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Sacituzumab govitecan (SG) is an antibody-drug conjugate (ADC) targeting TROP2, which has recently been approved for treatment-refractory metastatic urothelial cancer (UC). However, the variability of TROP2 expression across different bladder cancer (BC) subtypes, as well as after enfortumab vedotin (EV) exposure, remains unknown. Using gene expression data from four clinical cohorts with1400 patient samples of muscle-invasive BC and a BC tissue microarray, we found that TROP2 mRNA and protein are highly expressed across basal, luminal, and stroma-rich subtypes, but depleted in the neuroendocrine subtype. In addition, TROP2 mRNA levels are correlated with NECTIN4 mRNA but are more highly expressed than NECTIN4 mRNA in patient cohorts and BC cell lines. Moreover, CRISPR/Cas9-mediated knockdown of TROP2 demonstrates that its expression is one factor governing SG sensitivity. After prolonged EV exposure, cells can downregulate NECTIN4, leading to EV resistance, but retain TROP2 expression and remain sensitive to SG, suggesting nonoverlapping resistance mechanisms to these ADCs. While our findings warrant further validation, they have significant implications for biomarker development, patient selection, and treatment sequencing in the clinic as well as clinical trial design and stratification for metastatic BC patients. PATIENT SUMMARY: In this report, we investigated the expression levels of the drug target TROP2 across different molecular subtypes of bladder cancer in multiple patient cohorts and cell lines. We found high levels of TROP2 in most subtypes except in the neuroendocrine subtype. Overall, TROP2 gene expression is higher than NECTIN4 gene expression, and cells resistant to enfortumab vedotin (EV), a NECTIN4-targeting antibody-drug conjugate, remain sensitive to sacituzumab govitecan (SG). Our findings suggest that SG may be effective across most bladder cancer subtypes, including the bladder cancers previously treated with EV.

Granulocytic–macrophage colony-stimulating factor (GM-CSF) is used as an adjuvant in cancer vaccine trials and has the potential to enhance antitumor efficacy with immunotherapy; however, its immunologic effects are not fully understood. Here, we report results from a phase I study of neoadjuvant GM-CSF in patients with localized prostate cancer undergoing radical prostatectomy. Patients received subcutaneous injections of GM-CSF (250 μg/m2/day) daily for 2 weeks (cohort 1; n = 6), 3 weeks (cohort 2; n = 6), or 4 weeks (cohort 3; n = 6). Treatment was well tolerated with all grade 1 or 2 adverse events. Two patients had a decline in prostate-specific antigen (PSA) of more than 50%. GM-CSF treatment increased the numbers of circulating mature myeloid dendritic cells, proliferating conventional CD4 T cells, proliferating CD8 T cells, and to a lesser magnitude FoxP3+ regulatory CD4 T cells. Although GM-CSF treatment did not augment antigen-presenting cell localization to the prostate, treatment was associated with recruitment of CD8+ T cells to the tumor. These results suggest that systemic GM-CSF can modulate T-cell infiltration in the tumor microenvironment. Cancer Immunol Res; 4(11); 948–58. ©2016 AACR.

Association between NECTIN4 mRNA expression and clinical characteristics in the Seiler cohort for (A) sex (p

Duration of treatment exposure and PSA response since start of therapy for patients receiving systemic therapy with an AR signaling inhibitor prior to study entry

Purpose:Apalutamide is a next-generation androgen receptor (AR) inhibitor approved for patients with nonmetastatic castration-resistant prostate cancer (CRPC) and metastatic castration-sensitive prostate cancer. We evaluated the pharmacokinetics, safety, and antitumor activity of apalutamide combined with abiraterone acetate plus prednisone (AA-P) in patients with metastatic CRPC (mCRPC).Patients and Methods:Multicenter, open-label, phase Ib drug–drug interaction study conducted in 57 patients with mCRPC treated with 1,000 mg abiraterone acetate plus 10 mg prednisone daily beginning on cycle 1 day 1 (C1D1) and 240 mg apalutamide daily starting on C1D8 in 28-day cycles. Serial blood samples for pharmacokinetic analysis were collected on C1D7 and C2D8.Results:Systemic exposure to abiraterone, prednisone, and prednisolone decreased 14%, 61%, and 42%, respectively, when apalutamide was coadministered with AA-P. No increase in mineralocorticoid excess–related adverse events was observed. Patients without prior exposure to AR signaling inhibitors had longer median treatment duration and greater mean decrease in prostate-specific antigen (PSA) from baseline compared with those who had received prior therapy. Confirmed PSA reductions of ≥50% from baseline at any time were observed in 80% (12/15) of AR signaling inhibitor–naïve patients and 14% (6/42) of AR signaling inhibitor–treated patients.Conclusions:Treatment with apalutamide plus AA-P was well tolerated and showed evidence of antitumor activity in patients with mCRPC, including those with disease progression on AR signaling inhibitors. No clinically significant pharmacokinetic interaction was observed between abiraterone and apalutamide; however, apalutamide decreased exposure to prednisone. These data support development of 1,000 mg abiraterone acetate plus 10 mg prednisone daily with 240 mg apalutamide daily in patients with mCRPC.

The p values from pairwise comparisons amongst the molecular subtypes for each cohort using the Wilcoxon rank-sum test are shown.

Supplementary Figure 1. Trial schema

Supplementary Figure 3. Boxplots: PSA response versus baseline circulating androgen levels: DHEA, T and ASD

Purpose:Enfortumab vedotin (EV) is an antibody–drug conjugate (ADC) targeting NECTIN4 (encoded by the PVRL4/NECTIN4 gene) approved for treatment-refractory metastatic urothelial cancer. Factors that mediate sensitivity or resistance to EV are unknown. In this study, we sought to (i) examine heterogeneity of NECTIN4 gene expression across molecular subtypes of bladder cancer and (ii) determine whether NECTIN4 expression mediates EV sensitivity or resistance.Experimental Design:Molecular subtyping and NECTIN4 expression data from seven muscle-invasive bladder cancer clinical cohorts (n = 1,915 total specimens) were used to assess NECTIN4 expression across molecular subtypes. The outcome of the transcriptomic analysis was relative NECTIN4 expression in the consensus molecular subtypes of bladder cancer. Expression of NECTIN4 was validated in bladder cancer cell lines. NECTIN4 was stably overexpressed or knocked down in basal and luminal bladder cancer cell lines and EV drug sensitivity assays were performed, as measured by cell proliferation and clonogenic assays.Results:NECTIN4 expression is heterogenous across molecular subtypes of bladder cancer and significantly enriched in luminal subtypes. NECTIN4 expression is positively correlated with luminal markers GATA3, FOXA1, and PPARG across all cohorts. NECTIN4 expression is both necessary and sufficient for EV sensitivity in luminal and basal subtypes of urothelial bladder cancer cells. Downregulation of NECTIN4 leads to EV resistance.Conclusions:Sensitivity to EV is mediated by expression of NECTIN4, which is enriched in luminal subtypes of bladder cancer. These findings may have implications for biomarker development, patient selection, and the inclusion of molecular subtyping in ongoing and future EV clinical trials.See related commentary by Teo and Rosenberg, p. 4950

