certain bacterial species, such as Bifidobacterium spp. in predisposing clinical response in patients with mRCC receiving ICIs (Salgia et al Eur Urol 2020). Moreover, recent evidence from a phase I clinical trial suggests that the addition of CBM588, a live probiotic comprised primarily of Clostridium butyricum, can enhance clinical response in patients with mRCC receiving nivolumab plus ipilimumab without incurring added toxicity (Meza et al ASCO, 2021). Herein we present the study design of an ongoing phase I study evaluating the biological effect of CBM588 in combination with cabozantinib plus nivolumab in patients with mRCC. Methods: This is an open label, randomized, single institution phase 1 trial for patients with confirmed mRCC with clear cell, papillary, or sarcomatoid components, who have not received prior systemic therapy for metastatic disease. A total of 30 eligible patient will be randomized 1:2 to receive either cabo/nivo at the standard dose/schedule (40mg PO QD and 480mg IV /4wks, respectively) alone or with CBM588 dosed at 80mg PO bid. The primary objective of the study is to determine the biologic effect of CBM588 with cabo/nivo in the modulation of the gut microbiome. This will be done by assessing the changes in Bifidobacterium spp. abundance and Shannon index (a measure of microbiome diversity) in stool specimens. Stool will be collected for bacteriomic profiling at baseline and after 12 weeks of treatment. Metagenomic sequencing will be performed using previously published methods (Dizman et al Cancer Med 2020). Secondary objectives include determining the effect of CBM588 on (1) clinical efficacy, through overall survival, response rate, and progression-free survival; (2) systemic immunomodulation, through assessment of changes in circulating Tregs, circulating cytokines/chemokines, etc; and (3) toxicities. A two-group t-test with a one-sided type I error of 0.05 will be used to assess the study primary endpoint. Clinical trial information: NCT05122546 .

The recent classification of colon cancer into molecular subtypes revealed that patients with the poorest prognosis harbor tumors with the lowest levels of Wnt signaling. This is contrary to the general understanding that overactive Wnt signaling promotes tumor progression from early initiation stages through to the later stages including invasion and metastasis. Here, we directly test this assumption by reducing the activity of ß-catenin–dependent Wnt signaling in colon cancer cell lines at either an upstream or downstream step in the pathway. We determine that Wnt-reduced cancer cells exhibit a more aggressive disease phenotype, including increased mobility in vitro and disruptive invasion into mucosa and smooth muscle in an orthotopic mouse model. RNA sequencing reveals that interference with Wnt signaling leads to an upregulation of gene programs that favor cell migration and invasion and a downregulation of inflammation signatures in the tumor microenvironment. We identify a set of upregulated genes common among the Wnt perturbations that are predictive of poor patient outcomes in early-invasive colon cancer. Our findings suggest that while targeting Wnt signaling may reduce tumor burden, an inadvertent side effect is the emergence of invasive cancer. Implications: Decreased Wnt signaling in colon tumors leads to a more aggressive disease phenotype due to an upregulation of gene programs favoring cell migration in the tumor and downregulation of inflammation programs in the tumor microenvironment; these impacts must be carefully considered in developing Wnt-targeting therapies.

Background Infigratinib (BGJ398) is a potent, selective fibroblast growth factor receptor (FGFR) 1–3 inhibitor with significant activity in metastatic urothelial carcinoma (mUC) bearing FGFR3 alterations. It can cause hyperphosphatemia due to the “on-target” class effect of FGFR1 inhibition. Objective To investigate the relationship between hyperphosphatemia and treatment response in patients with mUC. Intervention Oral infigratinib 125 mg/d for 21 d every 28 d. Design, setting, and participants Data from patients treated with infigratinib in a phase I trial with platinum-refractory mUC and activating FGFR3 alterations were retrospectively analyzed for clinical efficacy in relation to serum hyperphosphatemia. The relationship between plasma infigratinib concentration and phosphorous levels was also assessed. Outcome measurements and statistical analysis Clinical outcomes were compared in groups with/without hyperphosphatemia. Results and limitations Of the 67 patients enrolled, 48 (71.6%) had hyperphosphatemia on one or more laboratory tests. Findings in patients with versus without hyperphosphatemia were the following: overall response rate 33.3% (95% confidence interval [CI] 20.4–48.4) versus 5.3% (95% CI 0.1–26.0); disease control rate 75.0% (95% CI 60.4–86.4) versus 36.8% (95% CI 16.3–61.6). This trend was maintained in a 1-mo landmark analysis. Pharmacokinetic/pharmacodynamic analysis showed that serum phosphorus levels and physiologic infigratinib concentrations were correlated positively. Key limitations include retrospective design, lack of comparator, and limited sample size. Conclusions This is the first published study to suggest that hyperphosphatemia caused by FGFR inhibitors, such as infigratinib, can be a surrogate biomarker for treatment response. These findings are consistent with other reported observations and will need to be validated further in a larger prospective trial. Patient summary Targeted therapy is a new paradigm in treating bladder cancer. In a study using infigratinib, a drug that targets mutations in a gene called fibroblast growth factor receptor 3 (FGFR3), we found that elevated levels of phosphorous were associated with greater clinical benefit. In the future, these data may help inform treatment strategies.

