【万字综述】Molecular Cancer: 免疫微环境与ICIs治疗EGFR突变NSCLC疗效的关系

2019年9月16日，南方医科大学张健教授团队在Molecular Cancer杂志(IF=10.679)上发表了一篇关于在NSCLC患者中EGFR突变与ICI治疗的机制研究的综述。EGFR突变的NSCLC患者中NSCLC患者对PD-L1轴抑制剂的疗效存在差异的具体机制尚未完全明确，可能涉及多方面因素，例如EGFR-TKIs或EGFR突变影响肿瘤微环境免疫状态。本综述旨在阐述EGFR突变与PD-L1轴抑制剂疗效影响的可能机制，为临床决策提供有价值信息。

Role of the dynamic tumor microenvironment in controversies regarding immune checkpoint inhibitors for the treatment of non-small cell lung cancer (NSCLC) with EGFR mutations

DOI: 10.1186/s12943-019-1062-7

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Section

1.全文摘要 (Abstract)
2.背景介绍 (Introduction)
3.PD-1/PD-1L抑制剂治疗EGFR突变NSCLC疗效差异的潜在机制

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3.1 EGFR突变影响NSCLC中TME
3.1.1 EGFR突变和调节性T细胞 (Tregs)
3.1.2 EGFR突变和肿瘤浸润淋巴细胞 (TILs)
3.1.3 EGFR突变和外泌体 (Exosomes)
3.1.4 膜性免疫调节分子和免疫抑制可溶性因子
3.1.4.1 EGFR突变和CD73
3.1.4.2 EGFR突变和主要组织相容性复合物 (MHC)
3.1.5 EGFR基因突变位点和免疫检查点抑制剂的有效性
3.1.6 EGFR突变和肿瘤突变负荷(TMB)
3.1.7 EGFR突变和PD-L1表达
3.1.8 T790M突变状态和免疫检查点抑制剂的有效性

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4. EGFR-TKIs的免疫调节作用

4.1 EGFR-TKIs的免疫调节作用4.2 EGFR-TKIs引起PD-L1表达的动态变化

5. 前景 (Future prospects)

6. 结论 (Conclusion)

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1. 全文摘要
   免疫治疗已成为非小细胞肺癌 (non-small cell lung cancer, NSCLC)的一线和二线治疗手段之一。适应性免疫信号传导和致癌基因驱动（如EGFR突变）信号传导可能参与调控NSCLC中PD-L1的表达。然而，免疫检查点抑制剂（ICIs）在晚期EGFR突变NSCLC的疗效反应的诸多研究结果存在矛盾。ICIs应答效应往往持久，甚至数年之后仍可有效。然而，仅小部分研究和临床研究显示EGFR突变NSCLC患者从ICIs治疗中获益。因此，EGFR突变的NSCLC患者中NSCLC患者对PD-L1轴抑制剂的疗效存在差异的具体机制尚未完全明确，可能涉及多方面因素，例如EGFR-TKIs或EGFR突变影响肿瘤微环境免疫状态。因此，更好地理解EGFR突变NSCLC患者与接收PD-L1轴抑制剂疗效之间的关系成为一个紧迫的主题。这些机制可能包括，如动态变化的肿瘤免疫微环境，动态表达的PD-L1，低肿瘤突变负荷，可能解释了EGFR突变与PD-L1轴抑制剂疗效之间的关系。为此，本文将阐述EGFR突变与PD-L1轴抑制剂疗效影响的可能机制，为临床决策提供有价值信息。

2. 背景介绍
肺癌是全球最常见的恶性肿瘤以及首位的癌症死亡原因，发病率和致死率分别可达11.6%和18.4%，根据世界癌症研究机构(IARC)的数据统计，至今全球约有210万名肺癌患者。2018年，全球肺癌新发病例约2,093,876例，死亡病例约1,761,007例，其中80%为NSCLC，由于早期不具备明显的临床症状，患者发现时已经错过最佳治疗时期，约75%的NSCLC确诊时属中晚期，致使其5年生存率不足15%。

