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通讯作者:

张鹏(1979-),男,河北沧州人,博士生导师,主要从事肺癌的复发转移和免疫机制研究。E-mail:zhangpeng1121@tongji.edu.cn

中图分类号:R734.2

文献标识码:A

文章编号:2096-8965(2021)04-0049-11

DOI:10.12287/j.issn.2096-8965.20210407

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目录contents

    摘要

    部分非小细胞肺癌患者在早期治愈后会发生远期复发或转移,这可能与治疗后患者体内仍存在影像学或是实验室方法检测不到的肿瘤病灶,即微小残留病灶相关。这些肿瘤复发的潜在来源与患者较差的预后有着紧密的联系,因此, 在非小细胞肺癌病程中对这些病灶的监测十分重要。目前,针对微小残留病灶的检测主要依靠于液体活检,包括了循环肿瘤DNA检测、循环肿瘤细胞检测等方法。通过无创的检测手段,残留的肿瘤病灶为我们提供了肿瘤的进展状况以及具体的分子信息,预测了患者的预后状况,并进一步指导后续治疗方案。在这篇综述中,我们将探讨微小残留病灶发生发展的机制与影响,并关注对其的监测在临床治疗过程中可能的应用前景。

    Abstract

    Long-term recurrences or metastases occur in some patients with non-small cell lung carcinoma after complete remission in the early stages. This phenomenon may be related to the presence of tumor lesions undetectable by radiographic or laboratory methods, which are called minimal residual disease. Poor prognosis of patients is closely associated with these potential sources of tumor recurrences, so it is essential to supervise these lesions during the treatment of non-small cell lung carcinoma. Currently, the detection of minimal residual disease mainly relies on liquid biopsy, including circulating tumor DNA detection, circulating tumor cell detection and other approaches. Through non-invasive detection methods, remaining tumor lesions provide us with the progression of the tumor and specific molecular information, predict the prognosis of patients, and further guide the follow-up treatment regimen. In this review, we will explore the mechanism and impact of the occurrence and development of minimal residual disease, and focus on its potential application value in the clinical treatment process.

  • 非小细胞肺癌 (Non-Small Cell Lung Carci⁃ noma,NSCLC) 是常见的肺癌类型,约占肺癌发病患者的85%[1]。随着靶向治疗以及免疫治疗的研究日臻成熟,NSCLC患者的预后也逐渐提升[2]。然而,部分早期NSCLC患者在肿瘤获得影像学完全缓解 (Complete Remission,CR) 后出现复发[3],这些患者肿瘤复发的原因引起了广泛的关注。目前,许多学者认为,在患者接受治疗后,尽管从影像学上似乎彻底清除了病灶,但仍有极少量未被检测到的肿瘤细胞残存或肿瘤细胞来源分子异常[4]。这些残存的肿瘤病灶被称之为微小残存病灶 (Minimal Residual Disease,MRD),也可称之为分子残存病灶 (Molecular Residual Disease)。MRD具有与原始肿瘤细胞相同或相似的表型和遗传特征,可能仍拥有导致肿瘤复发的能力,也可能肿瘤细胞干性遭到化疗、放疗等破坏而缺乏复发的能力,是患者远期癌症转移复发的潜在来源之一。为探究MRD对于NSCLC患者疾病进展的影响以及潜在的应用,本文从多个角度探讨MRD在NSCLC疾病进展中扮演的角色,并对其可能在NSCLC治疗上的应用进行了总结。

  • 1 MRD的来源与形成

  • MRD中并非所有的细胞都会导致肿瘤的复发,不同的细胞可能带来不同的后果。MRD中包括微小复发性残留灶 (Minimal Relapsable Cancer, M-REC)、微小非复发性残留灶 (Minimal NonRelapsable Cancer,MN-REC) 和微小残留灶前体 (Minimal Residual Precursors,MRPs) [5]。微小复发性残留灶是具有与恶性肿瘤细胞一致的遗传和功能特性的肿瘤细胞,能够进一步扩散并引起复发;微小残留灶前体是已经包含恶性肿瘤细胞内存在的遗传信息变化,但尚未完全转化为肿瘤细胞的细胞,这两种细胞都是肿瘤发展隐匿阶段MRD引发远期复发的可能之一。而微小非复发性残留灶则是被治疗所破坏,无法进一步扩散并形成复发的肿瘤细胞。除此之外,尚未完全降解的死亡肿瘤细胞或切除的肿瘤细胞产生的核酸和存在于不合理的位置或以不合理的浓度存在的可检测到的肿瘤相关代谢物也可能与肿瘤的复发相关。