BackgroundThe clinical significance of tumor-specific genomic alterations in metastatic renal cell carcinoma (mRCC) is emerging, with several studies suggesting an association between PBRM1 mutations and response with immunotherapy (IO). We sought to determine genomic predictors of differential response to vascular endothelial growth factor–tyrosine kinase inhibitors (VEGF-TKIs) and IO.MethodsConsecutive patients who underwent genomic profiling were identified; patients receiving either VEGF-TKIs or IO were included. Clinical tumor-normal whole exome sequencing and tumor whole transcriptome sequencing test were performed using a Clinical Laboratory Improvement Amendments (CLIA)-certified assay (Ashion Analytics; Phoenix, Arizona, USA). Genomic findings were compared between patients with clinical benefit (CB; complete/partial response or stable disease for >6 months) and no clinical benefit (NCB) in VEGF-TKI-treated patient cohort and IO-treated patient cohort.Results91 patients received genomic profiling and 58 patients received VEGF-TKI and/or IO therapy. 17 received sequenced treatment involving both VEGF-TKI and IO, resulting in 32 patients in the IO cohort and 43 patients in the VEGF-TKI cohort. The most commonly used IO and VEGF-TKIs were nivolumab (66%) and sunitinib (40%). The most frequently detected alterations in the overall cohort were in VHL (64%), PBRM1 (38%), SETD2 (24%), KDM5C (17%) and TERT (12%). TERT promoter mutations were associated with NCB in the IO cohort (p=0.038); transcriptomic analysis revealed multiple differentially regulated pathways downstream of TERT. TERT promoter mutations and PBRM1 mutations were found to be mutually exclusive. While PBRM1 mutations were more prevalent in patients with CB with IO and VEGF-TKIs, no statistically significant association was found.ConclusionsOur analysis found that TERT promoter mutations may be a negative predictor of outcome with IO and are mutually exclusive with PBRM1 loss-of-function mutations.

Prostate cancer is a significant public health burden and one of the most common cancers globally and in the United States. The current cornerstone of prostate cancer systemic treatment involves the suppression of androgen receptor (AR) signaling, either by reducing the body's testosterone production or inhibiting its binding to AR and its subsequent gene regulatory network driving carcinogenesis. This signaling pathway plays a central role in both hormone sensitive and castration resistant prostate cancer (CRPC), as evidenced by survival benefit when AR-targeted therapies are applied in the setting of CRPC. With the development of increasingly potent central and peripherally acting androgen targeting agents physicians treating prostate cancer can expect to treat their patients for a longer duration with a larger selection of effective agents. In this setting clinicians are now faced with questions of how to best tailor treatments for the prostate cancer patient to not only maximize overall survival but also optimize the quality of life and mitigate toxicity. In this manuscript we discuss the newer hormone therapy agents for prostate cancer and highlight what they indicate about optimizing medical castration, and the potential value of peripheral blockade.

57 Background: Gonadotropin-releasing hormone (GnRH) antagonists were developed in part to avoid testosterone flare to achieve faster and more consistent testosterone suppression. Relugolix (Rel) was given FDA approval for use in prostate cancer (PCa) based on the phase 3 HERO trial but some real-world concerns include compliance, affordability, and performance in patients transitioning from another GnRH agent or in combination with other therapeutic agents. The aim of our study was to evaluate the real-world implications of prescribing and/or switching to Rel. Methods: A single institution retrospective study was conducted on patients prescribed Rel. Treatment data including concomitant therapeutic agents were collected. Compliance data was measured via chart review and pharmacy dispensary records. Patients were classified as either newly castrated or transitioning from another agent. PSA and testosterone levels were tabulated. Reported adverse effects (AE), and reasons for discontinuation if applicable including financial toxicity were noted. Results: 50 patients were reviewed; 15 (30%) treated for adjuvant, 18 (36%) for biochemical recurrence, and 17 (34%) for metastatic PCa. 12% were on concomitant therapy with abiraterone, 4% with enzalutamide, and 2% with apalutamide. 80% reported compliance to Rel. 5 (10%) never filled the prescription. 30 (60%) were newly castrated (castration restart or naïve) while 16 (36%) were transitioned from another GnRH. No changes in PSA or testosterone were noted in patients switched form injected GnRH to Rel. The most common documented AE effects included hot flashes 24%, fatigue, 29%, and weight gain 9%. No unexpected toxicity was reported in combination with abiraterone. Of 11 patients who discontinued therapy, 45% did so due to cost, 36% due to AE, and 27% due to therapy completion. 4 of 16 (25%) of those who transitioned from injection GnRH felt symptoms were worse on Rel. Conclusions: In our early experience with Rel, patients did comply to therapy based on self-reporting, pharmacy fill dates, and laboratory evidence of castration. However, financial toxicity was and remains a significant barrier both accessing Rel and remaining on Rel therapy. While a differential toxicity was reported by those who switched from injection GnRH, sometimes leading to discontinuation, there were no new safety signals reported, especially in combination with abiraterone.

185 Background: Short-term effectiveness of COVID-19 vaccination is widely demonstrated, but the emerging real-world data suggest that immunity may wane over-time (Levin et al. NEJM 2021). Herein we aimed to explore the long-term efficacy of the COVID-19 vaccination among pts with genitourinary cancer. Methods: In this study, pts with genitourinary malignancies (prostate, kidney, and bladder cancers) who had not received COVID-19 vaccination were included. Blood samples were collected prior to and after one dose of either an adenovirus- or mRNA-based COVID-19 vaccine at the 2- and 6-month timepoints. Additional blood samples from pts receiving systemic treatment were collected at 3 consecutive therapy cycles following vaccination. Antibody titers were assessed using the SCoV-2 Detect IgG ELISA assay and results were reported as immune status ratios (ISR). T-cell receptor (TCR) repertoire sequencing was performed using the MiXCR software (MiLabs) and custom strips were used to assess TCR abundance and homology clustering. Results: A total of 183 pts were enrolled, and 136 pts provided baseline blood samples. Among these, 59 (8:51 F:M) provided samples for both the 2- and 6-month timepoints by the 10/6/2021 data cut-off. In this subset of pts, median age was 66 (range 48-85) and 33 (55.9%), 25 (42.4%), and 1 (1.7%) pts had prostate, kidney, and bladder cancer, respectively. A majority of the pts (93.2%) were on systemic treatment with 23.7% on immune checkpoint inhibitors, 18.6% on targeted agents, and 1.7% on chemotherapy. The most commonly administered vaccines were BNT162b2 (61.0%) followed by mRNA-1273 (37.3%) and Ad26.COV2.S (1.7%). The mean (±standard deviation) ISR values at baseline and 2 months were 0.68±1.59 and 6.62±1.75, respectively. At the 6-month timepoint, mean ISR was 5.46±1.61; this was significantly lower than the 2-month antibody titers (p < 0.0001), and reflects a reduction of 17.6%. Further data on TCR sequencing will be presented at the meeting. Conclusions: To our knowledge, this is the first data assessing the long-term serologic outcomes of COVID-19 vaccination in pts with cancer. Our data suggest waning immunity over time in cancer pts. Strategies to prolong host immunity against SARS COV-2 (e.g., booster vaccination) are likely warranted.

