| Summary: | Amplification of androgen receptor (<i>AR</i>) 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 <i>AR</i> gene in cell-free DNA, we developed a multiplex digital PCR (dPCR) assay that targeted <i>AR</i> enhancer (<i>AR</i>-En), <i>AR</i> exon 1 (<i>AR</i>-E1), <i>AR</i> exon 8 (<i>AR</i>-E8) and <i>OPHN1</i> (downstream of <i>AR</i>). We selected three relatively stable genes, <i>C2orf16</i>, <i>FAM111B</i>, and <i>GRIA3,</i> 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 <i>AR</i>-En, <i>AR-</i>E1, <i>AR</i>-E8 and <i>OPHN1</i> was observed in 28, 25, 24 and 19 of 108 mCRPC patients, respectively. Among the 41 patients with <i>AR</i> region amplification, 9 (21.9%) showed amplification at all four selected regions and 15 (36.6%) showed amplification at <i>AR</i>-En, <i>AR-E1,</i> and <i>AR-</i>E8. Six (14.6%) patients showed independent <i>AR</i>-En amplification, while the remaining 3 (7.3%) demonstrated <i>AR</i>-E8 amplification only. Kaplan–Meier analysis showed overall survival’s association with the amplification of <i>AR</i>-En (<i>p</i> = 0.02, HR = 1.68 (1.07–2.65)), <i>AR</i>-E8 (<i>p</i> = 0.02, HR = 1.78 (1.08–2.92)) and <i>AR</i>-En-E8 (the combination of <i>AR</i>-En and <i>AR</i>-E8 (<i>p</i> = 0.009, HR = 1.77 (1.15–2.73)). Multivariate models that included <i>AR</i>-En-E8 amplification and clinical factors significantly improved prognostic performance (<i>p</i> = 0.0001). With further validation, the multiplex dPCR assay may assist in prognostication of mCRPC patients.
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