Kohli, Manish

Abstract Introduction: Survival in mCRPC stage is heterogeneous and in this stage no molecular predictors of survival are known. Elevated circulating tumor cell (CTC) counts (>5/7.5 ml blood CellSearch® assay) is the only FDA cleared prognostic biomarker in mCRPC stage that predicts poor prognosis but is not routinely performed in clinical practice. We evaluated a novel plasma cfDNA based molecular algorithmic score which is based on copy number variations (CNVs) in multiple candidate genes as a prognostic predictor of survival and also compared it to CTCs. Methods: We obtained biospecimens in a prospective cohort study (NCT#01953640) conducted in mCRPC patients who demonstrated progression on androgen deprivation therapy and were candidates to initiate pre-chemotherapy abiraterone acetate/prednisone (AA/P). Biospecimen collection was performed prior to initiating AA/P and included plasma and CTCs, both obtained at the same time. All samples underwent uniform processing. Cellsearch® assay was used for enumerating CTC counts. Plasma cfDNA was extracted using Qiagen whole blood kit and low pass sequencing was performed in the extracted plasma cfDNA and germline DNA. An algorithmic score based on CNVs in 13 candidate genes was developed after calculating an Absolute Log2 Ratio (ALR) for each pre-selected gene. The sum of ALRs for these 13 genes were combined to make a “Plasma Genome Abnormality” (PGA) score which was evaluated for association with the primary endpoint of overall survival (OS) using Hazard ratio (HR) with significance set at p ≤ 0.05. Association of concurrently collected CTC counts with OS was also determined. The PGA score based survival was adjusted for CTC counts in the final analysis. Results: Plasma and CTCs were available for analysis in 72/90 enrolled patients between 05/2013 and 09/2015 with the last enrolled patient having completed 23.5 months follow-up. 45/72 patients have died of cancer progression and the median OS of the cohort is 26.5 months (IQR: 19-46). The median plasma cfDNA amount obtained was 7.2 (Range: 1.6-70.4)ng/ml plasma and the median CTC count is 1/7.5ml blood (range: 0-308). The median PGA score was 1.108 (Range: 0.56-6.96) and the CNV based PGA score was observed to be associated with OS (HR: 1.63; 95% C.I: 1.144, 2.308); p-value= 0.0066). Association of elevated CTC counts (≥ 5/7.5 ml blood) with OS showed a statistically significant trend (HR: 1.92; CI: 0.99 - 3.7; p-value 0.051). After adjusting for CTC counts, an increased PGA score remained predictive of poor survival (HR: 1.52; 95% CI: (1.04, 2.21); p-value 0.028). Conclusions: In this cohort the PGA score, a molecular algorithm predictor based on plasma cfDNA copy number variations in candidate genes was a better prognostic marker of survival than CTC counts and potentially offers a biomarker tool which could be adopted for wider clinical application. Citation Format: Manish Kohli, Meijun Du, Liang Wang, Chiang-Ching Huang. Prognostic association of plasma cell free DNA (cfDNA) copy number variation based algorithmic score with survival in metastatic castration resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4588.