下一代测序技术(next-generation sequencing, NGS)，高通量基因组分析平台及基因工程小鼠模型(Genetically engineered mouse model，GEMM) 的应运而生，使得NSCLC的治疗逐步进入基于分子标志物的个体化治疗时代。鉴定出肺癌的驱动基因如KRAS和BRAF突变后，在肺腺癌患者中发现了EGFR突变，靶向EGFR的酪氨酸激酶抑制剂(tyrosine kinase inhibitors，TKIs)的出现为改善EGFR突变NSCLC的治疗点燃希望。近年来，EGFR-TKIs已被临床指南推荐作为EGFR基因敏感突变并且不存在耐药基因的晚期NSCLC患者一线治疗药物。与化疗相比，EGFR-TKIs显示出更为优越的客观缓解率(objective response rate, ORR) (67.0% vs 40.8%)和中位无疾病进展时间(progression free survival, PFS) (10.9 vs. 7.4个月)等生存获益。然而，EGFR-TKIs可诱导晚期EGFR突变NSCLC的快速应答，但通常在9-14个月发生获得性耐药现象。对于EGFR-TKIs耐药后的治疗策略，仍是亟待解决的问题，这些都促使人们开始关注免疫治疗带来的潜在获益。最近，重新激活肿瘤特异性T细胞这一免疫治疗策略在NSCLC治疗领域中取得突破性进展。ICIs可以改善肿瘤患者免疫状态，可使疾病得到持续控制。ICIs如检查点程序死亡受体 (programmed death-1,PD-1)及其配体 (PD-1 ligand,PD-L1)正在成为晚期NSCLC的另一全新突破性治疗方式。例如，迄今最长的一项随访研究表明129名多次治疗失败的晚期NSCLC患者接受Nivolumab治疗后5年生存率为达26%，[其发生频率远高于非免疫治疗的NSCLC5年生存率(1%-8%)]。此外，临床前研究表明，EGFR突变信号可以上调肿瘤细胞上PD-L1表达，从而诱导T细胞凋亡并参与EGFR突变型NSCLC的免疫逃逸。此外，EGFR-TKIs可增强主要组织相容性复合体(major histocompatibility complex, MHC)-I和MHC-II的表达 ，以响应γ-干扰素(interferon-γ, IFN-γ)并增强T细胞介导的肿瘤杀伤作用，这为支持PD-1/PD-L1抑制剂与EGFR-TKIs联合治疗携带EGFR突变的NSCLC患者可能产生协同作用提供了理论基础。因此，这些是否意味着免疫治疗联合靶向治疗能提高疗效并使更多EGFR突变NSCLC患者获益。

近期多项相关研究正不断开展，探索EGFR突变患者接受免疫治疗联合靶向治疗的疗效及安全性。最近，多项临床试验显示EGFR突变患者无法从免疫治疗中获益，甚至疗效更差，并且有可能导致爆发“超进展”(hyperprogressive，HPD)，增大毒副作用的叠加。一项开放的III期CAURAL试验（NCT02454933）研究了Osimertinib联合Durvalumab与Osimertinib单药治疗EGFR-TKI治疗后EGFR T790M突变阳性的晚期NSCLC患者的疗效和安全性差异，结果显示联合用药组在ORR(64% vs 80%)、中位DOR(17.5 vs 21.4个月)和DCR(100% vs 93%)上无法从联合治疗中获益，甚至低于Osimertinib单药治疗组。在另一项Ib期TATTON研究评估了Osimertinib联合Durvalumab 治疗EGFR突变NSCLC的疗效。该研究将患者分为经治组(剂量扩增疗法)和EGFR-TKI初治组(剂量探索疗法)，结果显示EGFR-TKI经治T790M阳性患者和EGFR-TKI经治T790M阴性患者ORR分别为67%和21%，TKI初治患者ORR为70%。尽管该试验联合治疗有效率尚可，然而患者耐受性欠佳，尤其是间质性肺炎 (Interstitial lung Disease, ILD）的风险却显著升高。此外，一项开放、多中心的临床I/II期研究Keynote-021试验中，在使用Pembrolizumab联合Erlotinib一线治疗EGFR突变晚期NSCLC患者中，患者从ORR(41.7%)和中位PFS(19.5个月)均显著获益，其中位PFS明显优于第1代EGFR-TKIs单药的一线治疗(11.0个月），甚至优于Osimertinib单药一线治疗(19.17个月)。另一项I期CheckMate012试验试图比较Nivolumab联合Erlotinib二线治疗EGFR突变晚期NSCLC患者的临床疗效。结果显示，20例经治患者的ORR为15%，DCR为65%。中位PFS为5.1个月，中位OS为18.7个月。截止2017年12月1日，所有存活的8例（8/21,38.1%)患者生存期都达到5年以上。因此，多项基础研究与临床研究结果存在矛盾(表1)，PD-1/PD-L1抑制剂和EGFR-TKIs的联合应用备受争议。

近几年，临床前与临床研究均纷纷探索EGFR突变NSCLC患者免疫检查点治疗疗效 存在矛盾的可能机制。越来越多的研究发现ICIs的治疗效果与肿瘤免疫微环境（如T细胞亚群）、肿瘤免疫原性、特定基因的突变、拷贝数变异和肠道微生物丰度等因素有关。多项研究提示EGFR突变可能与抑制性免疫微环境、肿瘤突变负荷(tumor mutation burden, TMB)和PD-L1表达等相关。此外，EGFR-TKIs可能参与调节肿瘤免疫微环境。以上因素作为空间和时间上连续变量，它们之间的确切界值及相关性依旧尚未明确。上述研究结果可能作为初治或经治的EGFR突变NSCLC患者对免疫检查点抑制剂或EGFR-TKIs联合治疗的疗效存在矛盾的可能机制。