Figure legends for Figure S1 and Figure S2

Confirmed PSA response (as change from baseline) at any time for patients who received no prior therapy with AA-P or ENZ and patients who received therapy with AA-P only, ENZ only, or both AA-P and ENZ prior to study entry. Postbaseline PSA measurements were not available from two patients.

Supplementary Figure 2. Boxplot: PSA response versus time on duration of prior ketoconazole

Purpose: Patients previously treated with ketoconazole were excluded from phase III trials of abiraterone acetate due to potential overlapping mechanism of action. The purpose of this study was to determine the clinical utility of abiraterone and its impact on circulating androgens following ketoconazole.Experimental Design: Chemotherapy-naïve patients with progressive metastatic castration-resistant prostate cancer (mCRPC) and prior ketoconazole therapy ≥28 days received abiraterone acetate 1,000 mg daily and prednisone 5 mg twice daily. The primary endpoint was the proportion of patients with PSA response, defined as ≥30% PSA decline at 12 weeks. H0 = 0.30 versus H1 = 0.50 (α = 0.05, power = 0.83). Circulating androgen levels were measured using liquid chromatography tandem mass spectrometry.Results: Thirty-nine patients were included in the final analysis. Twenty (51%; 95% confidence interval, 36%–66%) patients had ≥30% PSA decline; the null hypothesis was rejected. Sixteen (41%) had ≥50% PSA decline. Median PFS (progression-free survival) was 16 weeks; median radiographic PFS (rPFS) was 36 weeks. Samples for measurement of baseline androgens were available in 37 patients. The PSA response proportion was 59% in 29 patients with DHEA ≥ limit of quantitation (LOQ), compared with 13% in 8 patients with DHEA < LOQ (P = 0.042). Median PFS was 6 and 16 weeks in DHEA < LOQ and DHEA ≥ LOQ patients, respectively (P = 0.017); median rPFS was 14 and 36 weeks in DHEA < LOQ and DHEA ≥ LOQ patients, respectively (P < 0.001).Conclusions: Abiraterone demonstrates modest clinical efficacy in mCRPC patients previously treated with ketoconazole. Patients with DHEA ≥ LOQ were more likely to demonstrate PSA responses and longer PFS. Analysis of circulating androgens merits further investigation as a biomarker for response to androgen synthesis inhibitor therapy. Clin Cancer Res; 20(24); 6269–76. ©2014 AACR.

XLS file - 49K, CpG sites methylated in 100% of CRPC.

XLS file - 15K, Candidate Gene List and Copy number:Expression Correlation for Frequently Aberrant Genes.

XLS file - 32K, Detailed TMPRSS2-ERG and TMPRSS2-ETS2 aCGH and Methylation Mapping.

XLS file - 15K, Genes with copy number aberrations in _>66% of samples (N=15).

PDF file - 7.7K, Hierarchical Clustering of CRPC Samples by Copy Number and Expression: Clustering of samples by: (A) whole genome copy number (x axis = aCGH probes sorted by chromosome, chromosome 1 on left, chromosome 23 on right, green=gain, red=loss). (B) expression level of frequently copy aberrant genes (x axis=gene expression probes red=overexpressed, blue=underexpressed.

Background Exercise may improve clinical and quality of life outcomes for men with prostate cancer. No randomized controlled trials (RCTs) have examined the feasibility, safety, and acceptability of remote exercise training in men with metastatic castrate‐resistant prostate cancer (mCRPC). Methods We conducted a pilot RCT (1:1:1 aerobic or resistance exercise 3x/week or usual care) to determine the feasibility, safety, and acceptability of remotely monitored exercise over 12 weeks in 25 men with mCRPC. A prescribed exercise program was based on baseline testing including high‐ and moderate‐intensity aerobic exercise or resistance exercise completed at a local exercise facility. Feasibility was based on attendance, adherence, and tolerance; safety on adverse events; and acceptability on participant interviews. Results Between March 2016 and March 2020, 25 patients were randomized (8 aerobic, 7 resistance, and 10 control). Twenty‐three men (82%) completed the 12‐week study. Men who completed the remote intervention attempted 90% and 96% of prescribed aerobic and resistance training sessions, respectively, and 86% and 88% of attempted sessions were completed as or more than prescribed. We observed changes in performance tests that corresponded with the exercise prescription. No safety concerns were identified. Ninety percent of participants interviewed were satisfied with the program and would recommend it to others. Conclusions Remotely monitored exercise training is feasible, safe, and acceptable in men with mCRPC; there was no difference in these outcomes by mode of exercise. Through this research, we provide direction and rationale for future studies of exercise and clinical outcomes in patients with metastatic prostate cancer., We conducted a 3‐arm pilot randomized controlled trial to determine the feasibility, safety, and acceptability of two modes of remotely monitored exercise (resistance or aerobic) over 12 weeks compared to usual care in 25 men with metastatic castrate‐resistant prostate cancer (mCRPC). We report that remotely monitored exercise training is feasible, safe, and acceptable in this patient population, with a high satisfaction rate. Through this research we provide direction and rationale for future studies of exercise and clinical outcomes for people with metastatic cancer.

