Feng, Felix Y.

133 Background: The genomic classifier, Decipher, has been validated to predict risk of metastasis after radical prostatectomy (RP). However, the cohort size and event rate in the previous studies did not allow for a thorough investigation into performance within individual clinicopathologic or treatment subgroups. In this study, we present the first meta-analysis of the performance of the 22-marker genomic classifier in men with prostate cancer (PCa) post-RP. Methods: MEDLINE, EMBASE, and the Decipher genomic resource information database were searched for published reports of men with PCa treated by RP between 2010 and 2016 where the benefit of the Decipher genomic classifier test was assessed. The primary end point was the ability of Decipher to independently improve prognostication of regional or distant metastasis over routine clinicopathologic factors. Meta-analysis was performed with random-effects modeling, and extent of heterogeneity between studies was determined with the I2 test. Results: Five studies (975 total patients, and 855 with individual patient genomic and clinicopathologic data) were eligible for analysis. The median follow-up was 8 years. All patients had clinical high-risk disease, yet 60.9%, 22.6%, and 16.5% of patients were classified as low, intermediate, and high-risk, respectively by Decipher and had 10-year cumulative incidence rates of metastases of 5.5%, 15.0% and 26.7% (p < 0.001), respectively. Adjusting for standard clinicopathologic variables, on multivariable analysis Decipher remained a statistically significant predictor of metastasis (hazard ratio [HR] 1.30 per 0.1 unit, 95% confidence interval [CI] 1.14-1.47, p < 0.001), and the summary HR for metastasis of Decipher across the 5 studies was 1.52 (95% CI 1.39-1.67) per 0.1 unit. Conclusions: The genomic classifier test, Decipher, has the ability to independently improve prognostication of men post-RP, as well as within nearly all clinicopathologic and treatment subgroups. Strong consideration should be given to incorporating the use of genomic testing in clinical decision making and clinical trials to better individualize treatment.

9 Background: Prostate cancer molecular subtypes based on ETS gene fusions and SPINK1 were originally identified through outlier gene expression profiling analysis. Such molecular subtypes may have utility in disease stratification and clonality assessment, complementing available purely prognostic tests. Hence, we determined the analytical validity of molecular subtyping in a large sample of PCa treated with radical prostatectomy. Methods: We analyzed Affymetrix Human Exon 1.0ST GeneChip expression profiles for 1,577 patients from 8 radical prostatectomy (RP) cohorts. Multi-feature random forest classifiers and outlier analysis were used to define microarray-based molecular subtypes. Results: A random forest (RF) classifier was trained and validated to predict ERG fusion status using a subset with known ERG rearrangement status defined by FISH, achieving >95% sensitivity and specificity in the validation subset. Less frequent rearrangements involving other ETS genes or SPINK1 over-expression were predicted based on gene expression outlier analysis. Across cohorts, 45%, 9% 8% and 38% of PCa were classified as ERG+, ETS+, SPINK+, and Triple Negative, respectively. Global gene expression analysis shows that the four subtypes could be collapsed into three entities (ERG+, ETS+ and SPINK+/Triple Negative) based on expression patterns and clinical characteristics similarity. A series of multivariable analyses further revealed, ERG+ to be associated with lower pre PSA and Gleason scores but more likely to have EPE and occur in patients with European American ancestry compared to the ETS+, SPINK+/Triple Negative tumors (p<0.001). In contrast, patients with ETS+ were more likely to have SVI compared to both ERG+ and SPINK/Triple Negative (p=0.01), while SPINK+/Triple Negative had higher Gleason scores and were more likely to occur in African Americans (p<0.001). Conclusions: The Decipher platform can accurately determine ERG rearrangement status and PCa molecular subtypes. Inclusion of molecular subtyping, such as m-ERG status, may enable additional precision medicine opportunities in prognostic tests

