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269 Background: The 22-gene Decipher genomic classifier (GC) is a prognostic biomarker that has been validated in phase III trials in high-risk localized, post-prostatectomy, and metastatic and non-metastatic castration-resistant prostate cancer. Herein, we report the first validation of the biopsy GC in intermediate-risk prostate cancer from the phase III randomized trial NRG/RTOG 0126. Methods: After National Cancer Institute approval, biopsy slides were collected from the NRG biobank from RTOG 0126, a phase III randomized trial of men with intermediate-risk prostate cancer randomized to 70.2 Gy versus 79.2 Gy of radiotherapy without the use of concomitant hormone therapy. RNA was extracted from the highest grade tumor foci and processed through a quality control (QC) pipeline prior to generation of the previously locked 22-gene GC model. After GC data was generated it was linked with clinical outcomes to assess prognostic performance. The primary endpoint for this ancillary project was disease progression, defined as biochemical failure, local failure, distant metastasis or prostate cancer-specific mortality, as well as use of salvage therapy. Secondary endpoints included the previous individual endpoints, metastasis-free survival, and overall survival. Independent GC prognostic performance was assessed using cause-specific Cox or competing risk adjusted Fine-Gray multivariable models that included randomization arm and prognostic stratification factors. Death without events were treated as competing risks. Results: A total of 215 patient samples passed QC of the 449 that had suitable cDNA for expression analysis. The median follow-up was 12.8 years (range 2.4-17.7), and 61% had Gleason 3+4, 24% had Gleason 4+3, and the median PSA was 7.2 ng/mL (IQR 5.0-10.2). On multivariable analysis the 22-gene GC (per 0.1 unit) was independently prognostic for disease progression (subdistribution hazard ratio [sHR] 1.13, 95%CI (1.01-1.26), p = 0.03), biochemical failure (sHR 1.23, 95%CI 1.10-1.37, p < 0.001), distant metastasis (sHR 1.28, 95%CI 1.06-1.54, p = 0.01), and PCSM (sHR 1.45, 95%CI 1.20-1.76, p < 0.001). In patients with lower GC scores the 10-year distant metastasis rate difference between the 70.2 Gy and 79.2 Gy was 5%, as compared with 26% for higher GC patients. Conclusions: This study represents the first validation of any biopsy-based gene expression classifier in intermediate-risk prostate cancer. Decipher is independently prognostic and can identify patients that have low rates of metastatic events despite not receiving concurrent hormone therapy, and can be used to help personalize therapy in this setting. Clinical trial information: NCT00033631.

265 Background: A recent study ( Fossati et al, 2018) found that higher lymph node count at radical prostatectomy was associated with improved outcomes in patients treated with salvage radiation for elevated prostate-specific antigen (PSA) after surgery. We sought to validate these results in NRG/RTOG 9601, a randomized controlled trial of men with pT2/T3 disease who underwent either radiation (RT) alone or RT+antiandrogen (bicalutamide) therapy for PSA elevation following radical prostatectomy from 1998-2003. Methods: We reviewed available pathology reports for all patients in NRG/RTOG 9601 to determine the nodal count at radical prostatectomy. Clinical data was as of 11/5/2015, same as the primary endpoint for the trial. Cox proportional hazards models were used to assess the effect of number of positive lymph nodes, treatment arm (RT alone or RT+bicalutamide), Gleason score, positive margins, and seminal vesicle invasion on the following endpoints: times to local and distant failure and overall and disease specific survival. Results: Out of the 760 patients originally eligible in the trial, 552 (73%, 276 in each arm) had complete data available. Median node count in the entire cohort was 6 (range 0-33, Q1-Q3 3-9). There were no significant differences between treatment arms in terms of patient demographic or clinical characteristics, including total lymph nodes removed in either arm (RT alone vs RT+bicalutamide median 5 vs 6, p = 0.11). There was no significant association between total lymph nodes and overall survival with both arms combined (HR = 1.00, 95% CI:0.97-1.03, p = 0.87) or in the individual arms alone (RT+Casodex: HR = 1.01, 95% CI:0.97-1.05, p = 0.65; RT+Placebo: HR = 0.98, 95% CI: 0.94-1.03, p = 0.45). There was also no significant association between total lymph nodes and disease-specific survival with both arms combined (HR = 1.00, 95% CI:0.95-1.04, p = 0.84) and in the arms alone (RT+Casodex: HR = 1.00, 95% CI:0.95-1.05, p = 0.92; RT+Placebo: HR = 0.99, 95% CI: 0.92-1.07, p = 0.86). In multivariable analysis performed on the two arms, Gleason score was the only feature associated with worse overall and disease-specific survival, seen only in the RT alone arm. Similar findings were seen when evaluating times to local and distant failure. Conclusions: Lymph node yield in NRG/RTOG 9601 did not show any association with adverse outcomes in the entire cohort, or in either treatment arm alone. The therapeutic benefit of an extensive lymph node dissection in this population remains uncertain. Clinical trial information: NCT00002874.

