Abstract 6698: Combinatorial genomic and epigenomic cell-free DNA analysis of high-risk metastatic castration resistant prostate cancer reveals prognostic liquid biopsy signatures
Authors: Alahi Irfan , Chauhan Pradeep S. , Shiang Alexander L. , Webster Jace , Dang Ha X. , Greiner Lilli , yang Breanna , Ledet Elisa M. , Babbra Ramandeep K. , Feng Wenjia , Harris Peter K. , Jaeger Ellen B. , Miller Patrick J. , Caputo Sydney A. , Santos Giordano Cittolin , Sartor Oliver , Pachynski Russell K. , Maher Christopher A. , Chaudhuri Aadel A. 
Published: April 2023

Abstract
Background: Androgen receptor signaling inhibitors (ARSI) such abiraterone and enzalutamide have significantly improved clinical outcomes in metastatic castrate-resistant prostate cancer (mCRPC) patients. However, patients with genomic alterations in the androgen receptor (AR) and its enhancer region do not respond well and acquire resistance to these inhibitors. Here, we applied our previously developed cell-free DNA (cfDNA) liquid biopsy assay (EnhanceAR-Seq) to detect these high-risk mCRPC patients prior to the administration of first-line AR-directed therapy and correlated with survival. We also interrogated the plasma methylome to identify differentially methylated regions (DMRs) across these high-risk patients. Methodology: We applied EnhanceAR-Seq to plasma cfDNA isolated from 99 mCRPC patients enrolled from two institutions (n=52 Tulane; n=47 WashU). Plasma samples were collected prior to ARSI initiation (n=63) or during treatment (n=36). We also performed Enzymatic Methyl-seq in pre-treatment plasma from 43 patients. We split these 43 patients into cell-free genomically high-risk and low-risk groups using our EnhanceAR-seq results, and conducted DMR analysis using metilene. To identify significant DMRs, we performed multiple hypothesis testing and required q&lt;0.05 using the Benjamini-Hochberg procedure, and further required at least 5 CpGs per DMR. Results: EnhanceAR-Seq detected AR/enhancer alterations in 35% of all plasma samples. Cell-free AR/enhancer detection was highly prognostic (PFS HR=2.80, p=0.0002; OS HR=2.6, p=0.01). When considering only pre-treatment plasma, AR/enhancer alterations detected in 44% (28/63) of samples correlated significantly with worse PFS (HR=2.21, p=0.009) and OS (HR=2.60, p=0.02). AR/enhancer alterations detected in 19% (7/36) of samples collected during ARSI were also associated with worse PFS (HR=15.6, p=0.0002) and OS (HR=8.09, p=0.05). Plasma methylome analysis revealed that for cell-free genomically high-risk mCRPC patients (based on AR/enhancer alterations detected in cfDNA), significantly hypomethylated DMRs were found in the AR promoter, upstream AR enhancer, and in AR-associated genes including FOXP1 and FOLH1. Significantly hypomethylated DMRs were also observed in DNA damage repair and cell cycle genes including MSH6, MSH3, FANCD2, CDK12 and RAD51B. Hypermethylated DMRs were seen in tumor suppressor genes including ZBTB16, BRCA2, WT1 and GNAS. Conclusions: AR/enhancer alterations detected in plasma cfDNA predicted inferior survival in mCRPC patients. Cell-free genomically high-risk mCRPC patients could be distinguished from low-risk patients based on distinct methylation signatures.
Citation Format: Irfan Alahi, Pradeep S. Chauhan, Alexander L. Shiang, Jace Webster, Ha X. Dang, Lilli Greiner, Breanna yang, Elisa M. Ledet, Ramandeep K. Babbra, Wenjia Feng, Peter K. Harris, Ellen B. Jaeger, Patrick J. Miller, Sydney A. Caputo, Giordano Cittolin Santos, Oliver Sartor, Russell K. Pachynski, Christopher A. Maher, Aadel A. Chaudhuri. Combinatorial genomic and epigenomic cell-free DNA analysis of high-risk metastatic castration resistant prostate cancer reveals prognostic liquid biopsy signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6698.