8 Background: A number of biomarkers related to cell cycle, angiogenesis or apoptosis have been found to be associated with patient outcome in tissue samples from men treated with first line radiation therapy in RTOG clinical trials using immunohistochemical staining and analysis. In a prior study, four biomarkers (Ki-67, MDM2, p16 and Cox-2) and clinical covariates were included in a model of distant metastasis (DM; Pollack et al, Clin Cancer Res, 2014 [epub ahead of print]) risk. The current study tested the hypothesis that these genes are prognostic for DM in men treated primarily with total prostatectomy using RNA expression profiling. Methods: RNA fromprostatectomy samples from Cleveland Clinic (CC, n=182); Mayo Clinic (MC)-I (n = 545) and II (n=235); Memorial Sloan Kettering Cancer Center (MSKCC, n=131); Erasmus Medical Center (EMC, n=48) and Thomas Jefferson University (TJU, n=130) were profiled using 1.4 million RNA features. A Cox proportional hazards model was built on the MC-I training set to combine the 4 biomarkers into a prognostic risk score (4BMSig). 4BMSig was subsequently evaluated for its prognostic significance separately and in combination with clinical risk factors (biopsy Gleason Score, cT-category and Preop-PSA) for DM. Results: 4BMSig was found to discriminate DM patients significantly for the MC-II (AUC = 0.66, p < 0.001), CCF (AUC = 0.68, p < 0.001), and MSKCC (AUC = 0.71, p = 0.04) datasets, and achieved borderline significance for EMC (AUC = 0.70, p = 0.06). 4BMSig did not discriminate DM in the TJU dataset (only 10 DM events). Pooled multivariable analysis (n = 726) with clinical covariates revealed that 4BMSig is a strong independent prognostic covariate for DM (p < 0.001) and prostate cancer specific mortality (p = 0.005). Conclusions: The four genes identified previously as being associated with DM in radiotherapy patients were incorporated herein into 4BMSig, which was found to have potential as a pretreatment prognostic DM risk assessment tool for men treated with prostatectomy. Further validation would consist of testing 4BMSig from RNA in diagnostic tissue from prostate cancer patients prior to prostatectomy.

7 Background: Improved prognostic biomarkers are needed for localized prostate cancer. We undertook an unbiased large-scale analysis of genes associated with metastatic progression and validated the prognostic ability of the top candidate gene. Methods: Prostate cancer samples from prostatectomy patients were analyzed for gene expression using a clinical-grade, high-density Affymetrix GeneChip platform, encompassing >1 million genomic loci and assessed in a CLIA-certified laboratory. Nomination of prognostic candidate genes was performed on a discovery cohort (n=545) and validated on 3 independent cohorts (n=463). Multivariate analyses were performed for the primary endpoint of metastasis. The top prostate-specific gene was further evaluated in 208 additional tumor samples with a novel RNA in-situ hybridization (ISH) assay and in urine samples from 230 patients using PCR. Results: Of all known genes, the long noncoding RNA SChLAP1 ranked first for elevated expression in patients with metastatic progression by receiver-operator-curve analyses. Validation in three independent cohorts confirmed the prognostic value of SChLAP1. On multivariate modeling, SChLAP1 expression independently predicted metastasis within 10 years (odds ratio (OR) = 2.45, 95% confidence interval (CI) 1.70 – 3.53), death within 10 years (OR = 1.93, 95% CI 1.31 – 2.85), and biochemical recurrence within 5 years (OR = 1.76, 95% CI 1.28 – 2.41) with ORs comparable to Gleason score. Evaluation of SChLAP1 expression by RNA ISH confirmed a significant association with disease recurrence (OR = 1.99, 95% CI 1.06 – 3.73). Evaluation of urine SChLAP1 levels demonstrated increased expression in patients at higher risk for disease progression. Conclusions: We perform the largest high-throughput, unbiased study of prostate cancer prognostic biomarkers to date and discover SChLAP1 as a top gene predictive of metastatic progression. We validate SChLAP1 extensively with a clinical-grade assay. We show feasibility of a RNA ISH assay and a non-invasive urine test for SChLAP1. Our results, spanning 1,446 patients from 6 independent patient cohorts, suggest that SChLAP1 represents a very promising biomarker for aggressive clinical course.

168 Background: The optimal timing of postoperative radiotherapy following radical prostatectomy (post-RP RT) is unclear. We hypothesized that a genomic classifier (GC) would provide prognostic and predictive insight into the development of clinical metastases in men receiving post-RP RT and inform decision-making. Methods: GC scores were calculated from 188 patients with pT3 or margin positive PCa, who received post-RP RT at Thomas Jefferson University and Mayo Clinic, between 1990 and 2009. The primary endpoint was clinical metastasis. Prognostic accuracy of the models were tested using c-index and decision curve analysis. Cox regression tested the relationship between GC and metastasis. Results: The cumulative incidence of metastasis at 5 years post-RT was 0%, 9%, and 29% for low, average, and high GC scores, respectively (p=0.002). In multivariable analysis, GC and pre-RP PSA were independent predictors of metastasis (both p<0.01). Within the low GC score (<0.4), there were no differences in the cumulative incidence of metastasis comparing those who received adjuvant or salvage RT (p=0.79). However, for patients with higher GC scores (≥0.4) cumulative incidence of metastasis at 5-year was 6% vs. 23% for patients treated with adjuvant vs. salvage RT (p<0.01). Conclusions: In patients treated with post-RP RT, GC is prognostic for the development of clinical metastasis beyond routine clinical/pathologic features. Though preliminary, patients with low GC are best treated with salvage radiation, while those with high GC benefit from adjuvant therapy. These findings provide the first rationale selection of timing of post-RP RT.

