Elsevier

European Urology

Volume 78, Issue 2, August 2020, Pages 173-180
European Urology

Platinum Priority – Prostate Cancer
Editorial by XXX on pp. x–y of this issue
Combined Cell-free DNA and RNA Profiling of the Androgen Receptor: Clinical Utility of a Novel Multianalyte Liquid Biopsy Assay for Metastatic Prostate Cancer

https://doi.org/10.1016/j.eururo.2020.03.044Get rights and content

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.

Introduction

Advances in therapeutic strategies have significantly improved both quantity and quality of life for metastatic castration-resistant prostate cancer (mCRPC) [1]. However, there remains a pressing need to identify predictive and prognostic biomarkers for contemporary systemic therapies. Identification of such biomarkers may hold great utility, informing optimal treatment selection, design of innovative clinical trials, and facilitating discussions with patients and caregivers around expected outcomes.

In the era of precision medicine, liquid biopsies have emerged as a minimally invasive alternative for interrogating the prostate tumour genome, either through the capture of intact circulating tumour cells (CTCs) or through the analysis of cell-free tumour nucleic acids [2]. Circulating assays have demonstrated strong congruence with tumour biopsies [3], whilst simultaneously encapsulating the complexity of intrapatient heterogeneity evident within the mCRPC genomic landscape.

As sustained androgen receptor (AR) signalling remains a key driver in mCRPC [4], considerable efforts have been made to profile AR aberrations using circulating nucleic acids. Resistance to novel AR pathway inhibitors (ARPIs; eg, abiraterone and enzalutamide) has been observed in patients harbouring AR copy number gain [5], [6], [7], [8], [9], somatic AR mutations [6], [7], [8], and constitutively active AR splice variants (AR-Vs) [10], [11]. Similarly, AR copy number gain has been associated with poor outcomes in patients receiving chemotherapy [12]. Importantly, nearly all assays have principally focused on genomic aberrations, which likely explain only a proportion of the phenotypic responses observed clinically. Transcriptomic analysis has been proposed as the bridge between genotype and phenotype where liquid biopsies are concerned [13], with obvious advantages to combining DNA and RNA sequencing data.

De Laere et al [14], [15] recently combined sequencing of the AR in plasma cell-free DNA (cfDNA) with RNA sequencing of AR-Vs expressed in CTCs. However, current isolation methods are unable to consistently detect CTCs in a high proportion of patients [10], [16], [17], highlighting the on-going need to develop and optimise CTC-independent platforms to detect AR-Vs as well as other transcriptomic resistance markers. Importantly, aberrations found specifically in cell-free RNA (cfRNA) have not been correlated previously with clinical outcomes in mCRPC patients receiving contemporary systemic treatments.

Here, we use a novel, high-sensitivity, multianalyte, next-generation sequencing assay to simultaneously profile the AR in pretreatment plasma cfDNA and cfRNA obtained from two independent cohorts of mCRPC patients. We found that concurrent DNA and RNA AR aberrations portend poor treatment outcomes, likely reflecting patients with aggressive intrinsic disease biology and resistance to existing therapies. Our data suggest that combined cfDNA and cfRNA sequencing may have high clinical value in mCRPC.

Section snippets

Study cohorts and sample processing

Patients with mCRPC were prospectively enrolled between September 2016 and August 2018 across two Australian institutions (Monash Health and Chris O’Brien Lifehouse). All patients provided written informed consent, with ethics approval obtained from each institution’s human research ethics committee. Peripheral blood (10 ml) was collected in a single EDTA-containing or dedicated cfDNA-stabilising tube (Streck, La Vista, Nebraska, USA) immediately prior to commencing systemic therapy (ARPIs or

Assay validation

Multiple steps were taken for analytical and orthogonal validation of the assay (Supplementary material). Serially diluted reference DNA was used to determine an assay limit of detection of 0.25%, or 0.1% for hotspot mutations. For AR-V7 detection, sensitivity was found to be 100% for samples with a minimum of 10 AR-V7 molecules, whilst no AR-Vs were detected in healthy controls. Regarding orthogonal validation, AR gains were validated using low-pass whole-genome sequencing. Similarly, AR-V7

Discussion

In this prospective multicentre study, we demonstrated the application of a novel, first-in-class liquid biopsy assay capable of simultaneously detecting both cfDNA and cfRNA aberrations in a single 10 ml blood tube from 67 mCRPC patients commencing contemporaneous systemic therapy. Using this assay to comprehensively profile both genomic and transcriptomic aberrations in the AR, we confirmed the ongoing critical impact that AR aberrations have in shaping the disease course in mCRPC.

Conclusions

We demonstrate the application of a first-in-class multianalyte liquid biopsy assay capable of simultaneously detecting AR genomic alterations in cfDNA and cfRNA with high sensitivity from a single blood tube. In two independent cohorts, we identified a novel poor prognosis subgroup harbouring concurrent AR gain and AR-V expression. These data support further evaluation of minimally invasive, multianalyte circulating nucleic assays in advanced prostate cancer patients, including integration

References (30)

  • AA Azad et al.

    Androgen receptor gene aberrations in circulating cell-free DNA: biomarkers of therapeutic resistance in castration-resistant prostate cancer

    Clin Cancer Res

    (2015)
  • A Romanel et al.

    Plasma AR and abiraterone-resistant prostate cancer

    Sci Transl Med

    (2015)
  • AW Wyatt et al.

    Genomic alterations in cell-free DNA and enzalutamide resistance in castration-resistant prostate cancer

    JAMA Oncol

    (2016)
  • ES Antonarakis et al.

    AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer

    N Engl J Med

    (2014)
  • M Cieslik et al.

    Cancer transcriptome profiling at the juncture of clinical translation

    Nat Rev Genet

    (2018)
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    These authors contributed equally to this work.

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