PURPOSE Cisplatin-based combination chemotherapy remains the standard of care for locally advanced or metastatic urothelial cancer (la/mUC); however, toxicity is substantial, responses are rarely durable, and many patients with la/mUC are ineligible. Each enfortumab vedotin and pembrolizumab have shown a survival benefit versus chemotherapy in UC, are not restricted by cisplatin eligibility, and warrant investigation as a first-line (1L) combination therapy in patients ineligible for cisplatin. METHODS In this ongoing phase Ib/II, multicenter, open-label study, 1L cisplatin-ineligible patients with la/mUC received enfortumab vedotin 1.25 mg/kg once daily on days 1 and 8 and pembrolizumab 200 mg (day 1) intravenously once daily in 3-week cycles. The primary end point was safety. Key secondary end points included confirmed objective response rate, duration of response (DOR), and overall survival (OS). RESULTS Forty-five patients received enfortumab vedotin plus pembrolizumab. The most common treatment-related adverse events (TRAEs) were peripheral sensory neuropathy (55.6%), fatigue (51.1%), and alopecia (48.9%). Twenty-nine patients (64.4%) had grade 3 or higher TRAEs; the most common were increased lipase (17.8%), maculopapular rash (11.1%), and fatigue (11.1%). One death (2.2%) was classified as a TRAE. The confirmed objective response rate after a median of nine cycles was 73.3% with a complete response rate of 15.6%. The median DOR and median OS were 25.6 months and 26.1 months, respectively. CONCLUSION Enfortumab vedotin plus pembrolizumab showed a manageable safety profile. Most patients experienced tumor shrinkage. The median DOR and median OS exceeding 2 years in a cisplatin-ineligible patient population make this a promising combination currently under investigation in a phase III study (ClinicalTrials.gov identifier: NCT04223856 ).

Despite the efficacy of tyrosine kinase inhibitors (TKIs) across multiple cancers, side effects including treatment-related diarrhea can impede a patient's ability to reach therapeutic doses or stay on therapy. Below, we present the case of a 72-year-old patient with metastatic papillary renal cell carcinoma recurrent despite nephrectomy. Over the course of treatment, the patient received multiple different tyrosine kinase inhibitors with varying efficacy. Treatment with the TKI cabozantinib after failure of two prior TKIs resulted in a clinical response with shrinkage of his nodal metastatic disease. However, the severe treatment-related diarrhea refractory to conventional management required both dose holds and dose reductions of cabozantinib. Off-label administration of crofelemer, a novel FDA-approved antidiarrheal agent, successfully controlled the treatment-related diarrhea and allowed resumption and partial dose increase of cabozantinib. This case suggests that crofelemer could be a viable therapeutic strategy to address TKI-induced diarrhea.

Objectives Serologic testing for antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in potential donors of coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) may not be performed until after blood donation. A hospital-based recruitment program for CCP may be an efficient way to identify potential donors prospectively Methods Patients who recovered from known or suspected COVID-19 were identified and recruited through medical record searches and public appeals in March and April 2020. Participants were screened with a modified donor history questionnaire and, if eligible, were asked for consent and tested for SARS-CoV-2 antibodies (IgG and IgM). Participants positive for SARS-CoV-2 IgG were referred for CCP collection. Results Of 179 patients screened, 128 completed serologic testing and 89 were referred for CCP donation. IgG antibodies to SARS-CoV-2 were detected in 23 of 51 participants with suspected COVID-19 and 66 of 77 participants with self-reported COVID-19 confirmed by polymerase chain reaction (PCR). The anti–SARS-CoV-2 IgG level met the US Food and Drug Administration criteria for “high-titer” CCP in 39% of participants confirmed by PCR, as measured by the Ortho VITROS IgG assay. A wide range of SARS-CoV-2 IgG levels were observed. Conclusions A hospital-based CCP donor recruitment program can prospectively identify potential CCP donors. Variability in SARS-CoV-2 IgG levels has implications for the selection of CCP units for transfusion.

Purpose: Apalutamide is a next-generation androgen receptor (AR) inhibitor approved for patients with nonmetastatic castration-resistant prostate cancer (CRPC) and metastatic castration-sensitive prostate cancer. We evaluated the pharmacokinetics, safety, and antitumor activity of apalutamide combined with abiraterone acetate plus prednisone (AA-P) in patients with metastatic CRPC (mCRPC). Patients and Methods: Multicenter, open-label, phase Ib drug–drug interaction study conducted in 57 patients with mCRPC treated with 1,000 mg abiraterone acetate plus 10 mg prednisone daily beginning on cycle 1 day 1 (C1D1) and 240 mg apalutamide daily starting on C1D8 in 28-day cycles. Serial blood samples for pharmacokinetic analysis were collected on C1D7 and C2D8. Results: Systemic exposure to abiraterone, prednisone, and prednisolone decreased 14%, 61%, and 42%, respectively, when apalutamide was coadministered with AA-P. No increase in mineralocorticoid excess–related adverse events was observed. Patients without prior exposure to AR signaling inhibitors had longer median treatment duration and greater mean decrease in prostate-specific antigen (PSA) from baseline compared with those who had received prior therapy. Confirmed PSA reductions of ≥50% from baseline at any time were observed in 80% (12/15) of AR signaling inhibitor–naïve patients and 14% (6/42) of AR signaling inhibitor–treated patients. Conclusions: Treatment with apalutamide plus AA-P was well tolerated and showed evidence of antitumor activity in patients with mCRPC, including those with disease progression on AR signaling inhibitors. No clinically significant pharmacokinetic interaction was observed between abiraterone and apalutamide; however, apalutamide decreased exposure to prednisone. These data support development of 1,000 mg abiraterone acetate plus 10 mg prednisone daily with 240 mg apalutamide daily in patients with mCRPC.