  • 微小复发性残留灶及其前体也被认为是肿瘤治疗后残存的为数不多的持续耐药细胞,这些细胞的出现与治疗或非治疗状态下肿瘤的进化相关[6, 7]。由于肿瘤微环境的微小差异以及空间和时间上的选择差异,单个肿瘤内也会存在多个具有不同分子特征的肿瘤亚克隆存在,这便是肿瘤内异质性[8, 9]。由于肿瘤突变负荷的压力与肿瘤微环境的影响,在不同突变驱动下,肿瘤自身发生了适应性的基因组与代谢改变,产生肿瘤自适应性的基因重编码[10]。先行发生耐药突变的肿瘤细胞在药物治疗过程中存活了下来,而部分适应性重编码的非耐药细胞在治疗给予的压力下也存活了下来,并在持续的治疗压力下自身进化成为耐药细胞[11, 12]。除此之外,肿瘤微环境在治疗中也会发生适应性的改变,并分泌如高水平的转化生长因子-β(Transforming Growth Factor-β, TGF-β) 等因子以避免对原先突变驱动的依赖,进而逃脱靶向药物的杀伤[13]。药物的剂量不足或是药物对肿瘤环境较差的浸润也可能使部分肿瘤细胞存活,形成残留病灶[14]

  • 肺部残留病灶出现后,影像学能够检测到的部分能够通过手术、射频消融或是更换药物等治疗策略予以进一步消除;而影像学未能检测到的部分则可能在肺部一直存在形成MRD。在持续的治疗压力下,MRD可能以休眠肿瘤的形式存在[15],以逃避药物或是患者自身免疫系统的杀伤,而在停止治疗或是其他致癌因素驱动后,MRD中的肿瘤细胞再次增殖、迁移,最终形成治疗后的远期复发与转移。

  • 2 MRD的检测

  • 目前,对于MRD的检测方法主要依靠液体活检。液体活检是通过对体液,主要是血液,同时也包括脑脊液和尿液进行检测[16],通过其中的细胞游离DNA (cell-free DNA,cfDNA)、循环肿瘤DNA (circulating tumor DNA,ctDNA)、循环肿瘤细胞 (Circulating Tumor Cells,CTCs)、外泌体等物质对患者的肿瘤病情发展进行更进一步的分析。尽管组织活检依然是对患者病情评估的金标准,但23%的患者会因为较低的组织数量或质量影响对病情的评估[17],同时可能出现的并发症也是组织活检需要面对的问题[18, 19]。相比之下,液体活检有着微创且高效的优势,能够在患者治疗期间持续地追踪患者的治疗状态,并对其治疗策略予以及时的指导。目前,针对MRD的液体活检研究主要集中于ctDNA和CTCs的检测上,其他检测如T细胞受体 (T Cell Receptor,TCR) 等也有少量研究但尚不成熟。

  • 2.1 循环肿瘤DNA检测

  • 在人的体液中,由于细胞坏死或凋亡,一些小片段的DNA得到释放,这些长度在200个碱基对以下的较为稳定存在的DNA就是cfDNA[20]。而在这其中,来源于肿瘤细胞的小片段DNA称之为ctDNA,约占cfDNA的不到1%[21]。cfDNA大多来自于破裂的非恶性细胞,而ctDNA则被认为来自于肿瘤细胞凋亡和坏死,因此也被认为是MRD的重要标志物之一[22]。肿瘤患者的血浆DNA含量往往比健康人要高得多[23],这可能与肿瘤对远处目标细胞的影响相关,进而导致了肿瘤的转移性扩散。

