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游离DNA检测是肿瘤监控的临床首选技术

2017-07-02 00:00来源:原版作者:Jerry Li

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。 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] 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 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]201661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21L858R)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cfDNAEFGR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%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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