Radiopharmaceuticals, including Ga-68-prostate specific membrane antigen (PSMA)-11 and F-18-Fluciclovine, are increasingly used to inform therapies for prostate cancer (CaP). Stereotactic body radiation therapy (SBRT) to PET-detected oligometastatic CaP has been shown to improve progression free survival (PFS) and delay androgen deprivation therapy (ADT) compared to observation. For men who subsequently develop oligorecurrent CaP, outcomes following second SBRT are unknown.A retrospective cohort study was conducted. Eligibility criteria included patients with oligometastatic (1-5 lesions) CaP detected on PSMA or Fluciclovine PET who underwent 2 consecutive SBRT courses to tracer-avid sites. Data on stage, tracer type, concurrent systemic therapy, and prostate-specific antigen (PSA) responses for first SBRT (SBRT1) and second SBRT (SBRT2) were collected. Outcomes included PSA decline ≥50% (PSA50), PFS after SBRT2, and ADT initiation or intensification-free survival after SBRT2. Factors potentially associated with PSA50 after SBRT2 was evaluated with multivariable logistic regression. Factors potentially associated with PFS and ADT initiation/intensification-free survival after SBRT2 were evaluated with separate multivariable Cox proportional-hazards models.Twenty-five patients were identified. At SBRT2, oligorecurrence was detected on PSMA and Fluciclovine PET in 17 (68%) and 8 (32%) patients, respectively. Fifteen (60%) patients had castration-sensitive disease and 10 (40%) had castration-resistant disease. After SBRT2, 16 (64%) achieved a PSA50 response, median PFS was 11.0mo, and median ADT initiation/intensification-free survival was 23.2mo. On multivariable analysis, maximum percent change in PSA after SBRT1 (OR 0.94, 95%CI 0.88-0.99, P = 0.046) and concurrent change in systemic therapy (OR 21.61, 95%CI 1.12-417.9, P = 0.042) were associated with PSA50 responses after SBRT2. PSA50 response after SBRT1 was associated with improved PFS (HR 0.36, 95%CI 0.00-0.42, P = 0.008) and ADT initiation/intensification-free survival (HR 0.07, 95%CI 0.01-0.68, P = 0.021) after SBRT2. From SBRT1 to last follow-up (median 48 months), 7 (28%) patients remained ADT-free.Serial SBRT for oligometastatic CaP detected on PSMA or Fluciclovine PET is feasible and can achieve PSA declines, with or without systemic therapy. Degree of biochemical response to first SBRT warrants further study as a potential predictor of PSA response, PFS, and ADT initiation/intensification-free survival following a subsequent SBRT course. This preliminary evidence provides rationale for larger, prospective studies of this strategy.

499 Background: In EV-103 Cohort K (NCT03288545), EV and P in combination (EV+P) showed encouraging antitumor activity and a manageable safety profile when used as 1L therapy in patients (pts) w/ la/mUC who are ineligible for cisplatin, a population w/ high unmet need. Here we report results of an analysis of prespecified Cohort K subgroups known to be associated w/ poor outcomes. Methods: Pts who are cisplatin-ineligible w/ previously untreated la/mUC were randomized 1:1 to EV (1.25 mg/kg) as monotherapy on Days 1 and 8 or in combination w/ P (200 mg) on Day 1 of 3-week cycles. Primary endpoint is cORR per RECIST v1.1 by blinded independent central review w/ no formal statistical comparison between arms. Secondary endpoints included duration of response and safety (e.g. treatment-related adverse events, TRAEs). The cORR analysis was performed in prespecified subgroups including age, ECOG PS, liver metastasis, PD-L1 expression status, metastatic disease site at baseline, and primary disease site of origin. Results: 149 pts were treated: EV+P n=76; EV n=73; cORRs across key subgroups for both EV+P and EV monotherapy are shown in the table. For EV+P overall cohort, cORR (95%CI): 64.5% (52.7, 75.1); median DOR was not reached. cORRs were consistent across subgroups for EV+P including those w/ ECOG PS score of 1-2: 62.8% (46.7, 77.0) and presence of liver metastasis: 53.8% (25.1, 80.8). Among TRAEs of special interest in the EV+P arm, skin reactions occurred in n=51 (67.1%); peripheral neuropathy occurred in n=46 (60.5%). For EV+P, 68.4% of pts had TRAEs leading to interruption of either EV or P; 48.7% of pts had TRAEs leading to EV dose reduction. Median duration of EV+P treatment was 11 cycles. Conclusions: EV+P showed promising cORR in 1L cisplatin-ineligible pts w/ la/mUC; activity was consistently observed across a range of pre-specified subgroups including those with poor prognosis. EV+P TRAEs were manageable w/ close monitoring and appropriate dose modifications w/ a meaningful duration of treatment. EV+P has the potential to address high unmet needs in 1L la/mUC and MIBC and is being further evaluated in 3 Phase 3 trials (NCT04223856, NCT04700124, NCT03924895). Clinical trial information: NCT03288545 . [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]

439 Background: The EV-103 Cohort K trial evaluated 1L EV+P or EV alone (NCT03288545) in pts with la/mUC who were cisplatin-ineligible. EV+P showed a clinically meaningful objective response rate (64.5%; 95% CI, 52.7–75.1) with a manageable safety profile. Because la/mUC is associated with symptoms with a frequent impact on quality of life (QOL) and functioning (Mamtani et al. JCO 2021), we describe the impact of EV+P or EV alone on QOL and symptoms. Methods: In this open-label, Phase 1b/2 trial, cisplatin-ineligible pts with la/mUC were randomized 1:1 to EV+P or EV alone. For exploratory PRO endpoints, pts completed EORTC QLQ-C30 and the BPI-SF at baseline, weekly for cycles 1–3, and once every cycle for the remainder of treatment period. The PRO analysis set included only pts who were treated and completed the questionnaire at baseline. Mixed effect models for repeated measures (MMRM; least squares [LS] mean, standard error [SE]) not adjusted for multiplicity, estimated change vs baseline until Week 24; established thresholds (EORTC QLQ-C30: 10-point change; BPI-SF: 2-point change) were applied to determine clinically meaningful change. Results: Of the 76 pts treated with EV+P, 65 were included in the PRO analysis set. EORTC QLQ-C30 and BPI-SF were completed by 100% and 95%, respectively, at baseline; compliance rates were ≥84% for both instruments through Week 24. In the EORTC QLQ-C30 MMRM analyses, QOL was maintained through Week 24 for EV+P; functioning and symptom scores remained stable over time, with improvements in emotional functioning, pain and sleep disturbance vs observed baseline. In the EV+P arm, clinically meaningful reductions in pain were seen at Week 12 (-12.64 [3.208]) vs baseline and persisted through Week 24 (-13.20 [3.406]). In the BPI-SF MMRM analyses, worst and average pain, pain interference and severity consistently showed improved scores from Week 4–24 for EV+P; clinically meaningful change in worst pain was seen at Week 21 (-2.08 [0.361]). Similar completion and compliance rates were seen for the EV alone PRO analysis set (n=63). EV alone demonstrated clinically meaningful improvements in pain at Week 24 in the EORTC QLQ-C30 (-10.63 [4.110]) and consistent small-to-moderate improvements in the BPI worst and average pain, and pain severity (0.5–1.0 points). Sleep disturbances demonstrated improvement, and other symptom scales, QOL and functional domains remained stable. Conclusions: PRO data showed that EV+P in cisplatin-ineligible pts with la/mUC was associated with preservation or improvement of QOL, functioning, and symptoms. Improvement in pain was demonstrated consistently in both PRO instruments and treatment arms. These PRO data complement the clinical outcomes of EV+P in 1L cisplatin-ineligible pts with la/mUC. Clinical trial information: NCT03288545 .