  • 目前已有多种ctDNA检测技术,主要分为两大类:第一类是非定向方法,如第二代测序技术 (Next-Generation Sequencing,NGS),目的是描述整个基因组的特征,并发现新的差异基因,从而改变对特定基因的治疗策略;而第二类检测方法如安全测序系统 (Safe Sequencing System,Safe-SeqS)、肿瘤特征性深度测序 (Cancer Personalized Profiling by Deep Sequencing,CAPP-seq)、数字化聚合酶链式反应 (digital Polymerase Chain Reaction,dPCR) 等技术则是检测一组预先设定的基因中的突变,从而探究该基因与肿瘤进展及治疗之间的联系。

  • 2.1.1 第二代测序技术

  • NGS是一种非定向的突变筛选方法,能够对多个患者样本的大量DNA模板进行高通量测序,可以包括整个基因组、外显子组或是转录组,而不仅限于一组特定基因的特定突变。由于其较广的基因覆盖面,NGS能够从大量突变基因中寻找与肿瘤相关的可疑基因,也能够通过对大量基因突变的筛选来给予临床治疗决策更大的帮助[24]。能够快速检测大量患者样本以及对低频突变的较高检出率也同样是NGS的优势。然而,尽管NGS能够检测到不同的突变位点,但其对于重排或是多拷贝的重复扩增灵敏度较低[25]。因此,将NGS与定向的检测方法相结合能够对ctDNA中的大量突变进行多重检测,在保证检测效率的同时也尽可能的提高了NGS检测的灵敏度[26]

  • 2.1.2 安全测序系统技术

  • Safe-SeqS是一种针对性的深度测序方法,主要分为两个步骤:首先是为每个DNA模板分子分配一个唯一的标识符,接着对每个唯一标记的模板进行扩增以放大其独特的标记,进而产生大量序列相同的子分子[27]。标识符一般是剪切后的DNA片段,可以分为内源性或外源性,使用不同的标识符可以同时评估多个基因或深入研究单个基因[28]。如果预先在模板分子中存在突变,在未出现测序错误的情况下该突变也会大量出现在扩增出的子分子中;而在原始模板中不发生的突变在扩增步骤中不会产生唯一的标识符,因此不会继续扩增,由此来区分真正的变异与那些由于测序人工产生的变异[27]。这一方法显著提高了测序的准确性,目前被用于确定体外合成的寡核苷酸的可靠性以及正常细胞核和线粒体基因组中的突变。

  • 2.1.3 肿瘤特征性深度测序技术

  • CAPP-seq是一种定向ctDNA测序方法,能够基于目前已有的公共数据库选择研究者感兴趣的反复突变的区域进行检测,能够识别超过95%的肿瘤突变[29]。这种方法针对肿瘤细胞DNA中反复出现的突变区域,借助生物信息学方法设计一个由DNA寡核苷酸组成的筛选标记,因此对特定的单个基因突变有较好的识别率和准确性。然而, ctDNA样本的质量和检测过程中所用试剂的潜在剂量偏差可能影响检测的精度[30],因而与集成数字化纠错技术 (integrated Digital Error Suppression, iDES) 相结合,对单链和双链DNA进行编码,不仅可以提高点突变的检出率,还可以减少假阳性率,提高检测的准确性[31]。目前,在iDES技术的帮助下,我们能够对肿瘤患者疾病进展前低至0.004%的ctDNA中的上皮生长因子受体 (Epider⁃ mal Growth Factor Receptor,EGFR) 基因突变进行检测,特异性可达99.9%。

  • 2.1.4 数字化聚合酶链式反应技术

  • dPCR采用荧光聚合酶链式反应扩增突变型或野生型DNA片段并进行差异标记,将一个聚合酶链式反应分成多个反应,每个小反应都根据荧光信号发射的强弱或缺失进行评分,不同颜色荧光信号的强度比例就代表等位基因突变率。dPCR能够对突变的类型进行检测,因而能够对潜在突变碱基进行更加精确地识别[32]。随着技术的发展,大规模并行测序使得dPCR可以逐个分析大量模板分子,而BEAMing (Beads, Emulsion, Amplification, Magnet⁃ ics) 技术将样本DNA变为每个包裹着1个DNA分子的液滴[33],进一步提高了dPCR的灵敏度,同时也减少了处理时间。