371 Background: The role of gut microbial composition as a determinant of clinical outcomes has been well established in several cancers, including metastatic renal cell carcinoma (mRCC) (Routy et al Science 2018). A growing body of evidence suggests that examining the metabolic function of microbial communities may provide a more insightful understanding of these associations (Helmink et al Nature Medicine 2019). Herein, we aimed to examine the effect of nivolumab/ipilimumab with or without CBM588 on clinical outcomes and gut microbiome functionality. Methods: Treatment naïve mRCC pts with clear cell and/or sarcomatoid histology and IMDC intermediate/high risk disease were enrolled and randomized into receiving nivolumab/ipilimumab or nivolumab/ipilimumab with CBM588 in 1:2 fashion. Whole metagenome sequencing was performed on stool samples collected at baseline and week 12. Generated MetaPhlan 3.0 data was run through HUMAnN 3.0 to identify differentially expressed metabolic pathways between two timepoints in each arm and with respect to treatment response. Results: A total of 30 pts were enrolled and randomized, and 29 pts were eligible for analysis as one patient was excluded as tumor tissue next-generation sequencing revealed genomic alterations pathognomonic for sarcoma after initiation of protocol-based therapy. Median age of the participants was 66 years, 21 pts (72%) were male, 10 pts (34%) had sarcomatoid features, and 29 pts (45%) had prior nephrectomy. Objective response was achieved in 58% and 20% of the pts in nivolumab/ipilimumab/CBM-588 and nivolumab/ipilimumab arm, respectively. Significant changes in 40 metabolic pathways (37 with upregulation and 3 with downregulation) in nivolumab/ipilimumab arm and 52 metabolic pathways (49 with downregulation and 3 with upregulation) in nivolumab/ipilimumab with CBM588 arm were identified. In detail, dTDP-β-L-rhamnose biosynthesis, L-lysine biosynthesis II and superpathway of pyrimidine ribonucleosides degradation pathways were found upregulated while O-antigen building blocks biosynthesis (E. coli) pathway was found downregulated after treatment with nivolumab/ipilimumab and CBM588 (p = 0.001, p = 0.007, p = 0.037, p = 0.005 respectively). Heatmaps detailing the dynamics of metabolic pathway expressions in regard to response in each arm will be presented. Conclusions: We observed an increase in the activity of the pathways associated with butyrate consumption and a resultant decrease in glycolytic dependence. Further, suppression of the pathogenic E. coli function was observed, suggesting a role for CBM588 in protection from pathogenic species. Our findings provide mechanistic evidence for the effect of the addition of CBM588 to nivolumab/ipilimumab on gut microbiome function and resultant improvement in clinical outcomes in mRCC. Clinical trial information: NCT03829111.

561 Background: Recently published data suggests that the presence of a TERT promoter mutation is predictive of superior overall survival (OS) in patients (pts) with advanced/metastatic bladder cancer (mUC) treated with an immune checkpoint inhibitor (ICI) (Kouchkovsky et al, JITC 2021). We aim to validate the results of this study in a large independent cohort. Methods: Pts with mUC treated at two tertiary cancer centers with available genomic data collected in the course of routine clinical care were identified retrospectively. Pts that had received at least one line of ICI therapy in the metastatic setting were selected. Demographic and treatment data were collected, with pts divided into two groups based on the presence or absence of TERT mutation status ( TERTm or TERTwt, respectively). We evaluated OS from diagnosis of at least muscle invasive disease, progression free survival (PFS), and objective response rate (ORR) with ICI therapy across the two groups. OS in our cohort was compared with findings from pts with bladder cancer in The Cancer Genome Atlas (TCGA) database. Results: From our combined data sets, a total of 166 pts had available genomic data, with 64 TERTm pts (52:12 M:F) and 58 TERTwt pts (32:26 M:F) meeting criteria for inclusion. Median age at diagnosis was 67 in both groups. The site of primary disease was bladder in 54 (84%) TERTm vs. 41 (71%) in TERTwt; 10 (16%) and 17 (29%) had upper tract disease, respectively. 47 (73%) TERTm pts and 40 (69%) TERTwt pts had pure urothelial disease; 17 (27%) and 18 (31%) pts had mixed/pure variant histology, respectively. 37 (58%) and 42 (72%) pts received first-line ICI therapy whereas 27 (42%) and 16 (28%) received subsequent-line therapy in TERTm and TERTwt, respectively. At the time of analysis, there were 24 (38%) patients alive in TERTm, and 23 (40%) patients alive in TERTwt. OS was 35 vs. 36 mos (95% CI 0.62-1.51, P=0.66) in TERTm and TERTwt, respectively. PFS on ICI therapy was 4.6 vs. 5.3 mos (95% CI 0.58-1.34, P≥0.99) in TERTm and TERTwt, respectively. ORR was 75% in TERTm and 50% in TERTwt (P=.004). OS in the TCGA database was 35 mos in TERTm and 47 in TERTwt (P=0.19) from a total of 311 and 127 pts, respectively. Conclusions: In contrast to previously published data, our data show no difference in OS and PFS on the basis of TERT mutational status in pts with mUC treated with ICI therapy. Further analysis from larger datasets is needed to reconcile the role of TERT mutations within this patient population.