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]

Enfortumab vedotin (EV) is a novel antibody-drug conjugate approved for advanced urothelial cancer (aUC) refractory to prior therapy. In the Urothelial Cancer Network to Investigate Therapeutic Experiences (UNITE) study, the authors looked at the experience with EV in patient subsets of interest for which activity had not been well defined in clinical trials.UNITE was a retrospective study of patients with aUC treated with recently approved agents. This initial analysis focused on patients treated with EV. Patient data were abstracted from chart reviews by investigators at each site. The observed response rate (ORR) was investigator-assessed for patients with at least 1 post-baseline scan or clear evidence of clinical progression. ORRs were compared across subsets of interest for patients treated with EV monotherapy.The initial UNITE analysis included 304 patients from 16 institutions; 260 of these patients were treated with EV monotherapy and included in the analyses. In the monotherapy cohort, the ORR was 52%, and it was40% in all reported subsets of interest, including patients with comorbidities previously excluded from clinical trials (baseline renal impairment, diabetes, and neuropathy) and patients with fibroblast growth factor receptor 3 (FGFR3) alterations. Progression-free survival and overall survival were 6.8 and 14.4 months, respectively. Patients with a pure urothelial histology had a higher ORR than patients with a variant histology component (58% vs 42%; P = .06).In a large retrospective cohort, responses to EV monotherapy were consistent with data previously reported in clinical trials and were also observed in various patient subsets, including patients with variant histology, patients with FGFR3 alterations, and patients previously excluded from clinical trials with an estimated glomerular filtration rate30 mL/min and significant comorbidities.Enfortumab vedotin, approved by the Food and Drug Administration in 2019, is an important new drug for the treatment of patients with advanced bladder cancer. This study looks at the effectiveness of enfortumab vedotin as it has been used at multiple centers since approval, and focuses on important patient populations previously excluded from clinical trials. These populations include patients with decreased kidney function, diabetes, and important mutations. Enfortumab vedotin is effective for treating these patients. Previously reported clinical trial data have been replicated in this real-world setting, and support the use of this drug in broader patient populations.

BackgroundMaintenance of function during cancer treatment is important to older adults. Characteristics associated with pretreatment life-space mobility and changes during non-small cell lung cancer (NSCLC) treatment remain unknown.MethodsThis mixed methods cohort study recruited adults age ≥65 with advanced NSCLC starting palliative chemotherapy, immunotherapy, and/or targeted therapy from a Comprehensive Cancer Center, Veterans Affairs, and safety-net clinic. Patients completed geriatric assessments including Life-Space Assessment (LSA) pretreatment and at 1, 2, 4, and 6 months after treatment initiation. LSA scores range from 0 to 120 (greater mobility); LSA

397 Background: Real-world data (RWD) is playing an increasingly important role in cancer research. Surrogate endpoints such as metastasis-free survival play an important role in prostate cancer research, leading to interest in its computational extraction, typically with use of International Classification of Disease (ICD) metastatic codes. While prior studies have suggested that ICD codes are valid for identification of patients (pts) with metastatic prostate cancer (MPC), delays in coding may impact their accuracy. The objective of this informatics-based study is to quantify the time delay between diagnosis of MPC and entry of ICD MPC-related code and its interaction with changing institutional healthcare processes. Methods: A single institutional EHR data warehouse was queried to identify a random sample of 100 pts with MPC diagnosis based on ICD codes (ICD10 C79 or ICD9 198.5) from 2013-2021 who were also seen in the genitourinary medical oncology program (GUMOP). Of note, in 6/2018, the GUMOP adopted EHR-specific MPC visit diagnosis identifiers (Dx ID) to improve MPC coding during clinic independent of ICD codes typically used by RWD researchers. Thus, the study cohort was designed to include pts whose first follow up after being diagnosed with MPC was before (n = 50) or after (n = 50) Dx ID implementation. Date of first MPC ICD code entry at any point in the EHR was compared against true date of MPC, based on physician review of definitive imaging or pathology. Data analysis was performed with Wilcox Signed rank test, bivariate analyses, and multivariable linear regression. Covariates included modality of diagnosis confirmation and timing with Dx ID implementation. Results: One hundred pts with MPC ICD coded in the EHR were included, with 29 pts diagnosed by PSMA PET and 71 by conventional imaging. Median time from true MPC diagnosis to first subsequent clinic follow up was < 1 month (IQR 0-2), while median time from true MPC diagnosis to entry of ICD MPC-related code was longer at 4mo (IQR 0-15). 5 pts had C79 applied for N1 disease and 10 pts for work-up of biochemical recurrence. On multivariable analysis of potential factors affecting time interval to MPC ICD entry, Dx ID implementation (b = -6.5 mo [95% CI -1.8 to -11.2], p = 0.007) and non-PSMA based diagnosis (b = -5.7 mo [95% CI -0.5 to -10.8], p = 0.03) were independently associated with shorter time to ICD coding. In subset analysis of the cohort after Dx ID implementation, use of both ICD and Dx ID to identify pts with MPC reduced the median time from true MPC diagnosis to EHR coding (1mo, IQR 0-6.3) compared to ICD alone (2mo, IQR 0-8) (p = 0.003). Conclusions: Timing of MPC ICD entry is highly variable and may carry biases derived from healthcare processes, including data entry and diagnostic testing. This may be improved with EHR workflow interventions. It is essential to have domain knowledge of clinical coding practices to improve information retrieval and recognize potential limitations and biases.