为此，我们试图比较和分析有关EGFR突变NSCLC患者对PD-1/PD-L1抑制剂或与EGFR-TKIs联合治疗的所有临床研究及临床前研究，探索使用PD-1/PD-L1抑制剂或与EGFR-TKI联合治疗EGFR突变NSCLC患者的可行性。本文探索了EGFR突变NSCLC患者的肿瘤免疫微环境，分析免疫治疗或联合靶向治疗的疗效相互矛盾的可能机制，并深入讨论如何提高EGFR突变NSCLC患者免疫治疗效果。有望为EGFR突变NSCLC患者发现和鉴定免疫治疗的生物标志物和治疗带来新的希望。

3. PD-1/PD-1L抑制剂治疗EGFR突变NSCLC疗效差异的潜在机制

3.1 EGFR突变影响NSCLC中TME

TME是肿瘤细胞赖以生存和发展的内环境。肿瘤免疫微环境对于制定肿瘤免疫治疗策略至关重要，而T淋巴细胞、髓源性细胞、细胞因子、外泌体构成了肿瘤微环境的免疫调节网络。随着肿瘤发展和免疫细胞的可塑性(plasticity)，T淋巴细胞通过免疫编辑从免疫监视转化为免疫逃逸，甚至发挥免疫抑制功能，如诱导调节性T细胞(regulatory T cell, Tregs)、上调髓源性抑制细胞(myeloid-derived suppressor cells, MDSC)。此外，TME中炎性细胞也可能作为介导肿瘤进展的重要机制。

最近多项研究显示EGFR突变的抑制性免疫作用，EGFR突变可调节肿瘤免疫微环境，如肿瘤浸润淋巴细胞（TILs），Tregs，MDSCs, 肿瘤相关的巨噬细胞(tumor-associated macrophages, TAMs) ，免疫相关的细胞因子和外泌体。这些临床前和临床研究结果提示EGFR突变NSCLC患者的肿瘤微环境免疫状态可能是独特并且不同于EGFR野生型患者。此外，EGFR突变可能通过影响TME来影响抗肿瘤效应 (图1)。

3.1.1 EGFR突变和调节性T细胞 (Tregs)

Tregs是一类抑制免疫反应性Th细胞亚群，其核心转录因子为Foxp3是功能性Treg的特异性标志。肿瘤内的Treg细胞分泌的转化生长因子-β(transforming growth factor-β, TGF-β)和白介素-10 (interleukin-10, IL-10)和IL-35能产生一个免疫抑制环境，有助于减弱CD4+ T细胞、CD8+ T细胞及NK细胞产生的抗肿瘤效应。

Huang等研究表明，EGFR(+)外泌体诱导耐受型DC细胞的可塑性转变，诱导DC细胞生成免疫耐受分子吲哚胺-2,3-双加氧酶（indoleamine2,3-dioxygenase, IDO)，IDO在CD3+ CD4+ CD25- T细胞转换为Treg细胞中发挥重要的作用，进而诱导免疫耐受和肿瘤免疫逃避。此外，研究表明双调蛋白(Amphiregulin，AREG)为EGFR配体之一，其在NSCLC患者血浆的表达水平与患者预后不良相关，并且作为肿瘤细胞中的外泌体中的特定分子在肿瘤增殖与存活中起促癌作用。Wang等研究发现，糖原合酶激酶-3β(glycogen synthase kinase 3β,GSK-3β)可直接或通过β-连环蛋白(β-catenin)和白细胞淋巴瘤/白血病xL(Bcl-xL)的表达间接下调Foxp3蛋白的表达，进而抑制Treg细胞功能。而在多种肿瘤细胞中表达均上调的双调蛋白(Amphiregulin, AREG)，可通过与Treg细胞上表达的EGFR结合激活EGFR信号抑制GSK-3β蛋白的活性，促进Foxp3蛋白翻译后的修饰过程，减少Foxp3蛋白降解以增强Foxp3蛋白表达，进而维持Treg细胞的抑制功能。而使用EGFR-TKI吉非替尼可以修复GSK-3β活性和减弱Treg细胞免疫抑制功能。此外，Mascia等研究表明敲除EGFR基因后可以明显抑制肿瘤细胞的生长和下调TME中Treg细胞的浸润程度。

