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洪媛媛
(2022-03-22 18:43):
#paper Hepatocellular Carcinoma Detection by Plasma Methylated DNA:
Discovery, Phase I Pilot, and Phase II Clinical Validation. Hepatology. 2019 March ; 69(3): 1180–1192. doi:10.1002/hep.30244. 这篇文献讲述了肝癌早筛甲基化marker的筛选过程。实验技术：RRBS、Q-RealTime PCR以及升级版的Q-RealTime PCR（TELQAS)。甲基化marker的筛选过程：1，dicovery/Technical validation， RRBS筛选肝癌组织 VS 正常肝组织，以及肝癌组织 VS血细胞差异的甲基化marker作为备选marker，然后测试备选marker在QPCR平台表现；2，Biological tissue validation作为独立的验证集，实验材料是肝癌组织 VS 正常肝组织，方法是QPCR；3，Phase 1 plasma study，在肝癌cfDNA和健康人cfDNA中测试上一步的甲基化marker，技术是TELQAS；4，Phase II plasma study作为血浆的独立验证集，技术是TELQAS。最终筛选得到6-marker panel (HOXA1, EMX1, AK055957, ECE1, PFKP and CLEC11A ）, AUC 达到 0.96 (95% CI, 0.93–0.99) ， 当健康人中特异性92% (86–96%)时，肝癌病人检测灵敏度 95% (88–98%) 。
Abstract:
Early detection improves hepatocellular carcinoma (HCC) outcomes, but better noninvasive surveillance tools are needed. We aimed to identify and validate methylated DNA markers (MDMs) for HCC detection. Reduced representation bisulfite …
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Early detection improves hepatocellular carcinoma (HCC) outcomes, but better noninvasive surveillance tools are needed. We aimed to identify and validate methylated DNA markers (MDMs) for HCC detection. Reduced representation bisulfite sequencing was performed on DNA extracted from 18 HCC and 35 control tissues. Candidate MDMs were confirmed by quantitative methylation-specific PCR in DNA from independent tissues (74 HCC, 29 controls). A phase I plasma pilot incorporated quantitative allele-specific real-time target and signal amplification assays on independent plasma-extracted DNA from 21 HCC cases and 30 controls with cirrhosis. A phase II plasma study was then performed in 95 HCC cases, 51 controls with cirrhosis, and 98 healthy controls using target enrichment long-probe quantitative amplified signal (TELQAS) assays. Recursive partitioning identified best MDM combinations. The entire MDM panel was statistically cross-validated by randomly splitting the data 2:1 for training and testing. Random forest (rForest) regression models performed on the training set predicted disease status in the testing set; median areas under the receiver operating characteristics curve (AUCs; and 95% confidence interval [CI]) were reported after 500 iterations. In phase II, a six-marker MDM panel (homeobox A1 [HOXA1], empty spiracles homeobox 1 [EMX1], AK055957, endothelin-converting enzyme 1 [ECE1], phosphofructokinase [PFKP], and C-type lectin domain containing 11A [CLEC11A]) normalized by beta-1,3-galactosyltransferase 6 (B3GALT6) level yielded a best-fit AUC of 0.96 (95% CI, 0.93-0.99) with HCC sensitivity of 95% (88%-98%) at specificity of 92% (86%-96%). The panel detected 3 of 4 (75%) stage 0, 39 of 42 (93%) stage A, 13 of 14 (93%) stage B, 28 of 28 (100%) stage C, and 7 of 7 (100%) stage D HCCs. The AUC value for alpha-fetoprotein (AFP) was 0.80 (0.74-0.87) compared to 0.94 (0.9-0.97) for the cross-validated MDM panel (P < 0.0001). Conclusion: MDMs identified in this study proved to accurately detect HCC by plasma testing. Further optimization and clinical testing of this promising approach are indicated.
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