58 Background: Distinguishing indolent from aggressive prostate cancer remains a key challenge for prostate cancer management decision-making. A growing number of biomarkers are now on the market to help address this need, but have rarely been examined together in the same patients to determine their relative value. Methods: We identified men with low-risk cancers defined by biopsy Gleason score ≤3+3, PSA ≤10ng/ml, and clinical stage ≤T2 who underwent immediate prostatectomy. We collected archived prostate tissue and pre-surgical plasma samples from N = 381 cases from UCSF and N = 279 cases from UW. Plasma was analyzed for TGFb1 and IL6SR, previously validated markers for prostate cancer prognosis, using ELISA. From prostate tissue, DNA and RNA were extracted and analyzed for the GEMCaP copy number variation score and the cell cycle progression (CCP) score, respectively, both well-validated and previously reported scores. Pathologic outcomes were minor (pGS 3+4 or pT3a) or major (pGS ≥4+3 or ≥pT3b) upgrading/upstaging (UGUS), and multinomial regression was performed to determine putative markers’ ability to predict these outcomes, controlling for PSA, percent of biopsy cores positive, age, and clinical site. Results: Overall, 357 men had no UGUS event at prostatectomy, 236 had a minor event, and 67 had a major event. Neither TGFb1 nor IL6SR statistically significantly predicted any UGUS or major UGUS. On the other hand, both CCP and GEMCaP were directly associated with minor UGUS on univariate analysis. On multivariable multinomial analysis including both scores, the CCP score predicted minor UGUS (OR 1.70, 95% CI 1.07-2.72, p = .03) and GEMCaP also predicted minor UGUS (OR 1.04, 95% CI 1.01-1.07, p < .01). Neither CCP nor GEMCaP predicted major UG/US, but the number of events was small. Noteworthy, both GEMCaP and the CCP score were statistically significant predictors of outcomes in the same model, suggesting they can complement each other by offering additional information. The other clinical parameters were not significant in this model. Conclusions: Biomarker signatures based on analysis of DNA and RNA significantly and independently predict adverse pathology among men with clinically low-risk prostate cancer undergoing prostatectomy.