3.1.2 EGFR突变和肿瘤浸润淋巴细胞 (TILs)

TIL是一类肿瘤浸润性且具有抗原效应的细胞群，可存在于肿瘤癌巢内和肿瘤间质中。CD8+ T细胞在肿瘤微环境肿瘤发展过程中是一种抗肿瘤免疫细胞，通过释放IFN-γ、穿孔素(perforin)和颗粒酶B(Granzyme B)等肿瘤毒性细胞因子达到杀死肿瘤细胞的作用，并且其数量决定其杀伤靶细胞的效率。多项研究表明NSCLC肿瘤微环境中高度浸润的CD8+ TIL与良好预后和治疗效果相关。Teng等通过建立肿瘤免疫微环境模型来预测免疫疗法的功效，对于肿瘤免疫微环境中TIL的存在或缺失和PD-L1表达的阳性或阴性来区分相应类型，结果显示免疫炎症型TME(PD-L1+ 和TIL+ )的患者从 PD-1/PD-L1抑制剂中获益的可能性最大。

Dong等发现EGFR突变型患者CD8+ TIL在肿瘤内的密度显著低于EGFR野生型患者(P=0.031)，此外，与EGFR野生组相比EGFR突变组双阴性(PD-L1-/ TIL-)比例显著高于双阳性(PD-L1+ /TIL+ )比例(Odd Ratio (OR): 1.79, 95% CI: 1.10–2.93; P = 0.02); 同样，与双阴性(PD-L1-/ TIL-)组相比双阳性(PD-L1+ /TIL+ )组具有更低水平的EGFR突变(P=0.005)。多重免疫荧光组织化学标记方法表明，通过鉴定Ki67(T细胞增殖状态)、颗粒酶B和CD3+ 等表达以进一步预测TILs的功能。Toki等通过使用Schalper建立的预测模型来探索EGFR突变与TILs功能相关性，结果表明EGFR突变组中28.6％(16/56)TILs表现为衰竭或休眠的免疫状态(高CD3，低Ki67，低颗粒酶B)；通过检测肿瘤细胞和基质细胞的PD-L1的表达状态和TILs功能后发现：无论是高表达PD-L1的肿瘤细胞或基质细胞均与活化TILs的高浸润显著相关(P= 0.0014 和 P=0.02)。此外，他们发现不同EGFR突变位点的免疫谱存在一定差异：与EGFR-19外显子缺失(EGFR-19Del)相比，EGFR-L858R型具有炎症型的肿瘤微环境，如高表达CD8+ T细胞 (P=0.03)和高表达的趋势CD3+ 和CD4+ (P= 0.11；P= 0.11)；相比，EGFR-L858R组较EGFR-19Del组具有高浸润的活化TILs，然而EGFR-L858R和EGFR-19Del在肿瘤细胞和基质细胞中PD-L1的表达并无统计学差异。

3.1.3 EGFR突变和外泌体 (Exosomes)

外泌体(Exosomes)是细胞分泌的微小膜囊泡，包含许多分子，如核酸(DNA，RNA，mRNA 和 miRNA) 、脂类和蛋白质。它作为信号载体介导细胞间通讯，并通过协同作用影响肿瘤细胞对药物的敏感性，与肿瘤转移的发生相关。肿瘤细胞来源的外泌体可以通过其内含 miRNA 影响远处靶细胞，改变局部微环境，形成“预转移龛” 发挥远程调控功能。Poggio等证明肿瘤细胞分泌带有PD-L1的外泌体导致免疫逃逸,通过与T细胞直接结合并抑制T细胞的功能，此外，外泌体PD-L1能够抑制淋巴结中T细胞的活性。研究者进一步研究发现外泌体的PD-L1对PD-L1抑制剂具有抵抗作用，通过使用CRISPR基因编辑技术敲除产生外泌体的相关基因后，外泌体缺陷肿瘤细胞能够引起系统性抗肿瘤免疫和免疫记忆，对免疫治疗后有着显著的疗效。此外，在抑制外泌体形成和PD-1/PD-L1抑制剂的联合治疗下，小鼠的生存时间明显较单一PD-1/PD-L1抑制剂疗法显著延长。

3.1.4 膜性免疫调节分子和免疫抑制可溶性因子

TME中膜性免疫调节分子的表达的变化和免疫抑制可溶性因子(如TGF-β，IL-10和腺苷(ADO)等的释放在在肿瘤进展的过程中发挥至关重要的作用。

3.1.4.1 EGFR突变和CD73

CD73是胞外5'-核苷酸酶，由糖基磷脂酰肌醇(GPI)锚定于细胞膜外表面，CD73在多种肿瘤中呈现高表达状态。它不仅参与嘌呤核苷酸的代谢和补救合成途径，而且作为一种重要的免疫信号负向调控分子，通过催化免疫抑制性介质腺苷(ADO)的形成，参与肿瘤的免疫逃逸。研究表明高表达的CD73与免疫抑制及NSCLC患者的预后不良有关。因此，更好地理解CD73，ADO与TME的调控体系尤为重要 (图1)。