Abstract Background: Abiraterone acetate/prednisone (AA/P) is used for treatment of metastatic castration-resistant prostate cancer (mCRPC), but response is variable and there are no known molecular predictive factors. Methods: We prospectively collected germline and plasma specimens from 72 mCRPC patients progressing on androgen-deprivation therapy (ADT) before initiating AA/P treatment (baseline) and serially after 12 weeks of treatment. After extracting cell-free DNA (cfDNA), we performed copy number analysis using low pass whole-genome sequencing approach. The primary endpoints were to determine association with response and survival. Cox regression and Kaplan-Meier survival analyses were used to associate the CNV with overall survival (OS) and progression-free survival (PFS) with statistical significance at p≤0.05. For determining primary resistance to AA/P, responders and nonresponders were defined as patients showing no progression or progression after 12 weeks treatment. T-test was used to evaluate the CNV with AA/P response at 12 weeks. Results: Whole-genome sequencing generated approximately 8.0 million mappable sequence reads per subject (total plasma samples sequenced, n=144). By comparing cfDNA to germline control DNA, our copy number analysis showed significant genomic abnormalities in 37 of 144 plasma samples. We observed the most frequent amplification of 1q, 8q, Xq and deletion of 2p, 6q,10q, 13q. Among these CNVs, amplification of MYC at 8q24.21, AR at Xq12, deletion of FOXP1 at 3p14, PTEN at 10q23.31, RB1 at 13q14.2, TP53 at 17P13.1, and gene fusions (TMPRSS2:ERG) were consistent with previous reports from tumor tissue-based studies. Survival analysis showed significant association of a fragment deletion at 13q14-32 with both OS and PFS (FDR= 0.0207/0.0141). The most significant fragment showing association with OS was the deletion of fragment at 2p22.1-p14 (FDR=0.00219). Genomic gains of MYC at 8q24.21 and NCOA2 at 8q13.3 were associated with PFS with FDR= 0.0085 and 0.0072, respectively. By comparing baseline CNVs between responder and nonresponders, we identified significant association of the deletion of PTEN at 10q23.31 (p= 0.0003), RB1 at 13q14.2 (p= 0.0008) and amplification of NCOA2 at 8q13.3 (p= 0.0030) with AA/P response/resistance at 12 weeks. Although statistical analysis showed the significant CNV change at AR locus between baseline and 12-week post-AA/P treatment (p=0.0002), the CNV change was not significantly different between responders and nonresponders (p=0.1697). Conclusions: cfDNA CNV may serve as a sensitive biomarker for prediction of treatment response to AA/P. Further detailed CNV and clinical association analyses are needed to fully validate the potential clinical utilities of cfDNA-based liquid biopsy for clinical management. Citation Format: Meijun Du, Manish Kohli, Chiang-Ching Huang, Liang Wang. Genomic alterations in plasma cell-free DNA predict treatment response to abiraterone acetate and prednisone and survival in metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1077.

Abstract Purpose: Deletions or mutations in PTEN and TP53 tumor suppressor genes have been linked to lineage plasticity in therapy-resistant prostate cancer. Fusion-driven overexpression of the oncogenic transcription factor ERG is observed in approximately 50% of all prostate cancers, many of which also harbor PTEN and TP53 alterations. However, the role of ERG in lineage plasticity of PTEN/TP53–altered tumors is unclear. Understanding the collective effect of multiple mutations within one tumor is essential to combat plasticity-driven therapy resistance. Experimental Design: We generated a Pten-negative/Trp53-mutated/ERG-overexpressing mouse model of prostate cancer and integrated RNA-sequencing with ERG chromatin immunoprecipitation-sequencing (ChIP-seq) to identify pathways regulated by ERG in the context of Pten/Trp53 alteration. We investigated ERG-dependent sensitivity to the antiandrogen enzalutamide and cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor palbociclib in human prostate cancer cell lines, xenografts, and allografted mouse tumors. Trends were evaluated in TCGA, SU2C, and Beltran 2016 published patient cohorts and a human tissue microarray. Results: Transgenic ERG expression in mice blocked Pten/Trp53 alteration–induced decrease of AR expression and downstream luminal epithelial genes. ERG directly suppressed expression of cell cycle–related genes, which induced RB hypophosphorylation and repressed E2F1-mediated expression of mesenchymal lineage regulators, thereby restricting adenocarcinoma plasticity and maintaining antiandrogen sensitivity. In ERG-negative tumors, CDK4/6 inhibition delayed tumor growth. Conclusions: Our studies identify a previously undefined function of ERG to restrict lineage plasticity and maintain antiandrogen sensitivity in PTEN/TP53–altered prostate cancer. Our findings suggest ERG fusion as a biomarker to guide treatment of PTEN/TP53-altered, RB1-intact prostate cancer. Clin Cancer Res; 24(18); 4551–65. ©2018 AACR.