69 Background: Over 15% of men with newly diagnosed prostate cancer (PCa) have high-risk features that raise the recurrence risk. Better biomarkers could allow for even earlier detection of biochemical recurrence (BCR) and inform adjuvant treatment decisions. Circulating tumor cells (CTCs) may represent the earliest form of metastases, however their role as biomarkers in men with localized PCa is not well defined. Here, we aim to enumerate and molecularly and genomically analyze CTCs using an enrichment-free, unbiased CTC identification technology from men with high-risk, localized PCa after radical prostatectomy (RP) and correlate the analysis with clinical outcomes. Methods: Blood samples from 37 patients with high-risk, localized PCa were obtained 2-5 mos post RP and shipped to Epic. All nucleated cells were subjected to immunofluorescent staining for cytokeratin (CK), CD45, and AR. CTCs were identified using algorithmic analysis. CK+ CTCs were enumerated and subsequently analyzed for AR expression and individually sequenced for copy number alterations (CNA). Patients were followed for BCR, defined as detectable PSA > 0.2ng/dL. Progression free survival (PFS) was calculated using Kaplan-Meier and Cox proportional hazards. Results: CTCs were detected in 81.1%(30/37) of patients with an average of 5.2 CTCs/ml (range: 0 – 22.9) detected per patient. AR expression was detected in 18.9% (7/37) of patients. Ninety nine CTCs from 14 patients were picked and sequenced. CNAs were identified in CTCs in commonly mutated genes in PCa, including MYC amplification and CHD1 deletions. Patients with higher traditional CTC (CK+) burdens exhibited a trend towards shorter PFS (hazard ratio: 1.65; 95% confidence interval: 0.7-3.86; p = 0.13). Conclusions: There was a high incidence of CTC detection after RP in patients with high-risk, localized PCa. A trend toward shorter PFS was seen in those with higher CTC burden. Genomic alterations were detectable in CTCs and consistent with established CNAs in PCa. With further testing in appropriately powered cohorts early CTC detection after primary therapy could represent an informative biomarker to stratify patients with high risk PCa.

74 Background: Among men with clinically low-risk prostate cancer, we have previously documented heterogeneity in terms of clinical characteristics and genomic risk scores. In this study, we aimed to study the underlying tumor biology of this patient population, by interrogating patterns of gene expression among men with clinically low-risk tumors. Methods: Prostate biopsies from 427 patients considered potentially suitable for active surveillance underwent central pathology review and genome-wide expression profiling. These cases were compared to 1290 higher-risk biopsy cases with diverse clinical features from a prospective genomic registry. Average genomic risk (AGR) was determined from 18 published prognostic signatures, and MSigDB Hallmark gene sets were analyzed using bootstrapped clustering methods. These sets were examined in relation to clinical variables and pathologic and biochemical outcomes using multivariable regression analysis. Results: 408 (96%) of biopsies passed RNA quality control. Based on average genomic risk quartiles defined by the high-risk multicenter cases, the UCSF low-risk patients were distributed across the quartiles as 219 (54%), 107 (26%), 61 (15%), and 21 (5%). Unsupervised clustering analysis of the Hallmark gene set scores revealed 3 clusters, which were enriched for the previously described PAM50 luminal A, luminal B and basal subtypes. These three clusters did not associate with existing clinical or known genomic risk characteristics, suggesting a novel and independent classification for low-risk prostate cancer. AGR was associated with both pathological (OR: 1.3, p < 0.001) and biochemical outcomes (OR: 1.5, p = 0.001 ) but the clusters were not. Conclusions: Prostate cancers that are largely homogeneously low-risk by traditional characteristics demonstrate substantial diversity at the level of genomic expression. Molecular sub-stratification of low-risk prostate cancer may facilitate better decision-making with respect to both timing and intensity of cancer surveillance and treatment.

132 Background: We evaluated the feasibility and efficacy of a decision support intervention designed to help men with low-risk prostate cancer consider active surveillance and standard treatments. The intervention incorporated a decision aid with coaching and question-listing. Our decision aid is the first to include data on long-term survival and side effects from men with prostate cancer undergoing active surveillance, surgery, and radiation. Methods: To develop the intervention, we conducted focus groups using the Nominal Group Technique. We used a survey instrument from the International Patient Decision Aids Standards to measure the stakeholder endorsement of our intervention. To test the intervention, we administered it to newly diagnosed men with low-risk prostate cancer (Gleason sum < = 3+4, stage < = T2N0M0, PSA < = 10 ng/ml) seen at UCSF. Before and after the intervention, we administered a survey with questions from the Decision Quality Instrument for Prostate Cancer. Our primary outcome was change in knowledge as assessed by two multiple-choice items: How many men diagnosed with early stage prostate cancer will eventually die of prostate cancer? How much would waiting 3 months to make a treatment decision affect chances of survival? Correct answers were: “Most will die of something else” and “A little or not at all.” Results: The development phase involved 6 patients, 1 family member, 2 physicians, and 5 other health care providers and four iterations of the intervention until consensus endorsement was reached. In the pilot test, 57 men consented, and 44 received the decision support intervention and completed surveys at both timepoints. Before the intervention, 30/44 (68%) got both questions right, compared to 36/44 (82%) after the intervention. 82% maintained or achieved perfect scores; 16% answered 1 or more incorrectly both before and after the intervention; and 2% answered both items correctly before, but 1 wrong after. Conclusions: This novel decision support intervention was feasible, and appeared to improve knowledge and informed decision-making. Data will guide the development of a larger scale randomized clinical trial to improve decision quality in men with prostate cancer, in the community. Clinical trial information: NCT02451345.