Park等根据CD73表达水平[CD73高表达(CD73-H)和CD73低表达(CD73-L)]进行亚组分析，结果显示与CD73-L组相比，CD73-H组高密度浸润的活化CD4 + T细胞（20％vs 41％，P < 0.01）和CD8 + T细胞（28％vs 47％，P < 0.01）更少。与CD73-H组相比，CD73-L组的OS和DFS更高（62 vs 44个月，P < 0.01；83 vs 34个月，P < 0.01）；此外，亚组分析提示EGFR突变与CD73的高表达有关（P = 0.03）。因此，Park等推测突变型EGFR NSCLC中CD73的过表达可能导致对免疫抑制疗法的不良反应，并提示CD73抑制剂与EGFR-TKIs或PD-1 / PD-L1抑制剂的组合可能是治疗耐药患者的潜在策略。相反，一项回顾性研究报道，在接受免疫疗法治疗的EGFR-TKI耐药组中，与CD73低表达相比，CD73过表达组中位PFS（16个月vs 1.2个月，P = 0.024）和ORR（66.7％vs 0 ％，P = 0.006）显著增加，而野生型EGFR患者的高和低CD73表达组之间中位PFS和ORR没有差异（中位PFS：2.8个月vs 2.8个月，P = 0.394。但是，目前的共识表明，EGFR突变的肿瘤细胞可能通过上调CD73，将ATP转化为ADO，进而活化ADO旁路以上调Tregs表达，改变肿瘤细胞和免疫细胞的功能，从而产生免疫抑制型肿瘤免疫微环境。由于结果的不一致性，CD73表达与免疫抑制性TME相关的确切机制仍不清楚。

3.1.4.2 EGFR突变和主要组织相容性复合物 (MHC)

MHC对肿瘤抗原呈递过程中发挥重要作用：MHC I类分子肿瘤抗原可组成细胞激活的第一信号，激活CD8+ T细胞，发挥抗肿瘤免疫效应。MHC II类分子与肿瘤抗原肽结合后，提呈给CD4+ T细胞，激活特异性CD4+ T杀伤细胞和辅助细胞。前者可特异性地杀伤肿瘤细胞，后者可通过分泌细胞因子提高和杀伤细胞的杀伤效应，参与机体抗肿瘤的正反馈调节。多项研究表明，MHC分子表达与免疫治疗的疗效相关。IFN-γ可以上调细胞表面MHC-I的表达。Watanabe等研究发现EGFR突变肿瘤细胞在IFN-γ存在的情况下其HLA-B表达水平仍低于EGFR野生型NSCLC细胞。近年来，多项研究表明EGFR突变不仅通过IFNγ信号通路下调MHCI和MHCII的表达，也能通过抑制对MHCII类分子反式激活因子(classII transactivator, CIITA)的诱导，进一步抑制MHCI和MHCII的表达。Watanabe等发现EGFR突变通过活化下游MEK/ERK信号通路进而抑制MHC-I表达 (图2)。

3.1.5 EGFR基因突变位点和免疫检查点抑制剂的有效性

EGFR基因突变主要发生在18-20号外显子，其中19号外显子缺失突变(p.E746-A750del)和21号外显子点突变(p.L858R)可占所有突变类型的85%以上，而这两者也是对EGFR-TKI有效有的最主要的两种敏感型突变。所有其他其他突变可称为罕见突变，如18号外显子的G719X突变(3%)和21号外显子的L861X突变(2%)是最常见罕见突变类型，G719X、L861X、19号外显子和20号外显子的嵌入突变也被认为对TKI治疗有效。多项研究表明EGFR罕见突变NSCLC患者较常见EGFR常见突变更能从免疫治疗中获益。

Yamada等对EGFR突变NSCLC按照罕见/常见突变位点(是否为19号外显子缺失突变和21号外显子L858点突变)进行亚组分析，结果表明EGFR罕见突变患者中位PFS(256天vs 50天,HR = 0.288 ;95% CI:0.13-0.63; P= 0.003)和中位疾病进展时间 (Time To Progress, TTP) (256天 vs 48天; HR =0.353; 95% CI:0.16‐0.77; P= 0.008)较EGFR常见突变患者得到显著延长。此外，最新CA209-003研究的5年生存者特征分析结果显示2例罕见EGFR突变患者(2/8,25%)生存期超过5年，突变位点分别为EGFR外显子20插入和外显子18错义突变G719A。此外，Yoshida等报道EGFR罕见突变患者较EGFR常见突变患者有着显著延长的PFS(P <0.05)。