374 Background: Treatment with N/I with the addition of CBM588, a live bacterial product comprised primarily of Clostridium butyricum, improved PFS and RR versus N/I alone when used as first line treatment for patients with mRCC (Meza et al ASCO 2021). Increased abundance of certain bacterial species in the gut microbiome have been associated with the development of treatment related adverse events (TREAs) in lung cancer patients receiving immunotherapy (Chau et al BMC Cancer 2021). However, this association has not yet been delineated in the setting of mRCC. Here, we present results of an exploratory analysis assessing the differences in stool microbiome composition between patients who experienced grade (G) 3/4 TREAs and those who did not. Methods: Patients were randomized 2:1 to receive N/I with or without CBM588. Stool collection for bacteriomic profiling was planned at baseline and after 12 weeks of therapy for all randomized patients. Whole metagenome sequencing was performed using previously published methods (Dizman et al Cancer Med 2021) and differences in microbiome composition were measured based on the occurrence of G 3/4 TREAs. Results: 30 patients were enrolled and 29 included in the study. At the time of data cutoff (April 15, 2021) the median follow up was 12.2 months (95% confidence intervals [CI], 10.6-13.8). Grade 3/4 TRAEs were experienced in 52, 50, and 53% of patients in the overall cohort, control, and intervention arms, respectively (p = NS). Among the most common G 3/4 TRAEs, subjects experienced fatigue, diarrhea, and hyperglycemia. Patients with a complete set of stool samples were included for the microbiome analysis (n = 26). In patients who experienced G 3/4 TRAEs, a significantly greater baseline abundance of Escherichia coli, Klebsiella spp. and Blautia spp. (p = 0.02, 0.03, 0.05) were seen when compared to those not experiencing G 3/4 TRAEs. In contrast, Bacteroides intestinalis and B. thetaiotamicron, were observed in significantly higher abundances in baseline stool specimens of patients who did not experience G 3/4 TREAs (p = 0.03 for both). No significant differences were seen for any of these species at the 12-week timepoint. Conclusions: We are among the first to investigate the differences in baseline stool microbiome in mRCC patients experiencing G 3/4 TRAEs while receiving immunotherapy. Our results suggest that certain taxa of bacteria are predictors of the development of serious TRAEs. Larger cohorts are needed to corroborate these findings. Clinical trial information: NCT03829111.

Breast cancer is the second leading cause of cancer-related deaths in women in the United States. Of these women, 5–10% have an inherited form of breast cancer with a mutation in a major gene, such as the breast cancer susceptibility genes 1 or 2 (BRCA1 or BRCA2). Triple negative (the most common subtype of BRCA1-associated breast cancers) and Her2-positive breast cancer patients have more frequently been observed to develop central nervous system (CNS) metastases compared to other molecular subtypes of breast cancers. However, it remains an open question if BRCA2-associated breast cancers also have a higher propensity to develop CNS metastases. Here we report a rare case of recurrent BRCA2-associated breast cancer which manifested as orbital metastases. At the time of this publication, this is one of the first cases of BRCA2-associated breast cancer to present with orbital metastases. In this article, we discuss the diagnostic challenges and review the literature regarding this rare presentation.

In the last couple of decades, breast conservation therapy, which utilizes a combination of surgery, radiotherapy, and endocrine or chemotherapy, has become the standard of care for treating early-stage breast cancer. This practice has been greatly beneficial in the improvement of the patient’s quality of life but has also led to the increased use of radiotherapy and associated soft-tissue sarcomas, with angiosarcoma being the most common malignancy. Radiation-associated angiosarcoma (RAS) of the breast is a rare phenomenon, which has been reported to occur in approximately 0.9 out of 1,000 cases, with a reported onset as late as 23 years following radiotherapy. Here we report 2 cases of RAS that occurred within 6 and 13 years following radiotherapy of their primary breast lesion. We discuss the diagnostic and therapeutic challenges regarding this disease and review the current literature. This case report serves as cautionary lessons on the importance of considering RAS of the breast in the differential diagnosis during evaluation for recurrent breast neoplasms. Ongoing clinical trials using combinations of vascular endothelial growth factor inhibitors and chemotherapy may provide future avenues of treatment for this difficult-to-treat disease.

Background: Preliminary studies have characterized potential adverse effects associated with COVID-19 vaccination in pts with cancer. However, biological characterization of vaccine response has yet to be performed. Methods: Eligible pts with advanced GU cancers (metastatic/unresectable prostate, bladder and renal cell carcinoma [RCC]) and had not yet received COVID-19 vaccination. Pts were consented to receive sequential blood draws prior to vaccination and at landmarks of 2, 6, and 12 mos following vaccination. Pts on systemic treatment had additional blood draws coinciding with their first 3 cycles of therapy following vaccination. Ab titers to SARS-CoV-2 were quantified via ELISA and reported as an immune status ratio (ISR). RNA was extracted from PBMC aliquots, converted into cDNA and TCR α/β sequences were selectively amplified. TCR abundance and homology clustering was performed using custom scripts. Results: As of May 14, 2021, 130 pts had consented to the study of whom 126 pts submitted baseline (BL) specimens. The current analysis focuses on 56 pts who submitted cycle 1 (C1) specimens. Among these, 29, 26, and 1 pts had RCC, prostate and bladder cancer, respectively;19 were on checkpoint inhibitor (CPI)-based regimens while 37 were on non-CPI regimens. BNT162b2 (Pfizer) was the most commonly administered vaccine in the cohort (n=29), followed by mRNA-1273 (Moderna;n=26). COVID-19 Ab titers increased significantly from BL to C1 across the cohort from 0.19 (interquartile range [IQR] 0.12-0.18) to 4.37 (IQR 0.2-6.60;P