588 Background: Genome-wide epigenetic events appear to play a role in the development and behavior of HPV+ cancers. The value of adjuvant therapy following chemoradiation for localized anal cancer (AC) remains unclear. Molecular prognostication to identify patients (pts) who may be at higher risk for recurrence would be valuable. The goal was to define methylomic profiles predictive of disease-free (DFS) and overall (OS) survival in pts with AC. Methods: Genomic DNA was extracted, processed and methylation status at ~450,000 CpG loci examined (Illumina HumanMethylation450 Array). A multistep bioinformatics methodology was applied to develop a prognostic methylomic classifier for OS and DFS: (1) feature selection for methylated regions (β-value interquartile range ≥ 0.2, ≥ 2 adjacent significant probes within a CpG Island and p < 0.05 by univariate Cox proportional hazards) (2) selected features were entered into a supervised principal component analysis (PCA) and 3 components (PC1, PC2, PC3) were derived (3) classifier was built using forward selection multivariate regression models [PC1, PC2, PC3 alone and in combination with clinical features (size: > T2 vs. ≤ T2, nodal status: N0 vs N+)] using a 10-fold cross-validation (4) final model prediction risk score was generated, dichotomized and evaluated for prognostic values in Cox regression analysis. Results: 121 AC specimens from RTOG 98-11 were examined. The methylomic-only classifier model trended towards statistical significance (log-rank p = 0.05; HR = 1.96; 95% CI 0.99-3.88) in DFS (PC1, PC3 selected). In the combined model with clinical features, the final classifier included T status and epigenetic features (PC1, PC3) and was strongly predictive for DFS (p < 0.0001, HR = 4.45; 2.02-9.76). Final OS classifier models [methylomic-only (p = 0.28 HR = 1.55; 0.70-3.44) or combined (p = 0.013 HR = 2.88; 1.20-6.89)] were not as accurate. Conclusions: Methylomic and clinical features synergize to predict DFS in AC. Multivariate modeling reveal independent contributions from clinical and methylomic variables. Epigenomic profiling may contribute to identification of high-risk pts who may benefit from adjuvant strategies. Support: U10CA180822, U10CA180868, U24CA196067

100 Background: We validated a biopsy-based assay as a predictor of PCa aggressiveness in two independent patient cohorts. Using GPS with NCCN risk group, the test provides an estimate of likelihood of favorable pathology (LFP) in low-intermediate risk PCa based on data from the first validation study. The second study re-confirmed the association between GPS and LFP. We combined information from both studies to provide more precise estimates of LFP, and examined predictive models using GPS with other clinical risk tools. Methods: Patient-specific MA provides precision-weighted predictions for individual patients using data from multiple studies (Crager and Tang, J. Appl. Stat. 2014). MA was performed on the two validation studies (732 patients total) using GPS (scale 0-100) with CAPRA score, NCCN risk group, or AUA/EAU risk group as predictors of LFP (Gleason score 3+3 or 3+4, organ-confined disease). Decision curves were calculated using the MA risk estimates. Results: MA provided more precise estimates of LFP with narrower confidence intervals (CIs) than either study alone (median width 24% narrower than the smaller of the two individual study CIs). GPS added significant predictive value for LFP to each of the 3 clinical classifiers. A model utilizing GPS and CAPRA provided the most risk discrimination. In decision-curve analysis, greater net benefit was shown when combining GPS with each clinical classifier compared with the clinical classifier alone. Over a wide range of threshold probabilities, incorporation of GPS would be expected to lead to fewer treatments of patients with favorable pathology without increasing the number of patients with adverse pathology left untreated. The proportion of men with MA-estimated LFP > 80% was 31% with GPS and CAPRA, and 23% with GPS and NCCN risk group; 11% were identified with LFP > 80% using NCCN risk group alone. Conclusions: Patient-specific MA of two independent studies provided more precise risk estimates reflecting the complete body of evidence. GPS adds predictive value to three clinical risk tools. The MA-estimated LFP identified more patients with an LFP > 80% than identified by clinical classifiers alone.

