Multiplex Digital PCR to Detect Amplifications of Specific Androgen Receptor Loci in Cell-Free DNA for Prognosis of Metastatic Castration-Resistant Prostate Cancer.

Meijun Du,Winston Tan,Manish Kohli,Liang Wang,Chiang-Ching Huang

doi:10.3390/cancers12082139

Abstract

Amplification of androgen receptor (AR) is a common genomic event in metastatic castration-resistant prostate cancer (mCRPC). To evaluate the prognostic value of the amplifications of specific loci in the AR gene in cell-free DNA, we developed a multiplex digital PCR (dPCR) assay that targeted AR enhancer (AR-En), AR exon 1 (AR-E1), AR exon 8 (AR-E8) and OPHN1 (downstream of AR). We selected three relatively stable genes, C2orf16, FAM111B, and GRIA3, as reference controls for copy number normalization. One hundred and eight mCRPC patients were recruited to test the association of specific AR loci amplification with clinical outcome. Using a normalized ratio ≥ 1.92 as cutoff, amplification of AR-En, AR-E1, AR-E8 and OPHN1 was observed in 28, 25, 24 and 19 of 108 mCRPC patients, respectively. Among the 41 patients with AR region amplification, 9 (21.9%) showed amplification at all four selected regions and 15 (36.6%) showed amplification at AR-En, AR-E1, and AR-E8. Six (14.6%) patients showed independent AR-En amplification, while the remaining 3 (7.3%) demonstrated AR-E8 amplification only. Kaplan–Meier analysis showed overall survival’s association with the amplification of AR-En (p = 0.02, HR = 1.68 (1.07–2.65)), AR-E8 (p = 0.02, HR = 1.78 (1.08–2.92)) and AR-En-E8 (the combination of AR-En and AR-E8 (p = 0.009, HR = 1.77 (1.15–2.73)). Multivariate models that included AR-En-E8 amplification and clinical factors significantly improved prognostic performance (p = 0.0001). With further validation, the multiplex dPCR assay may assist in prognostication of mCRPC patients.

Highlights

Circulating cell-free DNA has become a promising tool in molecular oncology, allowing the detection of molecular alterations associated with cancer biology, treatment response [1,2]Cancers 2020, 12, 2139; doi:10.3390/cancers12082139 www.mdpi.com/journal/cancersCancers 2020, 12, 2139 and overall survival (OS) [3,4,5,6]
We recently reported that OPHN1 gene, which is downstream of androgen receptor (AR), is amplified in metastatic castration-resistant prostate cancer (mCRPC) [25]
After failure of androgen-deprivation therapy (ADT), 59/108 patients received systemic chemotherapy

Summary

Introduction

Circulating cell-free DNA (cfDNA) has become a promising tool in molecular oncology, allowing the detection of molecular alterations associated with cancer biology, treatment response [1,2]Cancers 2020, 12, 2139; doi:10.3390/cancers12082139 www.mdpi.com/journal/cancersCancers 2020, 12, 2139 and overall survival (OS) [3,4,5,6]. Circulating cell-free DNA (cfDNA) has become a promising tool in molecular oncology, allowing the detection of molecular alterations associated with cancer biology, treatment response [1,2]. There still remain technical challenges to analyze small amounts of highly fragmented (150–200 bp) and diluted (nanograms per 1 mL plasma) cfDNA fractions that are attributable to tumors (circulating tumor DNA-ctDNA) within cfDNA that are shed from cancer cells and which generally comprise 1–2%. The sensitivity of sequencing techniques is limited by the availability of an adequate amount of ctDNA. To increase the sensitivity of detecting ctDNA in the blood stream, digital‘PCR (dPCR) has been developed, which allows the detection of mutant and wild type DNA fragments at ratios close to 1:100,000 (allelic frequency (AF) = 0.001%) [8,9,10]. By varying parameters that affect PCR efficiency and end-point fluorescence, dPCR has been used to detect multiple targets in one reaction with only two different fluorophores [11,12]

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