Design and development of a fully synthetic MLPA-based probe mix for detection of copy number alterations in prostate cancer formalin-fixed, paraffin-embedded tissue samples.

DNA copy number alterations (CNAs) are promising biomarkers to predict prostate cancer (PCa) outcome. However, fluorescence in situ hybridization (FISH) cannot assess complex CNA signatures due to low multiplexing capabilities. Multiplex ligation-dependent probe amplification (MLPA) can detect multiple CNAs in a single PCR assay, but PCa-specific probe mixes available commercially are lacking. Synthetic MLPA probes were designed to target 10 CNAs relevant to PCa: 5q15-21.1 (CHD1), 6q15 (MAP3K7), 8p21.2 (NKX3-1), 8q24.21 (MYC), 10q23.31 (PTEN), 12p13.1 (CDKN1B), 13q14.2 (RB1), 16p13.3 (PDPK1), 16q23.1 (GABARAPL2), and 17p13.1 (TP53) with 9 control probes. In cell lines, CNAs were detected when the percentage of cancer genome was as low as 30%. Compared to FISH in radical prostatectomy (RP) formalin-fixed paraffin-embedded (FFPE) samples (n=18: 15 cancers, 3 matched benign), the MLPA assay showed median sensitivity and specificity of 80% and 93%, respectively, across all CNAs assessed. In the validation set (n=40: 20 tumors sampled in two areas), the respective sensitivity and specificity of MLPA compared advantageously to FISH and digital droplet TaqMan PCR (ddPCR) when assessing PTEN deletion (FISH: 85% and 100%, ddPCR: 100% and 83%) and PDPK1 gain (FISH: 100% and 92%, ddPCR: 93% and 100%). In conclusion, this new PCa probe mix accurately identifies CNAs by MLPA across multiple genes using low quality and quantities (50 ng) of DNA extracted from clinical FFPE samples.