123 Background: We aimed to evaluate a recently proposed prognostic Gleason grading system among two distinct cohorts of men receiving treatment for localized PCa with extended clinical follow. Methods: We identified patients receiving treatment with radical prostatectomy (RP) in two settings: an academic referral center and the multi-center Cancer of the Prostate Strategic Urological Research Endeavor (CaPSURE) registry. We examined the independent prognostic ability of the proposed grade groups (3+3; 3+4; 4+3; 4+4; 9-10), adjusted for significant clinical and pathological characteristics, on disease recurrence, defined as two consecutive PSA values ≥ 0.2 ng/mL or receipt of salvage treatment, using Cox proportional hazards models. Adjusted pairwise comparisons were made between grade groups. Distant disease endpoints including metastatic progression and prostate cancer specific mortality (PCSM) were similarly evaluated within the CaPSURE cohort. Results: We identified 1,734 men receiving treatment at UCSF with immediate RP with pathologic assessment consistent with the 2005 ISUP modification, and 4,564 men within the CaPSURE database. Median follow up within the UCSF cohort was 33 months (interquartile range, IQR 16-58) and 91 months (IQR 50-135) within CaPSURE. Relative to Gleason grade 3+3, all prognostic Gleason grade groups were independently associated with risk of recurrence among the UCSF cohort (all p < 0.01). Adjusted pairwise comparisons of biopsy and pathologic Gleason groups, except 4+3 versus 4+4, also were statistically significant. Among the CaPSURE cohort, biopsy and pathologic prognostic Gleason grade groups were independently associated with risk of recurrence, metastatic progression and PCSM, though pairwise distinctions between Gleason 4+3 and 4+4 and Gleason 4+4 versus 9-10 were not significant. This study was limited by the comparatively smaller proportion of men receiving radical prostatectomy for higher Gleason grade ( > 4+4) disease. Conclusions: A proposed prognostic Gleason classification system stratifies men according to the risk of recurrence and downstream endpoints. However, the discrimination of outcomes among higher grade disease appeared less robust.

2564 Background: Sipuleucel-T is an FDA-approved autologous cellular immunotherapy for patients with asymptomatic or minimally symptomatic metastatic castrate resistant prostate cancer (mCRPC). To date, studies of sipuleucel-T in patients with mCRPC have studied immune response in peripheral blood. The effects of sipuleucel-T on prostate tumors are unknown. Methods: NeoACT (P07-1; NCT00715104) is an open-label, phase 2 study of patients with localized prostate cancer who received sipuleucel-T prior to radical prostatectomy (RP) to examine the immunologic effects of treatment on prostate tissue. Patients received 3 infusions of sipuleucel-T at approximately 2-week intervals, beginning 6-7 weeks prior to RP. The primary endpoint was the change in the frequency of lymphocytes between prostate biopsies (pre-treatment) and RP tissue (post-treatment), as assessed by immunohistochemistry (IHC). Results: The median age of the 42 enrolled patients was 61 years, and all had an ECOG performance status of 0. Thirty-eight patients received all 3 pre-RP sipuleucel-T infusions. To date, tissue IHC analysis has been completed on 32 patients. Treatment-related AEs were manageable and transient. Sipuleucel-T did not appear to impact surgery, as judged by operative complications, procedure time, and estimated blood loss. Frequent events that occurred ≤1 day after infusion (>10% of patients) were fatigue, headache, and myalgia. Significant increases (≥3 fold) in CD3+ and CD4+ T cell populations were observed at the tumor interface (where benign and malignant glands interface), compared with the pre-treatment biopsy, benign RP tissue, and tumor RP tissue (ANOVA post hoc Newman-Keuls test: p

