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

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.

Androgen deprivation‑induced OPHN1 amplification promotes castration‑resistant prostate cancer

“…Our results revealed that the expression of OPHN1 may promote cancer survival and invasion. Thus, the expression of OPHN1 could promote PCa progression, which is consistent with certain other studies on PCa ( 12 , 43 ). In addition, in gastric cancer, Dicken et al revealed that the expression of OPHN1 was related to lymphovascular invasion ( 44 ).…”

Clinical Applications of Liquid Biopsy in Prostate Cancer: From Screening to Predictive Biomarker

“…The most common genomic aberrations identified in PC are AR copy number variations (CNV), specifically AR amplification, and AR point mutations [ 67 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 ]. A meta-analysis of 16 studies representing more than 1000 patients found that AR gain is associated with a shorter PFS and OS in patients treated with ARSi, but it had no effect in patients treated with docetaxel in the first line or cabazitaxel in the second or third lines [ 80 ].…”

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

“…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 (<1%) [ 2 ]. Variation in detection frequency is attributed to varying patient cohorts and detection techniques with varying sensitivity and specificity using various biopsies [ 3 , 4 , 5 , 6 , 7 ]. AR amplification has commonly been associated with resistance developing against the third line ADT drug enzalutamide, which is a current therapy option for advanced PC patients [ 8 , 9 ].…”