  • 目前,ctDNA检测的优势在于较高的灵敏度和特异性,对于Ⅱ~Ⅳ期的NSCLC患者特异性可达96%[29],同时由于仅需检测血液样本,无创操作在避免创伤操作导致的并发症的同时也更易于对患者病情进行持续的监控。非定向检测方法有利于大规模的可疑基因的筛选,但这一优势也同样带来检测效率的降低。因此,结合定向突变检测技术对筛选出的基因多重检测往往能够提高ctDNA的检测效果。然而,血浆中ctDNA检测结果与真正病灶组织的一致性尚未完全确定[34],仍有研究认为ctDNA检测不能够完全代表患者的真实病情进展,而不同来源的cfDNA同样会带来混杂因素,使得检测结果更为复杂或是不准确[35]

  • 2.2 循环肿瘤细胞检测

  • CTCs是来源于原发肿瘤,获得脱离基底膜能力并通过组织基质进入外周血的肿瘤细胞,其特有的上皮特征标记和间质特征标记赋予了CTCs更强的可塑性,能够促进肿瘤细胞的迁移和入侵[36, 37]。 CTCs不仅能够作为肿瘤细胞群进行集体迁移,同时一同迁移的原发肿瘤基质细胞也能为CTCs增加侵袭能力[38, 39]。由于CTCs本身就来源于原发肿瘤部位,它能够更全面的反映肿瘤基因表达与代谢的变化。目前针对CTCs的检测主要是通过富集、检验和特征定性三个步骤,之后再对CTCs的分子遗传特征进行进一步的分析。

  • 2.2.1 循环肿瘤细胞的富集

  • 利用肿瘤细胞和外周血细胞之间的差异,包括细胞表面蛋白的差异表达或细胞的不同物理特征,就能够实现CTCs的高效富集。上皮细胞粘附分子 (Epithelial Cell Adhesion Molecule, EPCAM) 是最广泛应用于筛选CTCs的细胞表面蛋白之一[40],而其他上皮细胞表面抗原也正在被研究开发。但是,恶性CTCs经常失去其上皮抗原,并在上皮间充质转化 (Epithelial-to-Mesenchymal Transition, EMT) 期间获得间质标记特征[41],并且不是所有循环的上皮细胞都是恶性的,因此,方法敏感性和特异性不佳[42],需要通过阴性选择来避免,即使用抗体识别白细胞和血液中其他细胞进行排除以筛选CTCs[43]。阴性选择的缺点主要是由于需要排除的细胞组成过于复杂,分离出的CTCs群体的纯度低于正富集策略所能达到的纯度,因此一般在正富集后的细胞中进行以提高CTCs富集的纯度。

  • 不依赖细胞表面蛋白差异的CTCs富集技术是基于肿瘤细胞和非恶性血细胞的大小、密度、电荷和变形能力之间的物理差异进行筛选,然而这些特征在CTCs之间也是存在差异的,并且与非恶性细胞有所重叠,因此可能存在假阳性的状况。基于CTCs大小高于其它血细胞对CTCs进行分离,同时可以利用免疫细胞化学、免疫荧光或原位杂交等技术进行细胞形态分析,相比之下会更加可靠[44]。近年来,微流控技术的发展使得患者的CTCs能够与血液细胞通过尺寸校准分别捕获[45],这使得大部分实体肿瘤患者都能够通过外周血对CTCs进行检测[46],并且特殊的微过滤系统能够通过大小排除来捕获CTCs细胞簇[47]。通过微流控技术获得的CTCs样本纯度高,白细胞污染少,细胞易于保存以及后续的培养与分析[48]。除此之外,双向电泳 (Dielec⁃ trophoresis, DEP) 也能够根据肿瘤细胞和血细胞的不同电荷来分离CTCs[49]