The highly conserved Wnt signalling pathway plays an important role in embryonic development and disease pathogenesis, most notably cancer. The ‘canonical’ or β-catenin-dependent Wnt signal initiates at the cell plasma membrane with the binding of Wnt proteins to Frizzled:LRP5/LRP6 receptor complexes and is mediated by the translocation of the transcription co-activator protein, β-catenin, into the nucleus. β-Catenin then forms a complex with T-cell factor (TCF)/lymphoid enhancer binding factor (LEF) transcription factors to regulate multiple gene programmes. These programmes play roles in cell proliferation, migration, vasculogenesis, survival and metabolism. Mutations in Wnt signalling pathway components lead to constitutively active Wnt signalling that drives aberrant expression of these programmes and development of cancer. It has been a longstanding and challenging goal to develop therapies that can interfere with the TCF/LEF–β-catenin transcriptional complex. This review will focus on the (i) structural considerations for targeting the TCF/LEF–β-catenin and co-regulatory complexes in the nucleus, (ii) current molecules that directly target TCF/LEF–β-catenin activity and (iii) ideas for targeting newly discovered components of the TCF/LEF–β-catenin complex and/or downstream gene programmes regulated by these complexes. Linked Articles This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc

The recent classification of colon cancer into molecular subtypes revealed that patients with the poorest prognosis harbor tumors with the lowest levels of Wnt signaling. This is contrary to the long-standing understanding that overactive Wnt signaling promotes tumor progression from early initiation stages through to the later stages including invasion and metastasis. Here, we lower the levels of Wnt signaling in colon cancer via interference with two different steps in the pathway that lie upstream or downstream of the effector protein ß-catenin. We find that these Wnt-reduced cancer cells exhibit a more aggressive disease phenotype, including increased mobility in vitro and localized invasion in an orthotopic mouse model. RNA sequencing reveals that interference with Wnt signaling leads to an upregulation of gene programs that favor cell migration and invasion. We identify a set of upregulated genes common among the Wnt perturbations and find that elevated expression of these genes is strongly predictive of poor patient outcomes in early-invasive colon cancer. These genes may have clinical applications as patient biomarkers or new drug targets to be used in concert with existing therapies.One Sentence SummaryLow Wnt Signaling Leads to Invasive Tumor Phenotypes in Colorectal Cancer.

4507 Background: Cisplatin with gemcitabine (CG) remains the standard upfront chemotherapy regimen for metastatic urothelial cancer (mUC). Preclinical synergy was noted between cisplatin and berzosertib, a selective ATR inhibitor. The current study sought to determine if the combination of berzosertib and CG could improve clinical outcomes in mUC. Methods: An open-label, randomized study was conducted across 23 centers in the United States through the Experimental Therapeutics Clinical Trials Network of the National Cancer Institute. Key eligibility criteria included confirmed mUC, no prior cytotoxic therapy for metastatic disease, ≥ 12 months since perioperative therapy and eligibility for cisplatin based on standard criteria. Patients (pts) were randomized to receive either CG alone (control arm) or CG plus berzosertib (experimental arm). In the control arm, 70 mg/m2 of cisplatin was given on day 1 and gemcitabine at 1000 mg/m2 on days 1 and 8 of a 21-day cycle. In the experimental arm, 60 mg/m2 of cisplatin was given on day 1, gemcitabine at 875 mg/m2 on days 1 and 8 and berzosertib at 90 mg/m2 on days 2 and 9 of a 21-day cycle. The primary endpoint of the study was progression-free survival (PFS), with secondary endpoints including response rate (RR), overall survival (OS) and toxicity. Results: A total of 87 pts (median age 67; M:F 68:19) were randomized; 41 pts received CG alone while 46 received CG with berzosertib. Visceral metastases were present in 49% of pts and 52%, 45% and 3% of pts were Bajorin risk 0, 1 and 2, respectively. Median PFS was 8.0 months for both arms (Bajorin risk adjusted hazard ratio [HR] 1.22, 95% confidence interval [CI] 0.72-2.08). RR was 54%(4 CR, 21 PR) in the CG with berzosertib arm and 63% (4 CR, 22 PR) in CG alone arm (P = 0.66). Median OS was shorter with CG with berzosertib as compared to CG alone (14.4 versus 19.8 months; Bajorin risk adjusted HR 1.42, 95%CI 0.76-2.68). Notably higher rates of grade 3/4 thrombocytopenia (59% vs 39%) and neutropenia (37% vs 27%) were observed with CG plus berzosertib compared to CG alone. Higher rates of toxicity-related discontinuation were seen in the experimental arm (24% vs 15%), and the median cumulative cisplatin dose in the experimental arm was 250 mg/m2, as compared to 370 mg/m2 in the control arm (P < 0.001). Conclusions: No improvement in PFS was observed with the addition of berzosertib to CG, and a trend towards inferior survival was observed. These results suggest caution in reducing the starting dose of cytotoxic therapy to accommodate addition of a myelosuppressive agent, as in the experimental arm of this study. Clinical trial information: NCT02567409.

4513 Background: Recent evidence suggests that the gut microbiome is a potent mediator of immune checkpoint inhibitor (ICI) activity in metastatic renal cell carcinoma (mRCC), with both specific bacterial species and cumulative microbial diversity driving response (Routy et al Science 2018; Salgia et al Eur Urol 2020). We examined whether the butyrate-producing bacterium Clostridium butyricum, the key constituent of CBM-588, could modulate the gut microbiome in patients (pts) with mRCC receiving nivolumab/ipilimumab (N/I) and secondarily improve clinical outcome. Methods: An open-label, randomized study was conducted, with key eligibility criteria including confirmed clear cell and/or sarcomatoid mRCC, intermediate/poor risk by IMDC criteria and no systemic therapy for metastatic disease. Patients were randomized 2:1 to receive either N/I+CBM-588 or N/I alone. N/I was dosed at 3 mg/kg and 1 mg/kg IV every 3 weeks for 12 weeks, followed by N at 480 mg IV every 4 weeks. CBM-588 was dosed orally at 80 mg bid. Stool was collected for bacteriomic profiling at baseline and 12 weeks. Metagenomic sequencing was employed using previously published methods (Dizman et al Cancer Med 2020). The primary endpoint of the study was change in Bifidobacterium spp. from baseline to week 12. Secondary endpoints included change in microbial diversity and clinical outcomes including response rate (RR) and progression-free survival (PFS). Results: 30 pts were randomized between April 2019 and Nov 2020; 1 pt was excluded after genomic sequencing clarified a diagnosis of sarcoma. Among 29 evaluable patients (21:8 M:F), median age was 66, 10 pts (34%) had sarcomatoid features and 24 pts (83%) were intermediate risk. Metagenomic sequencing of paired stool specimens showed an 8-fold increase in B. bifidum and a 6-fold increase in B. adolescentis in pts receiving N/I+CBM-588 from baseline to week 12. C. butyricum was detected only in pts receiving CBM-588. Pathogenic species (e.g., Escherichia. coli and Klebsiella spp.) were more prevalent in pts not receiving CBM-588. RR was significantly higher among pts receiving N/I+CBM-588 vs N/I alone (59% vs 11%; P = 0.024). Median PFS was also prolonged with the addition of CBM-588 to N/I (NR vs 11 weeks; P < 0.001). No significant difference in grade 3/4 toxicities were observed between study arms. Conclusions: This is the first randomized, prospective study to suggest enhancement of ICI response with a live bacterial product. The observed clinical impact is corroborated by biologic findings supporting gut modulation by CBM-588. Clinical trial information: NCT03829111.