256 Background: B7-H3 (CD276), part of the B7 superfamily, has been shown to play an immunomodulatory role, however its regulation, receptor and mechanism of action remain unclear. Protein levels of B7-H3 have been previously shown to relate to prostate cancer outcomes and currently, humanized monoclonal antibodies are being developed for clinical use (MGA271, Macrogenics). Here we use genomic expression data to examine the relationship of B7-H3 to prostate cancer outcomes and molecular subtypes. Methods: Prostatectomy tissue from 905 patients were profiled using the Affymetrix HuEx 1.0 ST microarray. Kruskal-Wallis tests were used to identify significant associations of B7-H3 expression with clinico-pathologic variables, and survival analysis were used to evaluate the prognostic value of B7-H3. Pearson’s correlation analyses were also performed to assess the relationship of B7-H3 expression with molecular subtypes and individual transcripts. Androgen receptor (AR) occupancy of promoter regions was derived in silico from chromosomal immune-precipitation (ChIP) data. Results: B7-H3 expression was positively associated with Gleason score (p < 0.01) and tumor stage (p < 0.01). High B7-H3 expression also correlated with the development of metastasis and prostate cancer specific mortality (HR of 3.4 and 2.4 respectively, p < 0.05 for both), but this was not significant on multi-variable analysis. B7-H3 was positively associated with ERG+ disease (n = 670, r = 0.85, p < 0.05) and AR expression (n = 670, r = 0.46, p < 0.001). B7-H3 was found to be one of the most correlated genes with AR (95th percentile) and ChIP analysis revealed AR binding upstream of B7-H3, suggesting potential androgen dependent regulation. Conclusions: B7-H3 expression correlates with high Gleason grade and advanced prostate cancer stage with higher quartiles of expression portending poor oncologic outcomes in two independent prostatectomy cohorts. B7-H3 expression appears to relate to the androgen receptor.

12 Background: In 3 published randomized trials, adjuvant radiation therapy (ART) for prostate cancer (PCa) resulted in improved progression free survival. However, the impact on metastases and overall survival is unclear. To date, there have been no published prospective trials examining the impact of salvage radiation therapy (SRT) in this disease state. Hence, we conducted a retrospective, nonrandomized comparative study of ART, SRT, or no radiation following radical prostatectomy (RP) for men with pT3 disease or positive margins (adverse pathologic features, APF). Methods: 422 PCa men treated at 4 institutions with RP and having APF were analyzed with a primary end point of clinical metastasis. ART (n = 111), early SRT (n = 70) and delayed SRT (n = 83) were defined by PSA levels of < 0.2, 0.2 to 0.5, and ≥ 0.5 ng/mL, respectively, prior to RT initiation. Remaining 157 men who did not receive additional therapy prior to metastatic onset formed the no RT arm. Clinical-genomic risk was assessed by CAPRA-S and Decipher. Cox multivariable (MVA) model was used to evaluate the impact of treatment on outcome. Results: During study follow-up, 37 men developed metastasis with a median follow-up of 8 years. Both CAPRA-S and Decipher had independent predictive value on MVA for metastatic outcome (both p < 0.05). On MVA adjusting for clinical-genomic risk, delayed SRT and no RT had an HR of 4.31 (95%CI, 1.20-15.47) and 5.42 (95% CI, 1.59-18.44) for metastasis compared to ART. No significance difference was observed between early SRT and ART (p = 0.28). Men with low to intermediate CAPRA-S and low Decipher have a low rate of metastatic events regardless of treatment selection. In contrast, men with high CAPRA-S and Decipher benefit from ART, however the cumulative incidence of metastasis remains high. Conclusions: The decision as to the timing and need for additional local therapy following RP is nuanced and requires providers and men to balance risks of morbidity with improved oncologic outcomes. This analysis provides the most robust and accurate quantification of risk for these men. Post-RP treatment can be safely avoided for men who are low risk by clinical-genomic risk, whereas those at high risk should favor enrollment in clinical trials.

