Biomarkers of Castrate Resistance in Prostate Cancer: Androgen Receptor Amplification and T877A Mutation Detection by Multiplex Droplet Digital PCR.

Francis P Young,Mohammed Nimir,Therese M Becker,Bavanthi Balakrishnar,Yafeng Ma,Paul De Souza,Wei Chua,Thomas Opperman

doi:10.3390/jcm11010257

Abstract

Androgen Receptor (AR) alterations (amplification, point mutations, and splice variants) are master players in metastatic castration resistant prostate cancer (CRPC) progression and central therapeutic targets for patient management. Here, we have developed two multiplexed droplet digital PCR (ddPCR) assays to detect AR copy number (CN) and the key point mutation T877A. Overcoming challenges of determining gene amplification from liquid biopsies, these assays cross-validate each other to produce reliable AR amplification and mutation data from plasma cell free DNA (cfDNA) of advanced prostate cancer (PC) patients. Analyzing a mixed PC patient cohort consisting of CRPC and hormone sensitive prostate cancer (HSPC) patients showed that 19% (9/47) patients had AR CN amplification. As expected, only CRPC patients were positive for AR amplification, while interestingly the T877A mutation was identified in two patients still considered HSPC at the time. The ddPCR based analysis of AR alterations in cfDNA is highly economic, feasible, and informative to provide biomarker detection that may help to decide on the best follow-up therapy for CRPC patients.

Highlights

Prostate cancer (PC) remains one of the leading causes of cancer related deaths in the western world
androgen receptor (AR) amplification correlates with castration resistant prostate cancer (CRPC) and is found in approximately 20–60% of PC recurring during androgen deprivation therapy (ADT) while very rare in primary PC (
The AR-Amp-1/T877A assay multiplexes amplification and detection of the AR sequences encoding the T877A mutation by using a T877A specific probe (FAM conjugated) and the corresponding wild-type probe (HEX conjugated). Another set of primers and a probe (HEX conjugated) to detect the reference gene RPP30 located on human chromosome 10 is included to normalize for AR amplification status

Summary

Introduction

Prostate cancer (PC) remains one of the leading causes of cancer related deaths in the western world. PC responds generally well to androgen deprivation therapy (ADT). Eventually PC develops ADT resistance mechanisms, commonly via AR mutations, amplification, and splice variants [1]. There is an active area of developing generation ADTs with efficacy even in the context of these AR alterations, which warrants fast and practical identification of these alterations and will have the potential to underpin clinical trials to define patient eligibility and outcomes. AR amplification correlates with castration resistant prostate cancer (CRPC) and is found in approximately 20–60% of PC recurring during ADT while very rare in primary PC (

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