3.1.6 EGFR突变和肿瘤突变负荷(TMB)

TMB是评估肿瘤基因的外显子编码区每兆碱基发生置换、插入、缺失突变的总数；它是免疫抑制剂疗效预测的良好生物标志，可定量估计肿瘤基因组编码区的突变总数，TMB越高，肿瘤产生的新抗原越多，越易被免疫细胞识别，对免疫治疗有较好的客观缓解率，较长的无进展生存期和持久的临床响应。

Haratani等为研究EGFR突变NSCLC患者耐药后使用Nivolumab治疗疗效与TMB之间的关系，全外显子组测序(whole exomesequencing,WES)结果显示每位患者(n=9)的中位TMB为101(Mut/Mb)，且对药物有应答的患者(n=3)的TMB显著高于无反应患者(n=6)。此外，Dong等发现EGFR突变型(外显子19Del、L858R、L861Q、G719X、S768I)的中位TMB显著低于EGFR野生型TMB (56 vs 181 Mut/Mb); 此外，EGFR基因突变型与野生型的突变负荷中值比为突变负荷中位值分别为59:209 (Broad database)和162:197 (GLCI database)。

单个点突变如EGFR、BRAF和TP53突变通常是NSCLC早期克隆驱动事件，目前研究显示这类特定基因突变患者多数对免疫治疗反应不理想，而减少的TMB可能是EGFR突变对于免疫治疗疗效不佳的重要影响因素之一。目前关于EGFR突变与TMB的相关性研究还在进行中，而微卫星高度不稳定、错配修复基因缺失在免疫治疗中的作用也是未来研究中需要不断探索的部分。

3.1.7 EGFR突变和PD-L1表达

目前，EGFR突变与PD-L1表达调控仍然存在争议，但是体外实验及大中心研究均提示EGFR突变导致NSCLC中PD-L1表达异常升高。此外，近来有研究发现，Ras/RAF/MEK/ERK、PI3K/AKT/mTOR、JAK/STAT、NF-kB、GSK-3β等异常活化后可促进肿瘤细胞PD-L1表达(图2)。然而，近来多项临床研究得出相反结论，如PD-L1在EGFR野生型肺癌组织中高表达，EGFR突变和PD-L1表达呈负相关或无显著相关性。一项Meta分析显示EGFR野生型与突变型肿瘤PD-L1+ 表达率具有显著差异(44.1% vs 36.7%, P<0.05)。此外，另一项Meta分析得出相同结论，EGFR野生型肿瘤PD-L1+ 表达率明显高于EGFR突变型肿瘤(P=0.02)。为进一步证实，研究人员检测了NSCLC患者肿瘤样本的mRNA谱、PD-L1蛋白免疫组化（IHC）和反相蛋白阵列(reverse phase protein arrays, RPPA)，发现EGFR突变型肿瘤PD-L1表达明显低于EGFR野生型(P<0.05)。

不同试验研究结果相互矛盾可能与以下因素，如PD-L1检测技术(如不同的检测抗体、检测平台以及不同设定阳性阈值)、患者肿瘤异质性、患者肿瘤组织来源不同(如细胞学标本、存档标本与新鲜标本、原发部位或转移灶等)和TIL上亦可检测出PD-L1表达有关。此外，Noguchi等发现TAM上PD-L1的表达在肿瘤免疫逃逸发挥着重要的作用。肿瘤细胞上PD-L1表达是由IFN-γ和致癌驱动基因的突变介导的依赖性和暂时性表达，但免疫细胞上的PD-L1的诱导幅度更大，仅部分依赖于IFN-γ，并且其表达在监测时间内相对稳定。因此，许多研究人员认为PD-L1在免疫细胞上的表达是预测PD-1 / PD-L1抑制剂功效的更好的生物标志物。

3.1.8 T790M突变状态和免疫检查点抑制剂的有效性

针对EGFR敏感突变NSCLC患者，Gefitinib、Erlotinib和Afatinib作为一线EGFR突变靶向药物显示出较好的响应，但通常在9-14个月后出现获得性耐药现象，其中EGFR T790M突变作为最常见的耐药机制，可在约50-60%患者中检测到。Haratani等分析了EGFR-TKI治疗期间疾病进展后用Nnivolumab治疗的患者，结果显示，EGFR突变患者T790M(+)组和T790M(-)组患者的CD8+ TILs数量相似，但T790M(-)组PD-L1水平≥10%或≥50%的肿瘤细胞比例(20％vs 4％)和高密度浸润的CD8+ TIL(≥median) (12％vs 4％)共存现象多于T790M(+)组，T790M(-)组患者的FOXP3+ TILs计数显著低于T790M(+)组(P = 0.013)。Yamada等回顾性研究纳入27例EGFR-TKI耐药后使用ICIs治疗的患者，亚组分析表明T790M(-)患者较T790M(+)更能从PD-1抑制剂治疗中获益(中位PFS: 86天 vs 48天; P=0.03; 中位TTP:97天 vs 48天; P=0.03)。在对67例EGFR突变NSCLC患者分析结果显示T790M(+)肿瘤细胞中PD-L1表达明显低于T790M(-)肿瘤细胞 (P=0.0149)。