350 Background: In prostate cancer, overexpression of EZH2 is associated with disease recurrence, androgen independent growth, cell invasion, and metastases. We evaluated the prognostic significance of EZH2 gene expression in metastatic biopsies from men with CRPC. Methods: EZH2 (RNA) expression was evaluated in two serial metastatic site biopsies in patients (pts) prior to treatment with abiraterone acetate and prednisone (pre-AA/P) and after 12 weeks of treatment (post-AA/P). All pts were enrolled and prospectively followed for clinical outcomes. The primary endpoint was overall survival (OS), defined as the time from CRPC to death or last follow-up. Cox proportional hazard regression analysis was performed on EZH2 expression using normalized fragments per kilobase million (FPKM) > 1 for association with OS and time to treatment failure (TTF). Results: Of 92 enrolled pts, 59 pts had (pre-AA/P) gene expression using RNA-seq, 45 had paired pre and post-AA/P RNA-seq available for analysis. In pre-AA/P samples, the median EZH2 expression was 4.82 FPKMs (range 1.05-38.8). Elevated expression of EZH2 was associated with shorter OS [hazard ratio (HR) 2.58, p = 0.016]. Radiographic progression at 12 weeks was more frequent in high EZH2 tumors compared to low EZH2 tumors (48% vs 20%, p = 0.03, Fisher’s exact test) No statistically significant associations were identified between EZH2 expression and TTF. In paired post-AA/P biopsies, EZH2 expression increased in 13/45 tumors and decreased in 32/45 tumors. The median log2 fold change in EZH2 expression was -1.38 (range -6.7 to 3.1). In post-AA/P biopsies, no associations were identified between an increase in EZH2 expression and OS or TTF. Conclusions: EZH2 expression represents a potential novel prognostic biomarker in mCRPC and warrants further exploration. Clinical trial information: 01953640.

262 Background: Inherited and somatic aberrations in DNA repair genes in castrate resistant prostate cancer (CRPC) are associated with poor prognosis, but respond well to poly ADP ribose polymerase (PARP) inhibitors. We evaluated the prevalence and prognostic impact of harboring germline DNA repair variants in hormone sensitive prostate cancer (HSPC). Methods: Germline DNA from buffy coat was sequenced on HiSeq4000 with a median coverage of 200X for DNA repair variants in 20 genes in HSPC and CRPC patients (pts) enrolled in a hospital registry. Pts were divided into two groups; Group A: pts enrolled at the time of CRPC stage; Group B: treatment naïve HSPC stage pts. The primary endpoints were to determine any impact of harboring DNA repair variants on time to progression from HSPC to CRPC and, from CRPC to death. Group A pts were retrospectively analyzed for time to progression from HSPC to CRPC while Group B patients were followed prospectively for outcomes. Statistical analysis included Cox proportional hazard models and Wilcoxon Rank sum test with significance at p≤0.05. Results: In Group A, 51/562 CRPC pts (9.07%) had variants in the 20 genes (most frequently in BRCA2; n = 15). 44/51 pts with variants and 399/511 without variants had died. Median time of progression from HSPC to CRPC with/without variants was 22.1 vs. 25.1 months (mths); p-value = 0.679. Median time from CRPC to death with/without variants was 32.2 Vs. 27.7 mths (p = 0.6). In HSPC Group B, 14/100 pts were identified with germline variants in ATM (n = 5), CHEK2 (n = 3), BRCA1 (n = 2), BRCA2 (n = 2), RAD50 (n = 1), and MSH2 (n = 1). 31/100 have died and median time to progression from HSPC to CRPC with/without variants was 15.6 vs.11.8 mths, p-value = 0.76. Conclusions: Pts with germline DNA repair variants detected in HSPC stage were not associated with poor prognosis. Presence of additional somatic DNA repair gene aberrations in cell-free DNA, not investigated in this cohort may add to the prevalence of DNA repair gene variations in HSPC and together impact prognosis adversely so as to provide a rationale for PARP inhibitor therapy in select HSPC stage pts.