474 Background: Advanced/metastatic urothelial carcinoma (UC) is a significant public health burden with median overall survival of 15 months. Although, immune checkpoint inhibitors (ICI) have provided an additional second line treatment option, only 15-40% of patients will respond. There has been much effort in determining the mechanisms of immunotherapy resistance and predictive biomarkers to further improve these treatments. Methods: Pre-treatment genomic sequencing data derived from FFPE samples from the IMVIGOR210 clinical trial (n=298) was accessed for analysis. Briefly it was a single arm phase II clinical trial where advanced/metastatic UC patients refractory to platinum chemotherapy treatment received the ICI atezolizumab. This study has been published with detailed methods (PMID: 28950298). The raw sequencing data was pre-processed using standard QC measures and aligned to the human reference genome (hg38). The resulting outputs were then normalized and processed to generate the gene level counts for differential gene expression (DGE). We did DGE analysis comparing patients who had clinical benefit (CR, PR, SD) vs non-clinical benefit (ie. PD) to atezolizumab. The list of differentially expressed genes were then analyzed using various gene ontology, pathway and systems biology tools (IPA, Enrichr, and X2Kweb). Further subset analysis was done using gene-gene correlations (ie. PD-L1 and STAT3) and clinicopathologic features (eg. gender, race, smoking history). Results: Among the 298 patients in this study, there were 25 with CR, 43 with PR, 63 with SD, and 167 with PD based on clinical response to atezolizumab. Subgroup analysis for CR vs PD patients found that approximately 847 genes were differentially expressed with statistical significance (p ≤ 0.05). IPA analysis for this list of differentially expressed genes found among the top signaling pathways were “primary immunodeficiency” and “sirtuin signaling”. Further subset analysis of 39 genes (p ≤ 0.01) enriched in PD patients using Enrichr and X2kweb found that there was an overrepresentation of STAT3 signaling genes (hypergeometric p-val 6.32x104). Conclusions: Our results found that when the transcriptional profiles of CR vs PD there was differential gene expression in STAT3, primary immunodeficiency, and sirtuin signaling pathways. Of note it has been reported that STAT3 signaling can modulate immune activity and its expression is correlated with poor prognosis in urothelial carcinoma patients. These results warrant a larger study to see if STAT3 signaling is a potential biomarker for ICI resistance. If validated this may indicate that the STAT3 pathway is a potential therapeutic target to overcome ICI resistance and improve the efficacy of these agents.

Background: Along with the expanding treatment landscape of mRCC, a substantial need for ways to predict individual patients' (pts) benefit from different therapies has emerged. Recent studies highlight the clinical significance of tumor-specific genomic alterations, revealing a prognostic and/or predictive role of PBRM1 mutations with IO in mRCC (Miao et al Nature 2018). Employing a large institutional database, we aimed to identify genomic correlates of CB from IO and VEGF-TKIs in mRCC. Methods: Consecutive pts who underwent genomic profiling (GP) as part of routine clinical care at the City of Hope Comprehensive Cancer Center were retrospectively identified. GP included the GEM ExTra assay, a clinical tumor-normal whole exome sequencing and tumor whole transcriptome sequencing test performed at Ashion Analytics (Phoenix, AZ), a CAP-accredited, CLIA-certified laboratory. The clinical database included detailed information regarding demographics, treatment, response and survival outcomes. Pts who had complete response (CR), partial response (PR) or stable disease (SD) for >6 months were considered as having CB. Progressive disease (PD) as best response was considered no clinical benefit (NCB). Genomic findings were compared between pts with CB and NCB in the IO and VEGF-TKI cohorts. Results: Among the 58 (45:13 M:F) pts, 17 received sequencing treatment involving both a VEGF-TKI and IO, resulting in 32 pts in the IO cohort and 43 pts in the VEGF-TKI cohort. The most commonly used IO and VEGF-TKIs were nivolumab (59%) and sunitinib (40%). CB rate and median progression free survival were 59.4% (CR: 3.1%, PR: 18.8%, SD: 37.5%) and 15.6 months (95%CI NR-NR) in the IO cohort, and 86% and 14.2 months (95%CI 9.0 - 18.5) in the VEGF-TKI cohort. The most frequently detected alterations in the overall cohort were in VHL (64%), PBRM1 (38%), SETD2 (24%), KDM5C (17%) and TERT (12%). Interestingly, TERT promoter mutations and PBRM1 mutations were found to be mutually exclusive. In both IO and VEGF-TKI cohorts, tumor mutational burden did not differ between CB and NCB pts. No single genes were significantly associated with CB from VEGF-TKIs. While PBRM1 loss of function (LOF) was more common in IO pts with CB, and SETD2, KDM5C, TP53 alteration were more common in IO pts with NCB, there was no statistical significance observed (p values > 0.05). TERT promoter mutations were associated with NCB in the IO cohort (p=0.038). TERT promoter mutant tumors did not have CB with IO. Conclusion: Our analysis found that TERT promoter mutations are enriched in pts with NCB from IO and were mutually exclusive with PBRM1 LOF which had previously been shown to be an indicator of IO sensitivity. These results suggest that TERT promoter mutations may be a negative predictor of IO outcomes, which warrants future investigations with a larger sample size. Citation Format: Nazli Dizman, Sara Byron, Yung Lyou, Paulo Gustavo Bergerot, JoAnn Hsu, Andre-Philippe Sam, Denise Phan Trieu, Jeffrey Trent, Sumanta Kumar Pal. Identifying the genomic correlates of clinical benefit (CB) from immunotherapies (IO) and vascular endothelial growth factor-tyrosine kinase inhibitors (VEGF-TKI) in metastatic renal cell carcinoma (mRCC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1312.