BACKGROUND: Durvalumab has shown meaningful clinical activity in patients with metastatic urothelial carcinoma (mUC) in Study 1108 (NCT01693562). An important focus in treatment is health‐related quality of life (HRQOL). Here, patient‐reported outcomes (PROs) from Study 1108 and their relationship with inflammatory biomarkers are explored. METHODS: Disease‐related symptoms, functioning, and HRQOL were assessed with the Functional Assessment of Cancer Therapy–Bladder (FACT‐Bl) and the European Organisation for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire Core 30 (QLQ‐C30). Relationships between PRO improvements and the best changes in the tumor size, albumin level, and neutrophil‐lymphocyte ratio (NLR) were assessed with Spearman correlation analysis. RESULTS: The mean FACT‐Bl total score improved from 107.5 (standard deviation [SD], 23.0) at the baseline to 115.4 (SD, 22.6) on day 113, with similar increases found for the Trial Outcome Index (TOI) and Bladder Cancer Subscale (BLCS) scores. The mean FACT‐Bl total scores improved over time, and the FACT‐Bl TOI scores significantly improved by day 113 (P < .05). The mean EORTC QLQ‐C30 Global Health Status/Quality of Life score improved from 57.1 (SD, 24.8) at the baseline to 69.0 (SD, 21.4) on day 113; the functional scale and symptom scores (day 113) were higher than the baseline scores (P < .05) for EORTC Social Functioning. The FACT‐Bl total, BLCS, and TOI scores improved in 32.6%, 34.9%, and 32.6% of the patients by day 113; 26.3% to 37.8% of the patients exhibited improvements in EORTC QLQ‐C30 functional scores. The best tumor shrinkage and posttreatment improvements in serum albumin and NLR correlated with increases in FACT‐Bl total, TOI, and BLCS scores and in EORTC Physical Functioning and Role Functioning scores (P < .05). CONCLUSIONS: Durvalumab was associated with improvements in disease‐related symptoms, functioning, and HRQOL in patients with mUC. Improvements in systemic inflammation may contribute to PRO improvements in these patients.

Platinum-based chemotherapy has been the standard of care in advanced urothelial cancer, but long-term outcomes have remained poor. Immune checkpoint inhibitors, with their favorable toxicity profiles and noteworthy efficacy, have steered a new era in advanced urothelial cancer, with five agents targeting the PD-1/PD-L1 pathway approved by the U.S. Food and Drug Administration (FDA). However, most patients do not achieve response, whereas immunotherapy-related adverse events may cause morbidity, increased health care use, and—rarely—mortality. Therefore, there is an urgent need for additional therapeutic modalities across the disease spectrum. A plethora of clinical trials are ongoing in various disease settings, including chemotherapy regimens, radiotherapy, antibody-drug conjugates, agents targeting additional immune checkpoint pathways, vaccine, cytokines, adoptive cell therapies, as well as targeted and anti-angiogenic agents. Two agents, enfortumab vedotin and erdafitinib, have breakthrough designation by the FDA but are not approved yet (at the time of this paper's preparation). Novel combinations with various treatment modalities and optimal sequencing of active therapies are being investigated in prospective clinical trials. Evaluation of new treatments has met with substantial challenges for many reasons, for example, molecular heterogeneity, clonal evolution, and genomic instability. In the era of precision molecular medicine, and because patients do not respond uniformly to current therapies, there is a growing need for identification and validation of biomarkers that can accurately predict treatment response and assist in patient selection. Here, we review current updates and future directions of experimental therapeutics in urothelial cancer, including examples (but not an exhaustive list) of ongoing clinical trials.

BackgroundSipuleucel-T is a US Food and Drug Administration-approved autologous cellular immunotherapy that improves survival in patients with metastatic castration-resistant prostate cancer (mCRPC). We examined whether administering ipilimumab after sipuleucel-T could modify immune and/or clinical responses to this treatment.MethodsA total of 50 patients with mCRPC were enrolled into a clinical trial (NCT01804465, ClinicalTrials.gov) where they received ipilimumab either immediately or delayed 3 weeks following completion of sipuleucel-T treatment. Blood was collected at various timepoints of the study. Luminex assay for anti-prostatic acid phosphatase (PAP) and anti-PA2024-specific serum immunoglobulin G (IgG) and ELISpot for interferon-γ (IFN-γ) production against PAP and PA2024 were used to assess antigen-specific B and T cell responses, respectively. Clinical response was defined as >30% reduction in serum prostate-specific antigen levels compared with pretreatment levels. The frequency and state of circulating immune cells were determined by mass cytometry by time-of-flight and statistical scaffold analysis.ResultsWe found the combination to be well tolerated with no unexpected adverse events occurring. The timing of ipilimumab did not significantly alter the rates of antigen-specific B and T cell responses, the primary endpoint of the clinical trial. Clinical responses were observed in 6 of 50 patients, with 3 having responses lasting longer than 3 months. The timing of ipilimumab did not significantly associate with clinical response or toxicity. The combination treatment did induce CD4 and CD8 T cell activation that was most pronounced with the immediate schedule. Lower frequencies of CTLA-4 positive circulating T cells, even prior to treatment, were associated with better clinical outcomes. Interestingly, these differences in CTLA-4 expression were associated with prior localized radiation therapy (RT) to the prostate or prostatic fossa. Prior radiation treatment was also associated with improved radiographic progression-free survival.ConclusionCombining CTLA-4 blockade with sipuleucel-T resulted in modest clinical activity. The timing of CTLA-4 blockade following sipuleucel-T did not alter antigen-specific responses. Clinical responses were associated with both lower baseline frequencies of CTLA-4 expressing T cells and a history of RT. Prior cancer therapy may therefore result in long-lasting immune changes that influence responsiveness to immunotherapy with sipuleucel-T and anti-CTLA-4.