249 Background: Serum CGA has been identified as a candidate prognostic biomarker for mCRPC. In a two cohort study, we compared the prognostic value of serum CGA with a validated CTC assay. Methods: In the discovery cohort (DC), blood samples were collected from 256 men with mCRPC. In an independent validation cohort (VC), 92 men with mCRPC were enrolled in a biospecimen collection study. In both cohorts, men receiving proton pump inhibitors and those with non-castrate levels of testosterone ( > 50ng/dl) were excluded. Serum CGA was measured in a homogeneous automated immunofluorescent assay using time-resolved amplified cryptate emission. In the VC, CTC enumeration was performed using the FDA cleared CELLSEARCH CTC test prior to treatment with abiraterone acetate/ prednisone. Cox proportional hazard regression and Kaplan-Meier analysis were performed for associations with elevated CGA (above reference range), unfavorable (≥ 5) CTCs, and overall survival (OS). Results: In the DC, 200 men were eligible for analysis. The median age was 72 years (yrs), 81/200 pts had a Gleason score (GS) ≥ 8, 34/200 had an elevated CGA. At a median follow up of 2.2 yrs, 156/200 were deceased. In the subset of men with GS ≥ 8, elevated CGA was associated with shorter OS [hazard ratio (HR) 2.19, p = 0.017]. In the VC, 71 men were eligible for analysis. The median age was 71 yrs, 36/71 tumors were GS ≥ 8, 31/71 pts had an elevated CGA, 26/66 had unfavorable CTCs (≥ 5). At a median follow up of 1.8 yrs, 31/71 were deceased. Elevated CGA (HR 1.91, p = 0.043) and unfavorable CTC counts (HR 2.97, p = 0.0012) were adversely associated with OS. In the high GS group, both CTCs and CGA had the same area under the curve (AUC) of 0.72. Pts with elevated CTC and CGA had the poorest OS (HR 3.76, p = 0.008). Conclusions: Elevated serum CGA was negatively associated with OS in men with mCRPC. Serum CGA represents a prognostic biomarker that may complement CTC enumeration. Clinical trial information: NCT#01953640.

243 Background: AA and D have been shown in separate trials to increase overall survival in patients with mCSPC compared to Androgen Derivation Therapy (ADT). In the absence of head to head clinical trials and to provide clinical guidance, we performed an indirect comparison of AA and D using network meta-analysis. Methods: We performed a search of MEDLINE, EMBASE, Cochrane Library, and Cochrane Central Register of Controlled Trials to identify relevant clinical trials. Collected data included hazard ratio and confidence interval (CI) for Overall Survival (OS) and number of adverse events in each study arm. Risk for bias was assessed using the Cochrane Collaboration’s tool. Bayesian network meta-analysis was conducted using WinBUGS 1.4.3 software (MRC Biostatistics Unit, Cambridge, UK) to perform an indirect comparison of D and AA. Results: Five clinical trials were included in this analysis. Two trials (LATITUDE, STAMPEDE) compared AA to ADT and three trials (CHAARTED,STAMPEDE, GETUG-AFU 15 study) compared D to ADT. Results from both fixed effect and random effect network meta-analyses for the primary outcome (OS) revealed no statistical significance between AA and D (HR 0.81,95%CI 0.65-1.01; HR 0.81, 95%CI 0.40-1.82) respectively. Comparatively, abiraterone had statistically significant fewer events of anemia (OR 0.14,95%CI 0.08-0.23), neutropenia (OR 0.06,95%CI 0.03-0.12), peripheral edema (OR 0.21,95%CI 0.09-0.44), dyspnea (OR 0.22,95%CI 0.08-0.51), nausea (OR 0.09,95%CI 0.02-0.24), diarrhea (OR 0.06,95%CI 0.02-0.15), constipation (OR 0.25,95%CI 0.11-0.53), and fatigue (OR 0.12,95%CI 0.07-0.20). AA had statistically significant more events of hot flashes (OR 3.85, 95% CI2.33-6.25). For other adverse events, both drugs were statistically similar. Conclusions: There is no difference in OS using AA for longer periods in CSPC than a regimen of a limited number of cycles of D. There are significant differences in side effect profile of these drugs. Further analyses are needed to determine cost effectiveness of AA vs D under consideration of comparative efficacy and safety.