  • 2.2.2 循环肿瘤细胞的检验

  • 富集后的CTCs中可能仍然包含数百到数千个白细胞,因而需要使用可靠的方法来识别单个CTC。CTC识别的主要方法是使用针对细胞膜和细胞质抗原的抗体进行直接的免疫检测,其中包括上皮、间质、组织特异性和肿瘤相关标志物[40]。目前大多数CTC检验使用上皮细胞角蛋白作为CTC的标记物[50],通过荧光显微镜观察上皮细胞角蛋白荧光标记抗体染色的细胞[51],并通过CD45染色来排除其中的白细胞。此外,癌症特有的分子标记物以及组织特异性抗原或是肿瘤特有的基因突变等,也可以作为CTC的标记来用于识别检验[52]

  • 2.2.3 循环肿瘤细胞的特征定性

  • CTCs的分子特性可以在DNA、RNA和蛋白质水平上进一步探索,也可以通过将细胞注入免疫缺陷小鼠体内形成患者衍生的异种移植模型来研究CTCs的功能特性。近些年来,多种创新技术被开发出来分析CTCs的分子特征,如通过结合免疫染色和荧光原位杂交分析识别CTCs的基因组突变[53],或是通过对整个基因组进行扩增并使用NGS技术评估拷贝数突变和特定突变[54]。此外,实时定量聚合酶链式反应检测肿瘤细胞表达的组织特异性转录本具有高灵敏度,同时也可以评估来自同一患者的单个CTC之间的异质性[55]。但是由于RNA不如DNA稳定,在血液样本运输处理过程中可能发生降解,因此,这种方法依然面临不小的困难。对于蛋白质的研究,用抗体对感兴趣的蛋白质如增殖或凋亡的标记物进行免疫分型是最广泛使用的方法,但目前仅限于针对个别蛋白的识别评估[56]。而人源肿瘤异种移植 (Patient-Derived Tumor Xenograft, PDX) 模型的功能研究尽管能够揭示CTCs的功能特性并对抗癌治疗候选药物进行测试[57],但大量的CTCs细胞需求以及极低的CTCs移植成功率都大大限制了这一技术的应用,CTCs的体外培养也同样面临着类似的问题。

  • CTCs的检测优势在于无创检测的同时拥有与原发肿瘤组织的较好的一致性,并且能够从DNA、 RNA、蛋白质等多个层面去进行全面分析。但是,由于筛选过程中的标记特异性未必充足,检测的特异性难以提高[58],此外,由于技术要求,对CTCs的数量与质量的要求使得这项检测价格昂贵并在实际应用中有所受限[59]。因此,目前许多非基于EPCAM的CTCs检测方法如利用细胞表面波形蛋白的检测也在研究之中,以进一步针对不同的肿瘤进行特异性的检测,能够在未来对CTCs进行高灵敏度、高特异性、高易用性和广泛可用性的检测[60-62]

  • 2.3 T细胞受体检测

  • 现如今,免疫肿瘤学的发展让学者们意识到T细胞也能够成为肿瘤分析的对象。通过梯度密度离心以及流式细胞分选的方法能够从外周血中分离出不同种类的T细胞,以此来筛选外周血中的T细胞并进行功能分析[63]。在大多数T细胞中,TCR包括 α链和 β链两条链,而每条链又包含三个互补决定区 (Complementarity Determining Region, CDR)。 CDR1区和CDR2区的变异性由其种系所决定,而CDR3区的变异则与基因重组相关。CDR3区序列相当于TCR的身份证,对CDR3区测序能够进一步从免疫的角度分析肿瘤微环境中的变化[64, 65]。目前的研究显示,TCR文库的扩大与患者预后的联系不仅仅体现于肿瘤突变负荷的增加所带来的影响,也可能与患者所采取的治疗策略息息相关[66]。对细胞毒性T细胞的检测如今已经能够协助对患者病情的分析,而对调节T细胞的检测也可能为患者的免疫治疗策略制定带来一定的帮助[67]

  • 随着技术的发展与深入研究,液体活检技术也日益精进和完善。ctDNA、CTCs与TCR的检测各自拥有优势与不足。相比于CTCs,ctDNA检测更易于获得高质量的样本以进一步分析,因此其突变检出率要高于CTCs检测[68]。同时,CTCs检测中,背景中混杂的白细胞常常会带来野生型基因组DNA,进一步引起肿瘤DNA检测的偏倚。不过,由于ctDNA与CTCs来源不尽相同,在和相应肿瘤组织的匹配相符率上CTCs则更胜一筹,CTCs的检测结果或许更具有说服力。而TCR的检测更多的运用于对免疫治疗靶点的检测,进而预测免疫治疗应答及患者预后,而非对肿瘤突变基因进行检测以指导靶向的治疗策略。

