Prof. Joshua M. Lang of the Department of Medicine, University of Wisconsin in Madison, WI, US and colleagues used a novel liquid biopsy technology to collect mRNA from circulating tumour cells (CTCs) to measure expression of androgen receptor (AR) splice variants, AR targets, and neuroendocrine prostate cancer markers. A pattern of gene expression was identified by hierarchical cluster analysis from RNA extracted from CTCs; this cluster was shown to be prognostic for decreased overall survival (OS) in the training cohort and decreased progression-free survival (PFS) and overall survival (OS) in the validation cohort including patients from two clinical studies with AR signalling inhibitor (ARSI). The findings are published on 1 July 2021 in the Journal of Clinical Oncology.

The authors wrote in the background that comprehensive understanding of the molecular drivers of ARSI resistance and the ability to monitor this evolution over time is critical for early detection and appropriate treatment selection to improve patient outcomes.

They used a CTC liquid biopsy transcriptional assay capable of simultaneously measuring three resistance mechanisms. This multiplex panel includes AR splice variants (AR-V7 and AR-V9), a panel of AR-regulated genes (TMPRSS2, KLK2, KLK3, and FOLH1) that may indicate persistent or overactivation of AR transcriptional activity, and neuroendocrine prostate cancer associated genes (SYP and CHGA).

An institutional review board–approved prospective cohort (n=99) was used to identify patterns of gene expression. Two prospective multicentre phase II clinical studies of ARSIs for patients with castration-resistant prostate cancer (NCT01942837, enzalutamide, n=21] and NCT02025010, abiraterone, n=27) were used to further validate these findings.

Hierarchical clustering of CTC transcripts identified two distinct clusters. Cluster 2 exhibited increased expression of AR-regulated genes and was associated with worse OS. In particular, median OS was 8.6 versus 22.4 months (hazard ratio [HR] 3.45, 1.9 to 6.14; p < 0.01).

In multivariable analysis, cluster 2 was prognostic independent of other clinicopathologic variables. AR variant status was not significant when accounting for cluster 2.

Upon further validation in pooled multicentre phase II studies, cluster 2 was associated with worse OS, 15.2 months versus not reached (HR 8.43, 2.74 to 25.92, p < 0.01), prostate-specific antigen PFS 3.6 versus 12 months (HR 4.64, 1.53 to 14.11; p < 0.01), and radiographic PFS 2.7 versus 40.6 months (p < 0.01; HR 4.64, 1.82 to 17.41; p < 0.01).

The study team wrote that they demonstrated the first multiplex gene expression liquid biopsy assay that can assess multiple potential ARSI resistance mechanisms simultaneously in metastatic prostate cancer. Early identification of these molecular changes could help guide treatment decisions. The heterogeneous nature of their cohort, which included different disease states and therapies, suggests that these findings may be generalisable to the broader patient population.

In addition, they commented that the validation of prognostic clustering in two phase II studies with ARSIs suggests clinical utility; however, larger clinical studies are necessary. They are prospectively testing these findings in multiple ongoing clinical studies for patients with castration sensitive and castration resistant prostate cancer.

In terms of relevance of study findings, the authors wrote that detection of AR-V7 in CTCs has emerged as a biomarker that is prognostic for treatment resistance to ARSI and in their work they demonstrate that expression of AR-regulated genes in CTCs is also prognostic for survival and radiographic progression that may have broader ability to identify treatment resistance than AR-V7 alone.

The study was supported by awards and grants from Movember-Prostate Cancer Foundation, US National Institute of Health and Department of Defense.


Sperger JS, Emamekhoo H, McKay RR, et al. Prospective Evaluation of Clinical Outcomes Using a Multiplex Liquid Biopsy Targeting Diverse Resistance Mechanisms in Metastatic Prostate Cancer. JCO; Published online 1 July 2021. DOI: 10.1200/JCO.21.00169.