BackgroundImmune checkpoint inhibitors (ICI) can achieve durable responses in a subset of patients with locally advanced or metastatic urothelial carcinoma (aUC). The use of tumor genomic profiling in clinical practice may help suggest biomarkers to identify patients most likely to benefit from ICI.MethodsWe undertook a retrospective analysis of patients treated with an ICI for aUC at a large academic medical center. Patient clinical and histopathological variables were collected. Responses to treatment were assessed for all patients with at least one post-baseline scan or clear evidence of clinical progression following treatment start. Genomic profiling information was also collected for patients when available. Associations between patient clinical/genomic characteristics and objective response were assessed by logistic regression; associations between the characteristics and progression-free survival (PFS) and overall survival (OS) were examined by Cox regression. Multivariable analyses were performed to identify independent prognostic factors.ResultsWe identified 119 aUC patients treated with an ICI from December 2014 to January 2020. Genomic profiling was available for 78 patients. Overall response rate to ICI was 29%, and median OS (mOS) was 13.4 months. Favorable performance status at the start of therapy was associated with improved OS (HR 0.46, p=0.025) after accounting for other covariates. Similarly, the presence of a TERT promoter mutation was an independent predictor of improved PFS (HR 0.38, p=0.012) and OS (HR 0.32, p=0.037) among patients who had genomic profiling available. Patients with both a favorable performance status and a TERT promoter mutation had a particularly good prognosis with mOS of 21.1 months as compared with 7.5 months in all other patients (p=0.03).ConclusionsThe presence of a TERT promoter mutation was an independent predictor of improved OS in a cohort of aUC patients treated with an ICI who had genomic data available. Most of the clinical and laboratory variables previously shown to be prognostic in aUC patients treated with chemotherapy did not have prognostic value among patients treated with an ICI. Genomic profiling may provide important prognostic information and affect clinical decision making in this patient population. Validation of these findings in prospective patient cohorts is needed.

Prostate cancer is the most commonly diagnosed neoplasm in American men. Although existing biomarkers may detect localized prostate cancer, additional strategies are necessary for improving detection and identifying aggressive disease that may require further intervention. One promising, minimally invasive biomarker is cell-free DNA (cfDNA), which consist of short DNA fragments released into circulation by dying or lysed cells that may reflect underlying cancer. Here we investigated whether differences in cfDNA concentration and cfDNA fragment size could improve the sensitivity for detecting more advanced and aggressive prostate cancer. This study included 268 individuals: 34 healthy controls, 112 men with localized prostate cancer who underwent radical prostatectomy (RP), and 122 men with metastatic castration-resistant prostate cancer (mCRPC). Plasma cfDNA concentration and fragment size were quantified with the Qubit 3.0 and the 2100 Bioanalyzer. The potential relationship between cfDNA concentration or fragment size and localized or mCRPC prostate cancer was evaluated with descriptive statistics, logistic regression, and area under the curve analysis with cross-validation. Plasma cfDNA concentrations were elevated in mCRPC patients in comparison to localized disease (OR5ng/mL = 1.34, P = 0.027) or to being a control (OR5ng/mL = 1.69, P = 0.034). Decreased average fragment size was associated with an increased risk of localized disease compared to controls (OR5bp = 0.77, P = 0.0008). This study suggests that while cfDNA concentration can identify mCRPC patients, it is unable to distinguish between healthy individuals and patients with localized prostate cancer. In addition to PSA, average cfDNA fragment size may be an alternative that can differentiate between healthy individuals and those with localized disease, but the low sensitivity and specificity results in an imperfect diagnostic marker. While quantification of cfDNA may provide a quick, cost-effective approach to help guide treatment decisions in advanced disease, its use is limited in the setting of localized prostate cancer.

Author(s): Amara, Dominic; Wisel, Steven A; Braun, Hillary J; Collisson, Eric A; Friedlander, Terence; Worner, Giulia; Roll, Garret R; Hirose, Ryutaro; Stock, Peter G | Abstract: Stopping immunosuppression in a transplant patient with donor-derived malignancy offers the theoretical benefit that reconstitution of the patient's immune system will allow "rejection" of the malignancy, as the malignancy also originates from allogeneic tissue. However, this option exists with the caveat that the patient's allograft(s) will likely be rejected too. In simultaneous pancreas-kidney (SPK) recipients, the normal continued functioning and possible absence of malignancy in either the unaffected kidney or pancreas further complicate this decision.MethodsThe charts of 3 patients with donor-derived metastatic malignancies after SPK were retrospectively reviewed in detail. We provide treatment and management recommendations based on successful outcomes and a review of the existing literature.ResultsConsistent with a broad review of the literature, in all 3 cases, complete immunosuppression cessation, removal of both grafts, and in 1 case treatment with an immune checkpoint inhibitor to augment the immune response was successful. One patient is doing well 1 year after successfully undergoing kidney retransplantation, while a second patient is active on the waitlist for SPK retransplantation after no evidence of metastatic disease for 2 years.ConclusionThe successful management of metastatic donor-derived malignancies requires allograft removal, immunosuppression cessation, and adjuvant therapy that includes occasional use of checkpoint inhibitors to augment the immune response.

PURPOSE Cell-free DNA (cfDNA) may allow for minimally invasive identification of biologically relevant genomic alterations and genetically distinct tumor subclones. Although existing biomarkers may detect localized prostate cancer, additional strategies interrogating genomic heterogeneity are necessary for identifying and monitoring aggressive disease. In this study, we aimed to evaluate whether circulating tumor DNA can detect genomic alterations present in multiple regions of localized prostate tumor tissue. METHODS Low-pass whole-genome and targeted sequencing with a machine-learning guided 2.5-Mb targeted panel were used to identify single nucleotide variants, small insertions and deletions (indels), and copy-number alterations in cfDNA. The majority of this study focuses on the subset of 21 patients with localized disease, although 45 total individuals were evaluated, including 15 healthy controls and nine men with metastatic castration-resistant prostate cancer. Plasma cfDNA was barcoded with duplex unique molecular identifiers. For localized cases, matched tumor tissue was collected from multiple regions (one to nine samples per patient) for comparison. RESULTS Somatic tumor variants present in heterogeneous tumor foci from patients with localized disease were detected in cfDNA, and cfDNA mutational burden was found to track with disease severity. Somatic tissue alterations were identified in cfDNA, including nonsynonymous variants in FOXA1, PTEN, MED12, and ATM. Detection of these overlapping variants was associated with seminal vesicle invasion ( P = .019) and with the number of variants initially found in the matched tumor tissue samples ( P = .0005). CONCLUSION Our findings demonstrate the potential of targeted cfDNA sequencing to detect somatic tissue alterations in heterogeneous, localized prostate cancer, especially in a setting where matched tumor tissue may be unavailable (ie, active surveillance or treatment monitoring).