  • 3 MRD与NSCLC患者的预后相关性

  • 3.1 早期NSCLC

  • 对于可手术治疗的早期NSCLC患者,处于各分期的患者等位基因突变频率各不相同。当从外周血中检测到ctDNA时,处于Ⅰ、Ⅱ、Ⅲ期的NSCLC患者等位基因突变率中位数分别为0.31%、0.48%、 1.48%,而未检测到ctDNA的患者为0.07%、0.16%、 0.50%[69]。由此可见,未检测到MRD的患者等位基因突变率显著低于检测出MRD的患者。在TRAC⁃ ERx临床试验中,24名可手术治疗的早期NSCLC患者中,14名患者在术后出现了复发,而其中的13名在疾病复发之前检测到了ctDNA[70],他们的复发可能与ctDNA中检测到的单核苷酸变异有关。而在手术治疗后的第1天和第3天,外周血CTCs数量的反弹可能预示着复发及肿瘤的远处转移[71]。然而,手术之前的ctDNA与CTCs的检测似乎与患者未来的复发关系不大[72]。除了手术治疗外,使用化疗或其它药物辅助治疗的患者在初次治疗后,如果仍能够检出MRD,其中的94%将在未来发生肿瘤的复发[73]。由此可见,对于早期NSCLC患者,初次治疗后MRD的检出与之后可能发生的复发具有显著的相关性。而由于不同NSCLC病理类型之间肿瘤组织坏死的差异,肺鳞癌与肺腺癌患者ctDNA检出率分别为97%和19%[70]。这种显著的差异可能正是这两种NSCLC病理类型组织学差异和基因突变不同的结果[74],也可能是两者预后差异和对不同治疗方式应答不同的原因之一[75]

  • 3.2 晚期NSCLC

  • 目前对于晚期NSCLC患者的MRD研究并不多。对于采用化疗治疗的晚期NSCLC患者,82%的Ⅳ 期患者外周血中能够检测出ctDNA[23]。相比于治疗后检出的ctDNA,在治疗前的ctDNA水平似乎与患者的总生存期 (Overall Survival,OS) 关联更加密切[76, 77]。除此之外,在第一个化疗周期之后的Ⅲ~Ⅳ 期患者的7.5mL外周血中,CTCs若小于5个则预后可能会更好[78]。但这一结论并非绝对,因为晚期患者外周血中的CTCs数目易受治疗策略的影响。由于治疗方案的不同,患者CTCs的数量受到了持续的干预而不断变化,而在治疗前后CTCs显著下降的患者无进展生存时间 (Progression-Free Survival, PFS) 和OS均获得了显著的提升[68, 78]。可见,MRD与晚期NSCLC患者的PFS和OS明显相关,但也会受到不同治疗方案的影响而改变。

  • 4 MRD在NSCLC中的临床应用

  • MRD的存在影响了早期和晚期NSCLC患者的预后,增加了患者在治疗结束后病情复发的风险。因此,在患者疾病进展与治疗过程中对MRD的监测至关重要。相比于取样困难的组织活检,液体活检能够在无创条件下获知患者的疾病进展情况,样本更易获得,有利于对患者病情的连续监测,而在了解了患者的病情进展之后,对治疗策略的适当调整也能为患者带来不小的获益。

  • 4.1 通过MRD监测疾病进展及评估治疗效果

  • NSCLC患者死亡的一大重要原因是对于肿瘤的诊断以及对疾病进展的判断不够及时[79]。目前,对于肿瘤的早期诊断和病情的判断主要依靠的是影像学依据,而这些依据并不能全面而准确地反映患者的疾病进展。因此,有学者建议重复多次的液体活检能够成为对NSCLC患者疾病筛查监测的新手段[80]。在确诊NSCLC之后,患者每一个治疗周期或是新的治疗阶段都应采集血液样本并进行检测。如果患者的临床症状与外周血MRD检测结果一致,那么该检测结果或许能够替代影像学结果成为病情进展的依据;而如果患者出现新的临床症状而液体活检结果未发生改变,或是液体活检发现了新的差异但患者并无临床症状,医生都应根据具体状况更新对患者病情的判断,并酌情对治疗策略进行更改。

