Combined Cell-free DNA and RNA Profiling of the Androgen Receptor: Clinical Utility of a Novel Multianalyte Liquid Biopsy Assay for Metastatic Prostate Cancer

Abstract

Background: The androgen receptor (AR) remains a critical driver in metastatic castration-resistant prostate cancer (mCRPC). Profiling AR aberrations in both circulating DNA and RNA may identify key predictive and/or prognostic biomarkers in the context of contemporary systemic therapy.

Objective: To profile AR aberrations in circulating nucleic acids and correlate with clinical outcomes.

Design, setting, and participants: We prospectively enrolled 67 mCRPC patients commencing AR pathway inhibitors (ARPIs; n = 41) or taxane chemotherapy (n = 26). Using a first-in-class next-generation sequencing-based assay, we performed integrated cell-free DNA (cfDNA) and cell-free RNA (cfRNA) profiling from a single 10 ml blood tube.

Outcome measurements and statistical analysis: Kaplan-Meier survival estimates and multivariable Cox regression analyses were used to assess associations between clinical outcomes and the following AR aberrations: copy number variation, splice variants (AR-V7 and AR-V9) and somatic mutations.

Results and limitations: Cell-free DNA and cfRNA were successfully sequenced in 67 (100%) and 59 (88%) patients, respectively. Thirty-six (54%) patients had one or more AR aberrations. AR gain and cumulative number of AR aberrations were independently associated with clinical/radiographic progression-free survival (PFS; hazard ratio [HR] 3.2, p = 0.01 and HR 3.0 for 0 vs ≥2, p = 0.04) and overall survival (HR 2.8, p = 0.04 and HR 2.9 for 0 vs ≥2, p = 0.03). Notably, concurrent AR gain and AR splice variant expression (AR gain/AR-V+) was associated with shorter prostate-specific antigen PFS on both ARPIs (HR 6.7, p = 0.009) and chemotherapy (HR 3.9, p = 0.04). Importantly, key findings were validated in an independent cohort of mCRPC patients (n = 40), including shorter OS in AR gain/AR-V+ disease (HR 3.3, p = 0.02). Limitations include sample size and follow-up period.

Conclusions: We demonstrate the utility of a novel, multianalyte liquid biopsy assay capable of simultaneously detecting AR alterations in cfDNA and cfRNA. Concurrent profiling of cfDNA and cfRNA may provide vital insights into disease biology and resistance mechanisms in mCRPC.

Patient summary: In this study of men with advanced prostate cancer, DNA and RNA abnormalities in the androgen receptor detected in blood were associated with poor outcomes on available drug treatments. This information could be used to better guide treatment of advanced prostate cancer.

Keywords: Androgen receptor; Biomarker; Castrate resistant; Cell-free DNA; Cell-free RNA; Liquid biopsy; Prostate cancer.

Fig. 1 –

Landscape of AR aberrations identified in cell-free DNA and RNA across the Australian cohort. Asterisks denote ARPI therapy, whilst daggers denote taxane chemotherapy. Prior therapy at baseline is also shown. Orange tiles represent presence of aberration; missing cfRNA data are denoted by grey tiles. AR = androgen receptor; ARPI = AR pathway inhibitor; AR-V = AR splice variant; cfRNA = cell-free RNA.

Fig. 2 –

Kaplan-Meier analysis of PSA-PFS (top), clinical or radiographic PFS (middle), and overall survival (bottom), according to AR copy number status (left), the presence of at least one of AR gain, AR splice variant, or AR somatic mutation (centre), and the total number of AR aberrations (0, 1,≥2) present (right). Two and eight patients were excluded from analysis for any AR aberration and total AR aberrations, respectively, due to insufficient information on AR-V expression. AR = androgen receptor; AR-V = AR splice variant; Clin/rPFS = clinical/radiographic progression-free survival; OS = overall survival; PFS = progression-free survival; PSA = prostate-specific antigen.

Fig. 3 –

Kaplan-Meier analysis of (A) PSA-PFS, (B) clinical or radiographic PFS, and (C) overall survival, according to concurrent expression of both an AR-V and an AR copy number gain. Six patients were excluded from analysis due to insufficient information on AR-V expression. AR = androgen receptor; AR-V = AR splice variant; Clin/rPFS = clinical/radiographic progression-free survival; OS = overall survival; PFS = progression-free survival; PSA = prostate-specific antigen.