1.cfDNA技术成为液体活检的临床首选
在本期我们重点谈谈 cfDNA（又称cell free DNA或游离DNA）技术的发展和应用。 液体活检技术在近年来取得了长足进步，成为癌症的诊断和监测的首选方案[1-6]。医生和科学家们已经发现，由肿瘤细胞释放到血液中的cfDNA，在分子和病理水平上，能够比较准确地反映肿瘤本身的基因型态及其变化[1-6]。 相比液体活检领域的其他靶标，比如游离癌细胞,、游离RNA,、蛋白质和外泌体， cfDNA有着明显的优势，因为它获取更方便，结果更稳定，在临床上的使用也更为广泛。 所以在临床应用上，cfDNA是液体活检的首选技术。
我们必须承认，游离癌细胞技术对液体活检的基础研究，起到了根本的推动作用[1-2,7-9]。在前面几期的液体活检通讯里，我们已经看到，正是因为对游离癌细胞的认知拓展了人们对肿瘤生物学的认知。 而cfDNA作为实体瘤和游离癌细胞的基因代表，是临床应用上的首选，是医生和诊断工业共同认可的液体活检技术的代名词。
2. cfDNA液体活检覆盖各型癌症
众所周知，癌症不是一种疾病，而是多种疾病的组合，根据组织和器官来源来看，癌症有100多种，根据分子病理学分类癌症则是上千种或更多[10-14]。 常见和多发的癌症类型，在科研和临床方面，提供了很多病例和积累。 在这里我们看一下，哪些癌症类型可以用cfDNA技术来做诊断和监测。
在西方国家，以美国为例，最多发的癌症为肺癌、乳腺癌、肠癌、前列腺癌和黑色素癌等[10-11]。 到目前为止，针对cfDNA的液体活检技术，已经用于诊断和监测肺癌[15-18]、乳腺癌[19-21]、肠癌[22-23]、前列腺癌[24-25]和和黑色素癌[26-27]。至于发病率不高，但是致死率很高的癌症，比如胰腺癌，cfDNA的液体活检技术也有着有效的应用[28-29]。在中国的多发癌症，除了肺癌、乳腺癌、前列腺癌这些发病率已经和世界接轨的病种，还有具有中国特点的消化道相关的癌症，也是高发病种[13-14]。 以cfDNA为主的液体活检，在胃癌[30-31]、肝癌[32-33]和食道癌[34-35]领域，也已经成为来诊断和监测的有效手段。可以说，只要是有临床研究的癌症类型，cfDNA技术就已经或将很快覆盖了。
3. 液体活检的模型系统
当前的液体活检技术的模型是建立在肺癌的诊断和治疗系统上的， 这是由于肺癌这个病种有着成熟的科研、诊断、治疗和法规的路径[12,15,16,36-38]。液体活检的模型系统，是通过检测非小细胞肺癌患者血浆中的cfDNA来诊断EGFR突变型，从而指导肺癌的靶向治疗药物并监测药物的疗效。这个模型取得的成功，已经在衍射到其他的癌症病种领域。
肺癌有很高的发病率、死亡率，它的发病率在美国是第二位，在中国是第一位，死亡率在美国和中国都是第一位[10-14]。 除了吸烟者，很多从未吸烟的人因为遗传和环境的因素也患有肺癌。 正因为如此，针对肺癌的研究有很强的积累，人们已经知道在肺癌患者中，有85-90%的病例是非小细胞肺癌，所以对非小细胞肺癌的研究有很大投入，对基因突变型也有较为清楚的认识。
非小细胞肺癌的基因检测已经完全改变了它原来的分型和治疗方式。非小细胞肺癌不再被视为一个疾病，它是一类由不同分子亚型构成的异质性疾病。其结果是，针对性的应用分子靶向药物改善了无数患者的临床疗效[37-42]。在肺腺癌领域，EGFR基因突变和ALK基因重组意味着单纯化疗不再是首选治疗。液体活检目的仅仅是为了诊断， 而是要指导用药。肺癌患者如果在EGFR基因上有特定的突变（L858R，T790M），患者对靶向治疗的药物就会有很好的疗效。 常见的靶向治疗药物，包括tyrosine kinase inhibitor (TKI) 的第一代和新一代的药物。对非小细胞肺癌患者进行组织活检来测定 EGFR 和 ALK 状态已经是一个重要的诊疗步骤。肺癌的分子机理和诊断的方向相对于清晰，治疗方案和伴随诊断的路径也较为成熟，对肺癌患者的基因型分析，是诊断和治疗的重要一步，在这一步已经被cfDNA的液体活检技术覆盖。
