Mayo Clinic, Rochester, MN.
Exact Sciences Development Company, LLC, Madison, WI.
Hepatology. 2019 Mar;69(3):1180-1192. doi: 10.1002/hep.30244. Epub 2019 Feb 5.
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.
早期发现可改善肝细胞癌 (HCC) 的预后，但需要更好的非侵入性监测工具。我们旨在确定和验证用于 HCC 检测的甲基化 DNA 标记物 (MDM)。对来自 18 例 HCC 和 35 例对照组织的 DNA 进行了代表性重亚硫酸盐测序。在独立组织的 DNA 中通过定量甲基化特异性 PCR 对候选 MDM 进行了确认 (74 例 HCC，29 例对照)。在 21 例 HCC 病例和 30 例肝硬化对照的独立血浆提取 DNA 中，采用定量等位基因特异性实时靶标和信号扩增检测方法进行了 I 期血浆初步研究。然后，使用目标富集长探针定量扩增信号 (TELQAS) 检测方法在 95 例 HCC 病例、51 例肝硬化对照和 98 例健康对照中进行了 II 期血浆研究。递归分区确定了最佳 MDM 组合。通过将数据随机分成 2:1 的训练和测试，对整个 MDM 面板进行了统计学交叉验证。在训练集上执行随机森林 (rForest) 回归模型预测测试集中的疾病状态;在 500 次迭代后报告中位接收器操作特征曲线下的面积 (AUC;95%置信区间 [CI])。在 II 期，归一化后由六标志物 MDM 面板 (同源盒 A1 [HOXA1]、空螺旋孔同源盒 1 [EMX1]、AK055957、内皮素转换酶 1 [ECE1]、磷酸果糖激酶 [PFKP] 和 C 型凝集素结构域包含 11A [CLEC11A])组成的 B3GALT6 水平产生了最佳拟合 AUC 值为 0.96(95%CI，0.93-0.99)，HCC 敏感性为 95%(88%-98%)，特异性为 92%(86%-96%)。该面板检测到 4 个 0 期中的 3 个 (75%)、42 个 A 期中的 39 个 (93%)、14 个 B 期中的 13 个 (93%)、28 个 C 期中的 28 个 (100%)和 7 个 D 期中的 7 个 (100%) HCC。与交叉验证的 MDM 面板相比，甲胎蛋白 (AFP) 的 AUC 值为 0.80(0.74-0.87)，为 0.94(0.9-0.97)(P < 0.0001)。结论：本研究中鉴定的 MDM 通过血浆检测可准确检测 HCC。需要进一步优化和临床测试这种很有前途的方法。
