Division of Urology, Department of Surgery, McGill University and the Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada.
Department of Pathology, McGill University and the Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada.
J Mol Diagn. 2020 Oct;22(10):1246-1263. doi: 10.1016/j.jmoldx.2020.07.003. Epub 2020 Aug 4.
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 because of 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 cancer genome was as low as 30%. Compared with FISH in radical prostatectomy formalin-fixed, paraffin-embedded samples (n = 18: 15 cancers and 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 with FISH and TaqMan droplet digital 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%). 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 formalin-fixed, paraffin-embedded samples.
DNA 拷贝数改变 (CNAs) 是预测前列腺癌 (PCa) 结局的有前途的生物标志物。然而，由于低多重检测能力，荧光原位杂交 (FISH) 无法评估复杂的 CNA 特征。多重连接依赖性探针扩增 (MLPA) 可以在单个 PCR 测定中检测多个 CNA，但缺乏市售的特定于 PCa 的探针混合物。合成的 MLPA 探针被设计用于靶向 10 个与 PCa 相关的 CNA：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）和 17p13.1（TP53），以及 9 个对照探针。在细胞系中，当癌症基因组低至 30%时，即可检测到 CNA。与根治性前列腺切除术福尔马林固定、石蜡包埋样本中的 FISH 相比（n=18：15 例癌症和 3 例匹配的良性），MLPA 检测在评估的所有 CNA 中分别显示出 80%和 93%的中位敏感性和特异性。在验证组（n=40：20 个肿瘤在两个区域取样）中，与 FISH 和 TaqMan 液滴数字 PCR（ddPCR）相比，MLPA 评估 PTEN 缺失（FISH：85%和 100%；ddPCR：100%和 83%）和 PDPK1 增益（FISH：100%和 92%；ddPCR：93%和 100%）时具有优势。该新型 PCa 探针混合物通过 MLPA 使用从临床福尔马林固定、石蜡包埋样本中提取的低质量和低量（50ng）DNA 准确识别多个基因的 CNA。