4. 癌症的液体活检获得法规的认可
到目前为止，美国FDA批准的唯一的液体活检技术，是用罗氏公司的cobas EGFR突变测试v2[42-46]。2016年6月1日，FDA批准cobas EGFR突变测试v2使用血浆标本作为检测外显子19删除或外显子21（L858R）替代突变的伴随诊断测试表皮生长因子受体（EGFR）基因来鉴定适合用Tarceva®（厄洛替尼）治疗的转移性非小细胞肺癌患者。 在此之前，美国病理学会（CAP）的诊疗指南中，EGFR的突变测试还是传统的组织切片样本， 就是福尔马林固定石蜡包埋组织标本（FFPE）。 但是在cobas EGFR突变测试v2批准中，新用途是用于检测从血浆样品分离的cfDNA中的这些特定突变，以帮助医生鉴定可首先用TARCEVA（厄洛替尼）治疗的患者。这是FDA批准使用的第一个液体活检测试， 在法规角度上认可了EGFR和非小细胞肺癌系统在液态活检领域的模型和示范作用。
值得关注的是FDA对样本来源的推荐，在针对cobas EGFR突变测试v2的批示里， FDA提出测试的首选方法是血浆标本的液体活检，筛查结果如果是阴性则用组织活检确认[42]。 所以FDA的思路是用液体活检做一线的方法学，而传统的组织活检是二线方法学，用来做阴性样本的确认。迄今为止，针对血浆或血清的cfDNA检测技术已经得到了迅速的发展，其中以数字PCR技术[47-52]。和二代测序技术为代表[53-61]。
5. 液体活检指导肿瘤监控、治疗和预后
基于cfDNA的液体活检技术，在肿瘤的诊断和检测方面，能够准确地描述肿瘤负荷、肿瘤基因型， 以及肿瘤动态变化[62-71]。在最新的一个针对肠癌的报道中，癌症患者的血液cfDNA水平是14.23+/-6.33 ng/ml, 而健康人对照组的cfDNA水平是2.60+/-1.59 ng/ml, 差异非常显著[62]。 多项研究表明，肿瘤的总负荷和血液cfDNA的水平是直接相关的， cfDNA的整体水平反应了肿瘤水平[62-66]。
根据临床上最近10年的观察，cfDNA能够体现肿瘤的动态变化，在手术和化疗阶段，肿瘤cfDNA的水平也随之下降[67-71]。 在癌症的治疗过程中，药物的使用， 尤其是新型靶向药物的使用， 是以对癌症基因型的正确判断为前提， 而癌症基因型的检测， 越来越倾向用cfDNA技术为主的液体活检来完成[72-80]。 在疗程之后的监控，cfDNA更是起到了不开替代的作用， 这里所说监控既包括了靶向药物的疗效监控，也包括对常规抗癌手段（比如化疗和放疗）的监控，而这个监控阶段要持续几年，活检要求持续取样， 传统的组织活检难以做到，液体活检的优势在这里充分发挥。 对cfDNA的液体活检，不仅涵盖对药物疗效的确认，也能够及时监测到癌症复发， 以及抗药性的产生[81-86]。
6. 液体活检和组织活检的一致性
根据现有的治疗指南， 组织活检仍然是癌症诊断的金标准， 而液体活检在科研和临床界的蓬勃兴起，让我们意识到液体活检是组织活检的最佳替代。 那么液体活检究竟在多大程度上反映了组织活检的结果，两者的一致性如何，这个话题伴随着液体活检技术的发展在逐步深化， 已经积累了多项研究成果[87-104]。根据对cfDNA的EFGR分析，液体活检和组织活检相比，灵敏度、特异性、一致性（sensitivity, specificity and concordance）分别为65%，95%和80%， 这是综合了多家科研机构的上千例的数据的综合结论。 总体看， 液体活检的特异性很好，只要是它检出的阳性就很少是假阳性。 它的灵敏度随着不同方法学、不同的课题而不同，有着40%～90%的巨大区间。值得一提的是，现在液体活检在癌症领域的主要临床应用，不是早期诊断，而是监控已有病人，这和灵敏度问题不无关系。提高灵敏度将是近期液体活检在今后在技术上的一大发展点，液体活检也势必在癌症诊疗领域发挥更大的作用。
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