The recent novel coronavirus, named coronavirus disease 2019 (COVID-19), has developed into an international pandemic affecting millions of individuals with hundreds of thousands of deaths worldwide. The highly infectious nature and widespread prevalence of this disease create a new set of obstacles for the bladder cancer community in both delivering and receiving care. In this manuscript, we address the unique issues regarding treatment prioritization for the patient with bladder cancer and how we at City of Hope have adjusted our clinical practices using a team-based approach that utilizes shared decision making with all stakeholders (physicians, patients, caregivers) to optimize outcomes during this difficult time. In addition to taking standard precautions for minimizing COVID-19 risk of exposure for those entering a healthcare facility (screening all personnel upon entry and donning facemasks at all times), we suggest the following three measures: (1) delay post-treatment surveillance visits until there is a decrease in local COVID-19 cases, (2) continue curative intent treatments for localized bladder cancer with COVID-19 precautions (i.e., choosing gemcitabine/cisplatin (GC) over dose-dense methotrexate, vinblastine, doxorubicin, cisplatin (ddMVAC) neoadjuvant chemotherapy), and (3) increase the off-treatment period between cycles of palliative systemic therapy in metastatic urothelial carcinoma patients.

5038 Background: Infigratinib (BGJ398) is a potent and selective FGFR1–3 tyrosine kinase inhibitor (TKI), previously reported to be effective and well tolerated in patients with locally advanced/unresectable or metastatic urothelial carcinoma (mUC) bearing FGFR3 alterations [Pal et al. Cancer Discovery 2018]. However, this previous study did not examine differences in infigratinib activity based on number of prior lines of treatment (LOT). TKIs studied in other indications (e.g. VEGFRis in renal cell carcinoma) have shown consistent activity in both the first and later LOT. Given the effect seen with other TKIs, we sought to determine if infigratinib showed consistent treatment responses in patients with mUC according to LOT. Methods: Eligible patients had mUC and prior platinum-based chemotherapy unless contraindicated and activating FGFR3 mutations/fusions. Infigratinib was dosed at 125 mg orally daily (3 weeks on/1 week off). The primary endpoint of objective response rate (ORR: partial response [PR] and complete response [CR]) was assessed, as well as disease control rate (DCR: CR + PR + stable disease [SD]). After classification of LOT for each patient, subgroup analysis of response by LOT was performed. Results: 67 patients were enrolled; 81% received ≥1 prior LOT for mUC. 13 patients (19.4%) received infigratinib as 1st LOT for mUC due to ineligibility to receive platinum-based chemotherapy. ORR for the 67 patients was 25% (95% CI 15.5–37.5) and DCR was 64% (95% CI 51.5–75.5). The ORR with 1st-line infigratinib was 31% (95% CI 9.1–61.4) compared with 24% (95% CI 13.5–37.6) for 2nd and later (salvage) LOT. DCR was 46% (95% CI 19.2–74.9) for 1st-line and 69% (95% CI 54.4–80.5) for ≥2 LOT. 13 of the 59 patients with urothelial bladder carcinoma (UBC) received 1st-line treatment with an ORR of 31% (95% CI 9.1–61.4) and 46 patients in ≥2 LOT had an ORR of 20% (95% CI 9.4–33.9). All 8 patients with upper tract urothelial carcinoma (UTUC) received salvage therapy, with an ORR of 50% and a DCR of 100%. An analysis of other outcome measures (e.g. PFS, OS) will be presented. Conclusions: These results indicate that infigratinib has activity in patients with mUC regardless of LOT. Additionally, patients with UTUC showed a trend for better ORR and DCR. Taken together, these results support the ongoing adjuvant PROOF 302 study comparing infigratinib with placebo in patients with resected disease, assessing infigratinib in an even earlier setting in a UTUC-enriched population (NCT04197986). Clinical trial information: NCT01004224 .

5076 Background: Previous studies have delineated PBRM1 alterations as a prognostic indicator for response to nivolumab in mRCC (Miao et al Science 2018). However, further clinically-relevant biomarkers remain elusive in mRCC. Herein, we utilized genomic profiling to detect correlates of clinical benefit (CB) among patients (pts) with mRCC receiving VEGF-directed targeted therapy, immunotherapy, or both. Methods: Pts with mRCC who underwent clinical tumor-normal whole exome and whole transcriptome RNA sequencing (Ashion Analytics, Phoenix, AZ) were retrospectively identified at a single institution. Pts’ demographics and clinical variables including treatment type, treatment response, and survival outcomes were collected from an institutional mRCC database. Pts who had received immunotherapy or VEGF-directed therapy (or both) were eligible for analysis. CB was defined as pts who experienced a complete response, partial response, or maintained stable disease for at least 6 months on therapy. Two-tailed Fisher’s exact test was used to assess characteristics of genomic alterations and clinical benefit. Results: Out of 155 mRCC pts with genomic profiling, 58 pts with evaluable response and sufficient follow-up information were included in the analysis. The median age of the cohort was 63 years and 74% were male. The majority (81%) had clear cell histology. 43 pts received targeted therapy and 32 received immunotherapy (17 pts received both). No singular gene was associated with clinical benefit in the targeted therapy cohort. PBRM1 loss of function mutations were more frequent in pts on immunotherapy who experienced CB (p > 0.05). TERT promoter mutations were enriched in pts who experienced no CB on immunotherapy (p = 0.04). Differential gene expression analysis of the transcriptomes found 135 genes that were significantly upregulated in TERT-mutated samples, including SST, CYSLTR2, WNK2, and PTGES (adjusted p-value < 0.05). ENRICHR analysis found that MYC and KAT2A were key transcription factor pathways significantly enriched in pts with TERT mutation who experienced no CB from immunotherapy. Conclusions: These data support previous observations regarding the prognostic nature of PBRM1 in mRCC and offer novel findings associating TERT mutations with a lack of CB from immunotherapy. Our transcriptomic analysis implies that MYC-targeting agents and epigenetic modifiers (directed at KAT2A) could overcome immunotherapy resistance.