4.EGFR-TKIs的免疫调节作用

4.1 EGFR-TKIs影响NSCLC中TME

截至目前大多数基础研究和临床研究均表明EGFR-TKI通过以下途径，如EGFR-TKI可增强主要组织相容性复合体(major histocompatibility complex, MHC)-I和MHC-II的表达，提高免疫提呈作用；促使Foxp3降解来减弱Treg细胞的抑制功能；通过降低肿瘤微环境中Treg细胞浸润4并抑制肿瘤生长和增强细胞毒性T淋巴细胞(cytotoxic lymphocyte,CTL)介导的的抗肿瘤活性，减少T细胞凋亡，增加IFN-γ产生优化机体抗肿瘤活性，从而增强患者PD-1/PD-L1抑制剂获益几率。然而，这些机制却依旧无法完全解释EGFR突变患者使用免疫检查点抑制剂和EGFR-TKI的联合治疗方式疗效存在争议的原因。

最近，Jia等对在小鼠肺肿瘤模型(EGFRL858R or EGFR19DEL/T790M)中对使用EGFR-TKI后对小鼠的肿瘤免疫微环境的不同时期进行分析，结果显示EGFR-TKI治疗过程中EGFR突变肿瘤的TME呈现动态变化从肿瘤有益性免疫微环境(早期阶段)转变为肿瘤免疫抑制性微环境(晚期阶段)(图1;表2)。在EGFR-TKI治疗的早期阶段短期抑制肿瘤细胞的生长，并增加CD8+ T细胞、DC细胞和M1-like TAMs数量，减少Foxp3+ Tregs浸润和抑制M1型TAMs向M2型TAMs极化；然而，EGFR-TKI治疗的后期阶段，抗肿瘤效应细胞无明显变化甚至减少，血清中IL-10和CCL-2分泌增加，CCL2作为趋化因子配体可通过与其受体CCR2结合，进一步募集MDSCs至TME。此外，其他研究也发现EGFR-TKI可以上调CCL-2的表达。CCL2诱导T细胞分化为Th2细胞(抗炎功能)，上调并活化MDSCs的信号转导子和转录激活子-3(STAT3)，STAT3进一步介导Mo-MDSC扩增及活化，MDSC进一步发挥抗肿瘤免疫抑制作用，如产生免疫抑制分子IL-10, TGF-β 抑制抗肿瘤免疫细胞(T细胞、DC细胞和NK细胞)功能、诱导T细胞凋亡、增加Tregs数量和促进TAM巨噬细胞向M2表型极化。除了抑制免疫应答之外，MDSC还可以通过非免疫相关的机制促进肿瘤的侵袭和转移，包括分泌VEGF促进肿瘤血管生成，直接分化成肿瘤血管内皮细胞，释放各种金属基质蛋白酶(matrix metalloproteinase, MMP)促进肿瘤侵袭等方式。此外，IL-10不仅介导DC细胞和immature myeloid cells (IMCs) 中能够通过STAT3途径介导MDSCs的扩增和活化，还可以通过下调肿瘤细胞表面的HLA class I 的表达。因此，Jia及其同事推测肿瘤免疫微环境在EGFR-TKIs治疗期间存在短暂的窗口期，这一短暂并且可能最终消失的窗口期对于EGFR-TKIs与免疫检查点抑制剂的联合治疗方案可能是最有益的。以上研究提示我们，在肿瘤治疗的不同阶段TME可能呈现动态变化，进一步了解肿瘤微环境的动态变化对改善免疫治疗联合靶向治疗的疗效至关重要。

4.2 EGFR-TKIs引起PD-L1表达的动态变化

多项研究结果显示EGFR突变NSCLC患者在使用TKI治疗前后PD-L1表达水平发生动态变化，部分呈现PD-L1表达显著增加，而部分患者PD-L1表达下降。部分患者呈现PD-L1表达显著增加可能解释了一些患者EGFR-TKIs耐药后二线免疫治疗疗效较好的部分原因。Gainor等研究报道了EGFR-TKI使用前后PD-L1的变化情况，显示TKI治疗前PD-L1表达≥1%、≥5%和≥50%的EGFR突变NSCLC患者分别占24%、16%和11%，而TKI治疗耐药后分别占31%、29%和14%。28%(16/57)EGFR突变NSCLC患者在TKI耐药后PD-L1表达水平发生动态改变，其中12例(12/57, 21%)表达上调，4例(4/57,7%)表达下调。此外，PD-L1+ (表达≥5%)与高CD8+ TILs浸润(≥2级)同时出现的在与EGFR突变NSCLC患者治疗前后增加，但无统计学差异(2.1%vs11.6%;P=0.219)。另一项研究发现EGFR突变型肺癌患者在靶向治疗耐药后，8例(8/17, 38.9%)NSCLC患者肿瘤细胞的PD-L1表达出现上调，并且肿瘤组织发生间质上皮转化(Mesenchyal Epithelial Transition, MET)有关(P=0.028)。然而，PD-L1表达与对EGFR-TKIs耐药之间的关系和机制尚未明确。

