BACKGROUND Gene expression in circulating tumor cells (CTCs) can be used as a predictive liquid biopsy test in metastatic castration-resistant prostate cancer (mCRPC). We developed a novel 6-plex reverse transcription droplet digital PCR (RT-ddPCR) assay for the absolute quantification of 4 prostate cancer biomarkers, a reference gene, and a synthetic DNA external control (DNA-EC) in CTCs isolated from mCRPC patients. METHODS A novel 6-plex RT-ddPCR assay was developed for the simultaneous absolute quantification of AR-FL, AR-V7, PSA, and PSMA, HPRT (used as a reference gene), and a synthetic DNA-EC that was included for quality control. The assay was optimized and analytically validated using DNA synthetic standards for each transcript as positive controls. Epithelial cellular adhesion molecule (EpCAM)-positive CTC fractions isolated from 90 mCRPC patients and 11 healthy male donors were analyzed, and results were directly compared with reverse transcription quantitative PCR (RT-qPCR) for all markers in all samples. RESULTS Linear dynamic range, limit of detection, limit of quantification, intra- and interassay precision, and analytical specificity were determined for each marker. Application of the assay in EpCAM-positive CTC showed positivity for AR-FL (71/90; 78.9%), AR-V7 (28/90; 31.1%), PSA (41/90; 45.6%), PSMA (38/90; 42.2%), and HPRT (90/90; 100%); DNA-EC concentration was constant across all samples. Direct comparison with RT-qPCR for the same markers in the same samples revealed RT-ddPCR to have superior diagnostic sensitivity. CONCLUSIONS Our 6-plex RT-ddPCR assay was highly sensitive, specific, and reproducible, and enabled simultaneous and absolute quantification of 5 gene transcripts in minute amounts of CTC-derived cDNA. Application of this assay in clinical samples gave diagnostic sensitivity and specificity comparable to, or better than, RT-qPCR.

中文翻译：

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开发和分析验证 6 重逆转录液滴数字 PCR 测定法，用于对转移性去势抵抗性前列腺癌患者的循环肿瘤细胞中前列腺癌生物标志物的绝对定量。

背景 循环肿瘤细胞 (CTC) 中的基因表达可用作转移性去势抵抗性前列腺癌 (mCRPC) 的预测性液体活检试验。我们开发了一种新型 6 重逆转录液滴数字 PCR (RT-ddPCR) 测定法，用于对从 mCRPC 患者分离的 CTC 中的 4 种前列腺癌生物标志物、参考基因和合成 DNA 外部对照 (DNA-EC) 进行绝对定量。方法 开发了一种新的 6 重 RT-ddPCR 测定法，用于同时绝对定量 AR-FL、AR-V7、PSA 和 PSMA、HPRT（用作参考基因）和合成的 DNA-EC，用于质量控制。使用每个转录物的 DNA 合成标准作为阳性对照，优化和分析验证了该测定。分析了从 90 名 mCRPC 患者和 11 名健康男性供体中分离的上皮细胞粘附分子 (EpCAM) 阳性 CTC 组分，并将结果直接与所有样本中所有标记物的逆转录定量 PCR (RT-qPCR) 进行比较。结果 确定了每个标记的线性动态范围、检测限、定量限、测定内和测定间精度以及分析特异性。该检测在 EpCAM 阳性 CTC 中的应用显示 AR-FL (71/90; 78.9%)、AR-V7 (28/90; 31.1%)、PSA (41/90; 45.6%)、PSMA (38/ 90；42.2%）和 HPRT（90/90；100%）；所有样本的 DNA-EC 浓度都保持不变。与相同样本中相同标记的 RT-qPCR 直接比较显示 RT-ddPCR 具有更高的诊断灵敏度。结论 我们的 6 重 RT-ddPCR 检测非常灵敏，特异、可重现，并且能够同时和绝对定量微量 CTC 衍生的 cDNA 中的 5 个基因转录本。在临床样本中应用该检测方法可获得与 RT-qPCR 相当或更好的诊断灵敏度和特异性。