181 Background: Sipuleucel-T is an FDA-approved autologous cellular therapy that has been demonstrated to prolong overall survival in patients with asymptomatic or minimally symptomatic metastatic castrate resistant prostate cancer (mCRPC). To better understand the immunologic effects of sipuleucel-T, an open-label Phase 2 study (P07‐1; NCT00715104 ) of sipuleucel-T prior to radical prostatectomy (RP) was undertaken in patients with localized prostate cancer. Methods: Patients received 3 infusions of sipuleucel-T at approximately 2-week intervals, beginning 6–7 weeks prior to RP. Prostate biopsies (pre-treatment) and tissue from RP (post-treatment) were assessed for the presence of lymphocytes by immunohistochemistry (IHC). Results: The median age of the 42 enrolled patients was 61 years, and all had an ECOG performance status of 0. Thirty-eight patients received all 3 pre-RP infusions of sipuleucel-T. To date, tissue IHC analysis has been completed in 19 patients. Treatment-related AEs were manageable and reversible. Sipuleucel-T did not appear to impact surgery, as judged by operative complications, procedure time, and estimated blood loss. Frequent events that occurred ≤1 day after infusion (>10% of patients) were fatigue, headache, and myalgia. Significant increases (>2‐fold) in CD3+ and CD4+ T cells populations were observed at the tumor rim (where benign and malignant glands interface), compared with the pre-treatment biopsy (ANOVA post hoc Newman-Keuls test: p=0.0002, 0.0002, respectively). CD8+ T cells or CD56+ cells were not significantly increased at the tumor rim compared with benign biopsy regions. Conclusions: Neoadjuvant sipuleucel-T treatment appears to result in an increased frequency of T cells in prostate cancer tissue at the rim between the benign and malignant glands. These data suggest that sipuleucel-T may modulate the presence of lymphocytes at the prostate tumor site. Work is ongoing to more fully characterize the immune response within the prostate tumor tissue and in the peripheral blood.

483 Background: The mammalian target of rapamycin (mTOR) regulates cell growth by integrating nutrient and growth factor signaling and has been strongly implicated in cancer. Mutations in KRAS and the PI3K pathway are upstream of mTOR and are common in colorectal carcinoma (CRC). Currently approved mTOR inhibitors are derivatives of the natural product rapamycin and have shown little clinical efficacy in CRC. A new and potentially more efficacious class of ATP-competitive mTOR inhibitors has now entered clinical trials. Methods: Seeking a more representative preclinical model of CRC, we generated primary xenografts in nude mice of surgically resected specimens of human hepatic colorectal cancer metastases. We then treated xenograft tumors with the selective ATP-competitive mTOR inhibitor PP242 and monitored response by inhibition of tumor growth, changes in histopatholgy, and alterations in signaling pathways. Cell line experiments were performed to support observations made in the patient derived xenografts. Results: We demonstrate that in contrast to rapamycin, the mTOR inhibitor PP242 is highly effective at inhibiting tumor growth in both the primary xenograft model and in colorectal cancer cell lines. The inhibition of tumor growth in the xenografts and cell lines depended upon the strong inhibition of phosphorylation of mTOR substrate eIF4E binding protein 1 (4EBP1) but was not correlated with inhibition of phosphorylation of S6 kinase (S6K). Cells with mutant KRAS were relatively resistant to PP242 induced growth inhibition and this correlated with reduced inhibition of 4EBP1 phosphorylation. However, these effects were partially rescued in cells in which a co-mutation in PIK3CA resulted in AKT activation. Conclusions: Our studies reveal the first mTOR inhibitor resistant cell line, the genetic basis for its resistance and most importantly, these findings were revealed through the use of a primary xenograft mouse model which recapitulates morphologically the features of the tumors isolated from patients. We believe ATP-competitive inhibitors may be of limited clinical utility in mutant KRAS tumors, except for those that have concomitant activation of AKT.