  • 在连续对患者的外周血进行检测后,我们能够得到药物或手术治疗对其体内肿瘤负荷的影响趋势[59]。理想的情况下,患者的肿瘤细胞数会随着治疗的进行由最高峰变为根除或降至治愈水平。而在这之后由于治疗的结束,肿瘤负荷可能维持不变或是出现回升并引起复发。在围手术期中,存在MRD的患者人群接受辅助治疗的效果要优于其他患者,其无复发生存期较其他患者明显改善[81]。 MRD检测能够对NSCLC复发风险进行有效分层,并对患者术后12个月至15个月的复发状态进行准确预测[82]。在复发之前,如果能够在肿瘤细胞尚处于MRD诊断标准时便进行干预,在疾病进展的早期发现并予以治疗,将肿瘤负荷再次降至治愈水平从而避免肿瘤的复发与转移,将有利于患者获得更高的生存质量与更长的生存时间。在辅助治疗后, ctDNA的检测能够再次筛选出仍具有高复发风险的患者亚组,以便于临床医生制定更有效的治疗策略[83]

  • 除了监控肿瘤进展之外,在治疗后对MRD的检测可能能够提供患者休眠肿瘤细胞的实时信息,以此来判断肿瘤的异质性,是否有耐药细胞的存活或是非耐药自适应细胞的残留[84]。由此医生在制定治疗方案的同时可以对治疗的反应与效果进行一个大致的预测,以辅助后续治疗的制定与开展。同时,对MRD的深入研究或许能够发现患者休眠肿瘤细胞的驱动突变或是耐药机制,以此来寻求更佳的治愈机会。此外,将患者的CTCs转移至小鼠体内建立PDX模型,可以检测出不同治疗方案对小鼠的效果,进而为患者选择最优治疗方案提供依据[57]

  • 4.2 MRD在NSCLC靶向治疗中的应用

  • ctDNA的基因组突变分析与CTCs的转录变化分析都能够监测肿瘤在治疗压力下的驱动突变以及可能的进化方向,这有助于预测患者在使用哪些靶向药物时能够获益更大。在目前已发表的临床试验中,先行在血液中发现的EGFR突变帮助预测了患者对吉非替尼与厄洛替尼的应答,从而使患者获得了更好的预后[85, 86]。在治疗开始后,通过对患者的连续监测发现, EGFR突变率降低的越多,患者的PFS也会相应增加[87]。EGFR突变中的T790M突变常常对第一代EGFR抑制剂具有一定的耐药性,而对第三代抑制剂奥西替尼应答更佳[88]。血浆中的ctDNA检测对于T790M突变敏感度为70%,并且其检测结果对患者生存时间的预测与组织活检结果基本一致[89]。因此,ctDNA的检测提示这些患者选择奥西替尼进行治疗可能效果更好,但仍需警惕肿瘤细胞获得耐药性。而对于T790M突变阴性的患者,尽管第一代EGFR抑制剂与第三代抑制剂有着相似的应答率,但使用第三代抑制剂治疗似乎能够预防EGFR T790M突变的发生[90]

  • 在EGFR突变患者的治疗过程中,由于靶向药物给予的选择压力,患者肿瘤细胞有时会出现鼠类肉瘤病毒癌基因 (Kirsten Rat Sarcoma Viral Onco⁃ gene, KRAS) 突变。KRAS基因编码EGFR基因的下游信号,对EGFR基因的抑制会引发肿瘤内与肿瘤间KRAS基因的异质性突变[91]。因此,通过MRD对EGFR或KRAS的基因突变进行监测,能够指导EGFR靶向治疗在合适的时机暂停或是恢复,以避免新突变的产生进而影响靶向治疗的效果[92]