5. 前景

在EGFR突变NSCLC患者中联合应用EGFR-TKIs与免疫检查点抑制剂的治疗策略引起了人们的关注。多种联合治疗的研究结果相互矛盾，其单独或联合应用备受争议。值得注意的是，传统治疗手段(如化疗及靶向治疗)远不能满足EGFR突变NSCLC中的临床需求。一线EGFR-TKI治疗失败后，EGFR突变NSCLC患者治疗方案的选择至关重要。因此，新型治疗策略在NSCLC中的临床应用与探索之路仍任重道远。本文总结了EGFR-TKIs与免疫检查点抑制剂的治疗策略的潜在可能机制(图3)。

首先，II期临床研究（NCT0287994）结果表明不推荐将Pembrolizumab作为初治EGFR-TKIs且高表达PD-L1患者的一线治疗药物。

第二，对部分高表达PD-L1或EGFR罕见突变NSCLC患者而言，ICIs似乎是更有前景的治疗策略（可能由于肿瘤异质性或EGFR突变克隆）。

第三，肿瘤免疫微环境在EGFR-TKIs治疗期间存在短暂的窗口期，这一短暂并且可能最终消失的窗口期对于EGFR-TKIs与免疫检查点抑制剂的联合治疗方案可能是最有益的。

第四，高表达的PD-L1与对EGFR-TKIs耐药性之间可能存在关联，因此EGFR-TKIs可能不适合作为高表达PD-L1且未使用EGFR-TKI的一线治疗策略。

第五，血管内皮生长因子(vascular endothelial growth factor, VEGF) / VEGFR抑制剂与免疫检查点抑制剂的联合用药策略可能为EGFR-TKIs耐药的EGFR突变患者提供了新的选择。VEGF可促进免疫抑制性细胞的产生(如Tregs和MDSCs等)、限制DCs，抗原提呈细胞的成熟和效应T细胞的功能、阻碍肿瘤特异性T细胞和其他免疫效应细胞向肿瘤微环境的浸润和迁移，因此，VEGF抑制剂可通过拮抗上述多种途径增强机体的抗肿瘤免疫效应。然而，VEGF阻断和免疫调节作用的确切机制仍不清楚。一项随机，III期临床试验（IMpower150）结果显示，在EGFR突变NSCLC患者中，atezolizumab+贝伐珠单抗+标准化疗(TC)组与贝伐单抗+ TC组相比显示出了有希望的疗效（中位DOR：11.1个月vs 5.6个月）。

** 第六，**大多数EGFR突变晚期NSCLC患者同时具有多种致癌突变。

因此，推荐应用基因组诊断来进一步选择最合适的治疗策略。以上问题值得思考，例如：如何筛选不同的联合治疗策略的适宜人群、寻找预测联合治疗方案疗效的生物标志物等。

6. 结论

在全球，肺癌的确诊率、死亡率均占肿瘤的第一位，NSCLC占所有肺癌病例的85%以上，严重威胁着人们的健康。现今，免疫治疗成为NSCLC一个有前景的治疗策略。临床前研究表明EGFR突变通过PD-1/PD-L1途径介导肿瘤免疫逃逸，而EGFR-TKI下调肿瘤PD-L1表达。然而，EGFR突变NSCLC患者接受免疫检查点抑制剂治疗效果却不佳。但部分研究表明免疫检查点抑制剂对于EGFR突变患者的疗效仍然有效。本综述总结了EGFR突变NSCLC患者联合使用免疫治疗或联合EGFR-TKI治疗现状，EGFR突变基因可能造成抑制性免疫微环境、低TMB、动态表达PD-L1等免疫特征，EGFR-TKI使用过程期间机体肿瘤免疫微环境发生动态变化。这些矛盾和争议的出现均提示EGFR突变NSCLC患者免疫治疗或联合EGFR-TKI治疗尚处于早期阶段。因此，在这种情况下，如何在TME内诱导持久的抗肿瘤活性，如何最大限度地提高患者免疫治疗效果，研究人员和临床医生必须明确有效的疗效、毒性预测生物标志物、药物间的最佳剂量，最佳顺序和时间表。此外，仍需开展大量前瞻性实验探究何时将免疫疗法与靶向药物等其他疗法相结合，为EGFR突变患者寻找生存希望。

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