Non-small-cell lung cancer (NSCLC), the most common type of lung cancer accounting for 85% of the cases, is often diagnosed at advanced stages owing to the lack of efficient early diagnostic tools. 5-Hydroxymethylcytosine (5hmC) signatures in circulating cell-free DNA (cfDNA) that carries the cancer-specific epigenetic patterns may represent the valuable biomarkers for discriminating tumor and healthy individuals, and thus could be potentially useful for NSCLC diagnosis. Here, we employed a sensitive and reliable method to map genome-wide 5hmC in the cfDNA of Chinese NSCLC patients and detected a significant 5hmC gain in both the gene bodies and promoter regions in the blood samples from tumor patients compared with healthy controls. Specifically, we identified six potential biomarkers from 66 patients and 67 healthy controls (mean decrease accuracy >3.2, P?<?3.68E?19) using machine-learning-based tumor classifiers with high accuracy. Thus, the unique signature of 5hmC in tumor patient’s cfDNA identified in our study may provide valuable information in facilitating the development of new diagnostic and therapeutic modalities for NSCLC.
肺癌是全球发病率和死亡率最高的恶性肿瘤之一，其中约85%为非小细胞肺癌（NSCLC）。由于缺乏高灵敏度、准确度和可靠性的早期诊断方法，NSCLC患者被确诊时往往已经到了中晚期，其预后较差。因此，探索NSCLC早期诊断和预后评估的分子标志物成为当前肺癌研究的热点领域。近年来研究表明，血浆游离DNA（cell-free DNA，cfDNA）携带丰富的肿瘤细胞基因组突变及表观改变的相关信息，在肿瘤早期诊断、预后评估等方面具有重要的应用价值。5-羟甲基胞嘧啶(5-hydroxymethylcytosine, 5hmC)作为DNA去甲基化过程中的稳定中间产物，在基因表达和肿瘤发生发展调控中具有重要作用。本研究中，我们采用一种高灵敏度、特异性强的测序技术对66例NSCLC患者和67例健康志愿者cfDNA中微量5hmC修饰进行测序，并发现在NSCLC患者基因体区和启动子区域5hmC富集特异性增加。我们进一步鉴定到2459个差异修饰基因，并发现这些基因可有效地区分NSCLC患者和健康对照样本，同时揭示了这些基因主要富集于与癌症发生、发展密切相关的通路上，如cGMP-PKG、Rap及PI3K-Akt信号通路等。为进一步筛选cfDNA 5hmC候选肿瘤生物标志物，我们通过随机森林的机器学习方法鉴定出6个在NSCLC患者中5hmC修饰水平上升的潜在分子标志物。相比现有的临床肿瘤标志物，这6个基因具有更高的灵敏度和准确性。总之，本研究表明cfDNA的5hmC有望成为NSCLC的新型生物标志物，用于NSCLC的早期诊断、预后评估、耐药监测等，具有十分重要的临床应用价值。





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