  • 4.3 MRD在NSCLC免疫治疗中的应用

  • 程序性死亡蛋白1配体 (Programmed DeathLigand 1, PD-L1) 在肿瘤细胞中的表达往往能够预示患者对于免疫治疗的响应性,CTCs也同样如此。在Guibert等[93] 进行的临床研究中,对96名晚期NSCLC患者在化疗后使用免疫治疗,检出CTCs的患者有83%表现出PD-L1的高表达,而匹配对照组为41%。尽管目前CTCs的PD-L1表达水平是否与相应组织活检检测出的肿瘤PD-L1表达水平一致尚不明确,也有研究认为CTCs中表达的PD-L1高于组织样本[94],但CTCs中PD-L1表达阳性率高的患者对于抗PD-L1免疫治疗的应答显著低于阴性患者[95]。同时,在免疫治疗之后,外周血CTCs的PD-L1表达水平会下降,这往往预示着更好的治疗后的生存状况,同时也表明了对于免疫治疗药物较低的耐药性[96]

  • 另一方面,ctDNA水平的增加可能表明对免疫检查点抑制剂的抵抗。在使用德瓦鲁单抗 (Dur⁃ valumab) 治疗的NSCLC患者中,应答较好的患者在开始治疗后,ctDNA水平出现显著下降,而无响应者ctDNA水平没有显著变化或升高[97]。此外,通过ctDNA的检测对肿瘤突变负荷 (Tumor Mutation Burden, TMB) 进行评估,较高的TMB将导致肿瘤细胞异常增殖信号表达增加,可能更容易被T细胞进行识别[98, 99]

  • 4.4 MRD在NSCLC临床试验中的应用

  • MRD的存在与患者远期肿瘤复发转移相关,并与较差的预后存在关联,因此MRD的检测或许能够识别某些特定的患者,以便于在终止事件发生之前对临床试验的开展进行一定的预测[100]。目前针对MRD的临床试验设想主要有两类:一类是以MRD的检出为干预时间点,以验证MRD对于患者预后结果的影响;另一类是将MRD作为患者在临床试验中的代理终点,在患者复发或是死亡的临床终点之前预先对试验的结果进行预测[101]。第一类试验以MRD预测复发的高风险,并评估在此时各个治疗方案对于患者远期预后的影响,以此来判断治疗策略的正确与否,并指导在标准治疗方案完成之后是否需要额外治疗。而第二类临床试验利用MRD与复发的关联缩短临床试验周期,对于MRD检测阳性的患者直接以临床终点计,而对于MRD检测阴性的患者则继续随访。这样将部分特定患者的试验终点提前,不仅仅是对治疗临床效益的预测评估,而且是加速对药物审批的理论依据,从而减少新药的开发研究时间,有利于NSCLC的治疗新方案迅速取得进展。

  • 5 总结

  • 当接受治疗的NSCLC患者在影像学或是实验室方法中无法检测到肿瘤细胞时,该患者往往被认为已经治愈。然而,液体活检所发现的肿瘤来源分子异常往往预示着MRD的存在,而这也是患者远期可能的肿瘤复发或转移的潜在来源之一。目前对于MRD的检测研究集中于对ctDNA和CTCs的检测,这些方法相比于组织活检高效且无创,因而更有利于对患者治疗的持续监测,但其检测的灵敏度和特异性以及与肿瘤病灶组织的一致性仍有待商榷,从而限制了液体活检在临床上的进一步应用。检测到的MRD往往与NSCLC患者较差的预后相关,利用这一点对患者的病情进展及时采取治疗干预,能够帮助患者取得更好的生存获益。除此之外, MRD也能够提供一定的关于肿瘤来源的分子信息,有助于目前靶向治疗与免疫治疗等治疗方式的开展和评估。总而言之,MRD的发现一定程度上预示着NSCLC患者的不良预后,同时也可为患者的治疗提供及时的指导,为复发转移患者提供更多的治愈机会。MRD在NSCLC中的研究与应用仍有待进一步深入发展,未来关于MRD的更多理解将为NSCLC患者提供更多的生存获益,并助力NSCLC精准治疗的发展。

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