e18565 Background: Diagnosis of hereditary cancers by genetic testing (GT) has significant implications for treatment decisions for individuals and risk reduction among family members. Recent guidelines have expanded the eligibility criteria for GT. However, significant disparities in GT completion persist based on race, ethnicity, insurance status, and English proficiency. Thus, novel interventions are needed to provide diverse populations equitable access to GT. Methods: From March to October 2021, we conducted a single arm study of a genetic testing station (GTS) intervention in a safety net hospital. The GTS intervention included (1) screening of clinic schedules for eligible patients; (2) video-based GT education in English, Spanish, and Cantonese; (3) consent for GT and collection of a blood or saliva sample by a genetic counseling assistant; (4) telehealth-based post-test counseling with a genetic counselor (GC). Bilingual, culturally concordant staff developed the educational videos and study materials. Patients who met the NCCN criteria for GT were eligible. We compared this group to a historical cohort who initiated GT in the preceding nine months. The historical cohort underwent GT in a traditional model: (1) oncologist-initiated referral, (2) pretest counseling with a GC, (3) GT if appropriate, and (4) post-test counseling to review and discuss results. Our primary outcomes were accessibility and feasibility assessed by tracking the implementation process. Secondary outcomes included completion rates of GT and post-test GC visit, and time from enrollment to GT result disclosure. We evaluated all associations with Chi-square tests for categorical variables and Pearson’s Chi-square test of medians for time to post-test visit. Results: We approached 92 patients for GTS; 3 declined, 89 patients underwent the GTS intervention. Of 155 patients (GTS: n = 89, historical cohort: n = 66), 15% were Black, 28% Asian, 17% White, 31% Hispanic/Latinx, 2% Native Hawaiian/Pacific Islander, and 7% multiethnic/other; 51% spoke English, 27% Spanish, 17% Cantonese, and 5% other. In the GTS cohort, 85 (96%) of participants completed GT and 80 (90%) completed the post-test GC visit, compared to 46 (70%, p < 0.001) and 42 (64%, p < 0.001) respectively in the historical cohort. The median time to result disclosure was 30.5 days (IQR 23.8 - 38.8) for the GTS cohort and 80.5 days (IQR 37.5 - 137.5, p = 0.001) for the historical cohort. Conclusions: Implementation of GTS was feasible and acceptable in a safety net setting. GTS resulted in more patients completing GT and post-test counseling and significantly reduced the time to result disclosure, which has the potential to impact treatment decisions and outcomes. As GT eligibility criteria broaden, innovative interventions, workflows, and tailored educational resources for diverse populations are needed to ensure equitable access to GT.

BackgroundCell-free DNA’s (cfDNA) use as a biomarker in cancer is challenging due to genetic heterogeneity of malignancies and rarity of tumor-derived molecules. Here we describe and demonstrate a novel machine-learning guided panel design strategy for improving the detection of tumor variants in cfDNA. Using this approach, we first generated a model to classify and score candidate variants for inclusion on a prostate cancer targeted sequencing panel. We then used this panel to screen tumor variants from prostate cancer patients with localized disease in bothin silicoand hybrid capture settings.MethodsWhole Genome Sequence (WGS) data from 550 prostate tumors was analyzed to build a targeted sequencing panel of single point and small (in silicoagainst prostate tumor sequences from 5 patients to assess performance against commonly used alternative panel designs. The panel’s ability to detect tumor-derived cfDNA variants was then assessed using prospectively collected cfDNA and tumor foci from a test set 18 prostate cancer patients with localized disease undergoing radical proctectomy.ResultsThe panel generated from this approach identified as top candidates mutations in known driver genes (e.g. HRAS) and prostate cancer related transcription factor binding sites (e.g. MYC, AR). It outperformed two commonly used designs in detecting somatic mutations found in the cfDNA of 5 prostate cancer patients when analyzed in anin silicosetting. Additionally, hybrid capture and 2,500X sequencing of cfDNA molecules using the panel resulted in detection of tumor variants in all 18 patients of a test set, where 15 of the 18 patients had detected variants found in multiple foci.ConclusionMachine learning-prioritized targeted sequencing panels may prove useful for broad and sensitive variant detection in the cfDNA of heterogeneous diseases. This strategy has implications for disease detection and monitoring when applied to the cfDNA isolated from prostate cancer patients.

PurposeProstate cancer is the most commonly diagnosed neoplasm in American men. Although existing biomarkers may detect localized prostate cancer, additional strategies are necessary for improving detection and identifying aggressive disease that may require further intervention. One promising, minimally invasive biomarker is cell-free DNA (cfDNA), which consist of short DNA fragments released into circulation by dying or lysed cells that may reflect underlying cancer. Here we investigated whether differences in cfDNA concentration and cfDNA fragment size could improve the sensitivity for detecting more advanced and aggressive prostate cancer.Materials and MethodsThis study included 268 individuals: 34 healthy controls, 112 men with localized prostate cancer who underwent radical prostatectomy (RP), and 122 men with metastatic castration-resistant prostate cancer (mCRPC). Plasma cfDNA concentration and fragment size were quantified with the Qubit 3.0 and the 2100 Bioanalyzer. The potential relationship between cfDNA concentration or fragment size and localized or mCRPC prostate cancer was evaluated with descriptive statistics, logistic regression, and area under the curve analysis with cross-validation.ResultsPlasma cfDNA concentrations were elevated in mCRPC patients in comparison to localized disease (OR5 ng/mL= 1.34, P = 0.027) or to being a control (OR5 ng/mL= 1.69, P = 0.034). Decreased average fragment size was associated with an increased risk of localized disease compared to controls (OR5bp= 0.77, P = 0.0008).ConclusionThis study suggests that cfDNA concentration and average cfDNA fragment size may provide a quick, cost-effective approach to help determine which patients will benefit most from further screening and/or disease monitoring to help improve prostate cancer outcomes.

Summary Neoantigens arise from mutations in cancer cells and are important targets of T cell-mediated anti-tumor immunity. Here, we report the first open-label, phase Ib clinical trial of a personalized neoantigen-based vaccine, NEO-PV-01, in combination with PD-1 blockade in patients with advanced melanoma, non-small cell lung cancer, or bladder cancer. This analysis of 82 patients demonstrated that the regimen was safe, with no treatment-related serious adverse events observed. De novo neoantigen-specific CD4+ and CD8+ T cell responses were observed post-vaccination in all of the patients. The vaccine-induced T cells had a cytotoxic phenotype and were capable of trafficking to the tumor and mediating cell killing. In addition, epitope spread to neoantigens not included in the vaccine was detected post-vaccination. These data support the safety and immunogenicity of this regimen in patients with advanced solid tumors (Clinicaltrials.gov: NCT02897765).