106 Background: Androgen deprivation therapy (ADT) is one of the main treatment options for locally advanced and metastatic prostate cancer. Neuroendocrine prostate cancer (NEPC) is inherently less sensitive or even resistant to ADT. NEPC can be observed de novo (e.g., small cell prostate cancer) but more commonly arises after exposure to ADT. We hypothesized that a gene expression signature of NEPC when measured in primary tumor specimens (RP) of prostatic adenocarcinoma may be useful for predicting patients with innate resistance to ADT. Methods: Expression profiles of 1023 PCa patients treated with RP were obtained from the Decipher GRID database. These were split into training (n=529) and validation (n=494) sets and stratified by the receipt of adjuvant ADT (n=243) or no adjuvant ADT (n=780). A literature review of ADT resistance and neuroendocrine genes identified 1,557 genes as candidates. This set was further filtered, using logistic regression to select a 52-gene ADT resistance signature (ARS). ARS was trained using a generalized linear model with lasso regularization. Survival c-index and Kaplan Meier was used to compare survival differences between treated and untreated patients with high and low ARS scores (defined by median split). Results: In validation cohorts, the ARS was predictive of metastasis in cohorts receiving adjuvant ADT (10-year metastasis free survival c-index of 0.69 (95% CI 0.59-0.78) as compared to 0.45 (95% CI 0.29-0.61) in patients not treated with ADT). Similarly in a separate cohort of untreated patients that received no ADT until after metastatic onset, ARS was not prognostic (c-index 0.53). Among ADT treated patients, those with low ARS scores had a 10 year MFS of 87%, versus 70% in those with high ARS scores (p<0.001). In the subset of men who received ADT after metastatic onset and who developed castrate-resistant prostate cancer (CRPC, n = 41), median time to treatment failure was 1 year in patients with high ARS compared to 2 years for those with low ARS scores (p=0.07). Conclusions: A 52-gene ADT resistance signature was developed which showed significant differences in metastasis-free survival among adjuvant hormone treated but not untreated patients.

Abstract Serum levels of miR-194 have been reported to predict prostate cancer recurrence after surgery, but its functional contributions to this disease have not been studied. Herein, it is demonstrated that miR-194 is a driver of prostate cancer metastasis. Prostate tissue levels of miR-194 were associated with disease aggressiveness and poor outcome. Ectopic delivery of miR-194 stimulated migration, invasion, and epithelial–mesenchymal transition in human prostate cancer cell lines, and stable overexpression of miR-194 enhanced metastasis of intravenous and intraprostatic tumor xenografts. Conversely, inhibition of miR-194 activity suppressed the invasive capacity of prostate cancer cell lines in vitro and in vivo. Mechanistic investigations identified the ubiquitin ligase suppressor of cytokine signaling 2 (SOCS2) as a direct, biologically relevant target of miR-194 in prostate cancer. Low levels of SOCS2 correlated strongly with disease recurrence and metastasis in clinical specimens. SOCS2 downregulation recapitulated miR-194–driven metastatic phenotypes, whereas overexpression of a nontargetable SOCS2 reduced miR-194–stimulated invasion. Targeting of SOCS2 by miR-194 resulted in derepression of the oncogenic kinases FLT3 and JAK2, leading to enhanced ERK and STAT3 signaling. Pharmacologic inhibition of ERK and JAK/STAT pathways reversed miR-194–driven phenotypes. The GATA2 transcription factor was identified as an upstream regulator of miR-194, consistent with a strong concordance between GATA2 and miR-194 levels in clinical specimens. Overall, these results offer new insights into the molecular mechanisms of metastatic progression in prostate cancer. Cancer Res; 77(4); 1021–34. ©2016 AACR.

Purpose We aimed to update a previously published, multi-institutional nomogram of outcomes for salvage radiotherapy (SRT) following radical prostatectomy (RP) for prostate cancer, including patients treated in the contemporary era. Methods Individual data from node-negative patients with a detectable post-RP prostate-specific antigen (PSA) treated with SRT with or without concurrent androgen-deprivation therapy (ADT) were obtained from 10 academic institutions. Freedom from biochemical failure (FFBF) and distant metastases (DM) rates were estimated, and predictive nomograms were generated. Results Overall, 2,460 patients with a median follow-up of 5 years were included; 599 patients (24%) had a Gleason score (GS) ≤ 6, 1,387 (56%) had a GS of 7, 244 (10%) had a GS of 8, and 230 (9%) had a GS of 9 to 10. There were 1,370 patients (56%) with extraprostatic extension (EPE), 452 (18%) with seminal vesicle invasion (SVI), 1,434 (58%) with positive surgical margins, and 390 (16%) who received ADT (median, 6 months). The median pre-SRT PSA was 0.5 ng/mL (interquartile range, 0.3 to 1.1). The 5-yr FFBF rate was 56% overall, 71% for those with a pre-SRT PSA level of 0.01 to 0.2 ng/mL (n = 441), 63% for those with a PSA of 0.21 to 0.50 ng/mL (n = 822), 54% for those with a PSA of 0.51 to 1.0 ng/mL (n = 533), 43% for those with a PSA of 1.01 to 2.0 ng/mL (n = 341), and 37% for those with a PSA > 2.0 ng/mL (n = 323); P < .001. On multivariable analysis, pre-SRT PSA, GS, EPE, SVI, surgical margins, ADT use, and SRT dose were associated with FFBF. Pre-SRT PSA, GS, SVI, surgical margins, and ADT use were associated with DM, whereas EPE and SRT dose were not. The nomogram concordance indices were 0.68 (FFBF) and 0.74 (DM). Conclusion Early SRT at low PSA levels after RP is associated with improved FFBF and DM rates. Contemporary nomograms can estimate individual patient outcomes after SRT in the modern era.