Platinum Priority – Prostate CancerEditorial by XXX on pp. x–y of this issueCombined Cell-free DNA and RNA Profiling of the Androgen Receptor: Clinical Utility of a Novel Multianalyte Liquid Biopsy Assay for Metastatic 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
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2022, European UrologyCitation Excerpt :Adverse events (AEs) and serious AEs (according to Common Terminology Criteria for Adverse Events version 4.03) were collected up to 30 and 90 d after the last dose of avelumab, respectively. Peripheral blood was collected from a subset of patients for exploratory biomarker analysis of plasma cell-free DNA/RNA (cfDNA/cfRNA) using the PredicineCARE and PredicineRNA liquid biopsy panels (Predicine Inc., Hayward, CA, USA) as previously described [19–21]. Analysis of cfRNA was restricted to the AR splice variants AR-V7 and AR-V9, recognised for their strong association with pathogenicity [22].
Circulating RNAs in prostate cancer patients
2022, Cancer LettersCitation Excerpt :Blood cfRNA levels of both AR and AR-V7 have recently been utilized in multiparametric liquid biopsies to follow mCRPC during time and at progression upon the use of specific treatments such as PARP-inhibitor [56]. In another study, Fettke et al. integrated expression of circulating AR and its variants (AR-V7 and AR-V9) in a cohort of mCRPC patients undergoing treatment with AR pathway inhibitors or chemotherapy to associate the presence of AR (and/or its analyzed variants) with poor outcome [39]. Furthermore, AR full length and AR-V7 mRNAs were detected in urinary EVs collected from CRPC patients, thereby demonstrating the concrete possibility of using the detection of such transcripts in both urine and blood liquid biopsies [57].
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These authors contributed equally to this work.