概述
肿瘤细胞游离DNA（cfDNA）存在于癌症患者的血浆中。目前大多数关于循环肿瘤DNA（ctDNA）应用的研究都涉及突变的检测。cfDNA的分析经常在无创检测导致耐药机制的突变以及癌症患者的治疗和疾病监测的背景下讨论。事实上，这一领域已经取得了实质性的进展，开发出了能够达到高灵敏度并能探询大量基因的方法。然而，有趣的是，cfDNA还可以用于分析DNA的不同特征，如甲基化状态、大小片段模式、转录组学和病毒载量，这为癌症患者液体活检样本的分析开辟了新的途径。本文就实体恶性肿瘤患者cfDNA突变检测的新观点和挑战作一综述。
肺癌病友交流群
肿瘤细胞游离DNA（即无细胞DNA，英文缩写cfDNA）是指源自体液中任何细胞类型的细胞外DNA分子（双链DNA和线粒体DNA）。cfDNA已于1948年在患病和健康个体的血液中被检测到。cfDNA分析目前已用于产前诊断，其临床应用也在癌症、器官移植、自身免疫性疾病、创伤、心肌梗死和败血症等领域进行了评估。但是，我们对cfDNA的结构和起源，细胞释放机制和清除的了解仍是初步的。尽管大多数cfDNA分子起源于造血系统，但人们对确定健康和病理条件下不同器官对cfDNA总量的相对贡献有着极大的兴趣。不仅有多种释放机制，包括凋亡，衰老，肥大症，NETNET，吞噬作用和坏死，还包括主动分泌-包括与细胞外囊泡的结合或由其他机制（如红细胞释放成熟核，线粒体DNA分泌或重要的NETosis）诱导。另一方面，不同的参数控制着cfDNA分子的降解和消除：循环中的酶促裂解，通过肝脏消除核小体复合物，并在较小程度上通过肾脏除去DNA片段。cfDNA生物学的这些基本方面的描述超出了本文讨论的范围，故不做过多阐述。
cfDNA的肿瘤来源部分，通常称为循环肿瘤DNA（ctDNA），由于其作为癌症患者微创肿瘤生物标志物的巨大潜力，在过去十年中受到了广泛关注。至于cfDNA，肿瘤生物学与ctDNA释放之间的相关性仍未得到很好的理解，并且可能不仅仅依赖于垂死细胞的数量。不仅肿瘤的体积和代谢，而且其扩散速率也与血浆中ctDNA的含量呈正相关。
关于ctDNA在肿瘤学已发表研究阐明的潜在用途中，绝大多数提及的是在癌症患者血浆或血清中检测到的特定突变。简言之，ctDNA突变检测有可能用于早期癌症检测，判断组织起源、预后，监测疗效和评估治疗的潜在耐药性，或检测微小残留疾病。但是，表观遗传学改变在癌症发展中比体细胞突变更为常见。尽管ctDNA的突变分析显示了许多临床应用，但对cfDNA的评估超出了点突变的检测范围，包括对染色体重排，拷贝数畸变，甲基化，片段化和基因表达的研究，因此也越来越引起人们的关注（图1）。
图1：ctDNA的不同特征及其潜在的临床意义。
该图总结了可从cfDNA不同特征的研究中获得的与肿瘤相关的临床信息。ctDNA上可检测到的体细胞基因组畸变包括突变，染色体重排和拷贝数畸变。ctDNA的其他特征是特定的表观遗传畸变，例如甲基化模式或不同的DNA片段长度。通过读取核小体间的深度覆盖范围，也可以从ctDNA分析中获得有关肿瘤特异性转录的信息。在病毒引起的肿瘤（例如EBV相关的鼻咽癌或HPV相关的头颈部肿瘤）中，病毒序列的定量评估具有诊断意义。
显然，一些肿瘤类型和身体部位向血液中释放的ctDNA量较低。在这里，用于分子分析的非血液来源的ctDNA已经变得很有价值。很明显，在原发性脑肿瘤，如胶质瘤、中枢神经系统淋巴瘤和一些儿科实体瘤中，脑脊液（CSF）显示出比外周血更高的敏感性。同样，对于一些上呼吸道系统的肿瘤，唾液、痰或胸腔积液也可能是血液的良好替代物，最近的一些研究结果发现，尿液、粪便和精液等其他体液或样本，也适用于不同的液体活检方法。
结论与观点
越来越多的数据表明，除了从癌症患者血浆中获得的cfDNA突变外，还可能获得更多的信息，例如从片段模式或甲基化状态的分析中获得的信息，这些信息对于基因表达的调控尤其有用。人类恶性肿瘤细胞表现出DNA甲基化模式的普遍变化，从而导致基因表达的紊乱或基因组的不稳定性。从癌症治疗的潜在临床前景来看，从早期癌症检测到估计预后和监测治疗反应，破译这些异常的表观遗传修饰是至关重要的。对cfDNA的研究也显示了早期检测与病毒相关癌症的临床潜力，与实体肿瘤中复杂的体细胞突变谱相比，利用不同致病性病毒dna的复杂性会较低些。然而，剩下的挑战将是区分暂时性病毒感染和导致癌症的持续性感染。病毒ctDNA序列的检测还可以为血浆cfDNA的生物学和动力学提供重要的基础信息。对接受鼻咽切除术的鼻咽癌患者血浆中EBV负荷的连续监测显示，血浆中EBV cfDNA的清除率符合一级衰变动力学模型，中位半衰期仅为139 min。数据显示，EBV-DNA的清除非常迅速，因此与基线测量相比，手术后抽血可能是疾病复发的更好预测指标。将cfDNA分析引入癌症诊断的一个重要前提是将现有cfDNA技术的分析前和分析变量标准化。为此，已经建立了包括来自学术界、商界的合作伙伴在内的国际财团组织，例如CANCER-ID或ELBS，并且已经进行了环实验（在多个地点并行使用相同的样品或方法）。此外，越来越多的数据表明，其他基于唾液、脑脊液或尿液的非血源性液体活检方法可用于未来的临床试验。最后，值得一提的是，其他液体活检分析物，如循环肿瘤细胞、循环microRNAs、肿瘤培养血小板或肿瘤相关蛋白，可能提供肿瘤演变和癌症患者治疗反应的补充信息，开发一个复杂的多分析物生物标志物小组，这将需要复杂的生物信息学工具，如机器学习算法，将大大有助于癌症患者个体的无创治疗。
综上所述，10年前提出的液体活检的概念为癌症诊断开辟了新的途径，现在需要进行具有既定疗效指标的介入性临床试验，以进一步证明ctDNA和其他生物标记物的临床效用。
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转载自：
Keller, L., Belloum, Y., Wikman, H. et al.
Clinical relevance of blood-based ctDNA analysis: mutation detection and beyond.
Br J Cancer 124, 345–358 (2021).
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