576 Background: Infigratinib (BGJ398) is a potent and selective FGFR1–3 inhibitor with significant clinical activity in aUC bearing FGFR3 alterations. A common adverse event is hyperphosphatemia, a class effect associated with FGFR1 inhibition. We sought to better understand the relationship between hyperphosphatemia and response to infigratinib in patients with aUC. Methods: Eligible patients had aUC with activating FGFR3 mutations/fusions and had received prior platinum-based chemotherapy, unless contraindicated. Patients received infigratinib 125 mg orally daily (3w on/1w off) until disease progression or unacceptable toxicity. Calcium and phosphate levels within normal limits were required at enrollment. Efficacy was assessed by overall response rate (ORR) and disease control rate (DCR) based on RECIST 1.0 criteria. All patients received prophylaxis with the oral phosphate binder sevelamer. Hyperphosphatemia was defined as serum phosphorous >5.5 mg/dL, consistent with the threshold for action in the protocol. Results: Of the 67 patients enrolled, 48 (71.6%) had hyperphosphatemia on ≥1 post-baseline lab test. Efficacy findings in patients with vs without hyperphosphatemia were: ORR 33.3% (95% CI 20.4–48.4) vs 5.3% (95% CI 0.1–26.0), mPFS 4.9 months (95% CI 3.65–5.98) vs 1.84 months (95% CI 1.28–3.48), and mOS 8.74 months (95% CI 5.72–13.67) vs 7.62 months (95% CI 2.53–15.57). Median treatment length was 4.1 vs 1.4 months and mDOR was 5.0 vs 3.7 months for patients with vs without hyperphosphatemia, respectively. A landmark analysis at the 1-month mark was performed, and hyperphosphatemia (Y/N) was determined based on lab tests within the first month. The differences in efficacy outcomes were still observed. Conclusions: Hyperphosphatemia is a well-described class effect and pharmacodynamic biomarker of FGFR inhibitors, including infigratinib, and is generally reversible/easily managed with diet and phosphate binders. Our data support prior observations with FGFR inhibitors, suggesting that hyperphosphatemia is associated with treatment response and is not negatively associated with treatment length. Clinical trial information: NCT01004224.

Background The predictors of recurrence in ER- DCIS and the role of tumor microenvironment (TME) is unclear. Correlation of clinico-pathologic features of ER- DCIS with molecular markers and PD-L1 expression has not been done. Therefore, we under took this retrospective study comparing the clinical outcomes between ER+ and ER- DCIS and correlated this with the expression of PD-L1 in the tumor cells and the Tumor infiltrating lymphocytes. We hypothesize that increased expression of PD-L1 in ER- DCIS and TILs predict recurrence. The aim of this study was to identify biomarkers in ER- DCIS associated with increased risk of recurrence or progression to metastatic disease. Methods 50 patients with ER+ and ER- DCIS were identified retrospectively. Clinico-pathologic data was correlated with survival outcomes and local or distant recurrence. Information collected was the size, margin status, nuclear grade, ki-67 expression, architectural pattern, necrosis, and molecular phenotype. 25 cases of ER- DCIS are presented in the abstract. The rest will be presented at the meeting. ER+ and ER- DCIS will be stained for PD-L1, and lymphocyte markers CD3, CD4, CD8, FoxP3, and CD20 for the TILs in ER+ and ER- DCIS. The clinical, radiological and Immuno-pathologic features of ER+ DCIS will be compared with ER negative DCIS. Results 3/25 (12%) had disease recurrence. Two (67%) had recurrence with metastatic triple negative breast cancer with brain, hepatic and osseous metastasis within 5 years. High grade was associated with the development of systemic disease. 1/3 (33%) had recurrence as ER negative DCIS. 2/3 (67%) had recurrence as ER+ tumor. The rate of recurrence and distant metastasis was seen to be higher in high grade ER negative DCIS. Conclusions ER- DCIS has an aggressive phenotype and a distinct biology. It is more likely to present as loco-regional or metastatic disease. Identification of genomic biomarkers, PD-L1 analysis in the tumor and the TILs is currently being undertaken. The molecular drivers and immunologic markers of recurrence in ER negative DCIS continue to be an active area of exploration. Further characterization of ER- DCIS immune microenvironment may identify useful targets for immune-based therapy and breast cancer prevention. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Proliferation and differentiation are tightly controlled during neural development. In the embryonic neural plate, primary neurogenesis is driven by the proneural pathway. Here we report the characterization of Maturin, a novel, evolutionarily conserved protein that is required for normal primary neurogenesis. Maturin is detected throughout the early nervous system, yet it is most strongly expressed in differentiating neurons of the embryonic fish, frog and mouse nervous systems. Maturin expression can be induced by the proneural transcription factors Neurog2, Neurod1, and Ebf3. Maturin overexpression promotes neurogenesis, while loss-of-function inhibits the differentiation of neuronal progenitors, resulting in neural plate expansion. Maturin knockdown blocks the ability of Neurog2, Neurod1, and Ebf3 to drive ectopic neurogenesis. Maturin and Pak3, are both required for, and can synergize to promote differentiation of the primary neurons in vivo. Together, our results suggest that Maturin functions during primary neurogenesis and is required for the proneural pathway to regulate neural differentiation.