Clinicopathological Characteristics and Therapeutic Effect of Patients with Non-small Cell Lung Cancer and Uncommon EGFR Mutations
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摘要:目的
探讨表皮生长因子受体(EGFR)基因罕见突变非小细胞肺癌(NSCLC)患者的临床病理特征及治疗效果。
方法采用荧光定量PCR法检测674例NSCLC中EGFR基因状态, 分析EGFR罕见突变与临床病理特征的相关性。
结果EGFR突变阳性率为47.92%, 其中EGFR罕见突变率为5.19%, 包括ex18 G719 A/S/C (G719X)(1.63%)、ex20ins (1.04%)、ex21 L861Q (0.74%)、含一个以上EGFR突变位点的复合突变(1.78%)。相关性分析显示, EGFR罕见突变更多见于女性、无吸烟史、高-中分化、腺癌患者, 易发生脑、骨转移(均P < 0.05);与常见敏感突变相比, 各临床病理特征比较差异均无统计学意义(均P > 0.05)。35例EGFR罕见突变患者中有31例获得随访资料, 中位随访时间10个月, 其中23例为晚期患者, 8例G719X突变晚期患者中, 7例一线使用EGFR-酪氨酸激酶抑制剂(EGFR-TKIs)(其中5例为阿法替尼), 中位PFS为12个月, 1例采用培美曲塞加卡铂化疗方案, PFS为7个月, 低于TKI组; 4例L861Q晚期患者中1例未治疗, 其余3例一线使用TKI, 中位PFS为8个月, 其中使用阿法替尼及贝伐珠单抗靶向治疗的患者, 随访11个月疾病仍稳定; 2例EGFR ex20ins晚期患者行化疗及贝伐珠单抗治疗; 9例复合突变晚期患者使用TKI治疗, 其中5例含T790M的复合突变患者使用三代TKI, 中位PFS大于10个月。
结论EGFR各罕见突变类型与临床病理特征的相关性不尽相同, 晚期EGFR罕见突变(除EGFR ex20ins外)患者, 临床一线治疗一般选择TKI, 其中具有G719X、L861Q突变的晚期NSCLC患者, 推荐使用阿法替尼; 含T790M的复合突变患者三代TKI有显著疗效。
Abstract:ObjectiveTo investigate the clinicopathological characteristics and treatment effect of patients with non-small cell lung cancer (NSCLC) and uncommon epidermal growth factor receptor (EGFR) gene mutations.
MethodsReal-time fluorescence quantitative PCR was used to detect the mutation of EGFR in 674 samples of patients with NSCLC.The correlation between uncommon EGFR mutations and clinicopathological characteristics was analyzed.
ResultsThe EGFR mutation rate was 47.92%, of which the incidence of uncommon EGFR mutations was 5.19%, showed the presence of ex18 G719 A/S/C (G719X)(1.63%), ex20ins (1.04%), ex21 L861Q (0.74%), and compound mutations (1.78%).Correlation analysis showed that uncommon EGFR mutations were more common in women, non-smokers, patients with high-medium differentiation and adenocarcinoma, and patients were more prone to brain and bone metastasis (all P < 0.05).NSCLC with uncommon EGFR mutations showed no significant differences in clinical and pathological features compared with those with common sensitive mutations (all P > 0.05).Follow-up information was available on 31 patients, with a median follow-up time of 10 months, of which 23 were in advanced stage.Among eight patients with G719X mutation in late stage, seven patients used EGFR tyrosine kinase inhibitor (EGFR TKIs)(five of them used afatinib) in the first line and had a median PFS of 12 months; one patient received chemotherapy with pemetrexed and carboplatin and had PFS of seven months, which was lower than that of the TKI group.Among four patients with L861Q mutation in late stage, one patient was untreated and the three remaining were treated with TKI in the first line and had a median PFS of eight months.The patient who was treated with afatinib and bevacizumab was still stable after 11 months of follow-up.Two patients with EGFR ex20ins in advanced stage were treated with chemotherapy and bevacizumab.Nine patients with compound mutations in advanced stage were treated with TKI; among which, five patients harboring T790M compound mutations were treated with third-generation TKI and had a median PFS of more than 10 months.
ConclusionThe correlation between specific uncommon EGFR mutation and clinical pathological characteristics varies.For advanced patients with uncommon EGFR mutations (except for ex20ins), TKI is generally chosen as the first-line clinical treatment.Afatinib is recommended for advanced NSCLC patients with G719X and L861Q mutations.Third-generation TKI has significant efficacy in patients with complex mutations containing T790M.
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0 引言
多发性骨髓瘤(multiple myeloma, MM)是继非霍奇金淋巴瘤后第二常见的血液浆细胞恶性肿瘤,其发生发展与免疫功能密切相关[1]。基于疾病异质性及耐药性,尽管新型免疫疗法如蛋白酶体抑制剂、免疫调节剂、CAR-T细胞治疗以及CD38单克隆抗体Daratumumab等疗法延长了患者生存期,目前MM仍无法治愈,深入探索MM免疫系统特征将有助于新型免疫治疗的发展。
程序性死亡受体-1(programmed death-1, PD-1)/程序性死亡配体-1/2(programmed death-Ligand 1/2, PD-L1/2)通路是目前抗肿瘤新免疫疗法研究热点之一。作为负性共刺激分子,PD-1主要表达于激活和(或)衰竭的T细胞、B细胞、NK和抗原提呈细胞[2],PD-L1在各种实体瘤及免疫细胞亚群表达[3],PD-L2主要在激活的树突细胞、巨噬细胞和肥大细胞表达[4]。在MM骨髓微环境中PD-1与PD-L1/2结合可导致肿瘤细胞免疫逃逸、迁移和增殖[1-2, 5]。免疫检查点抑制剂治疗黑色素瘤等实体瘤的临床试验已获得了良好的反应,其在血液系统疾病的研究较晚,近期在晚期MM患者的应用取得了一定的临床疗效,免疫检查点抑制剂作为治疗MM的新手段逐渐被人们认识。
目前各研究小组对MM患者免疫细胞及肿瘤细胞PD-1、PD-L1及PD-L2的表达水平及临床特征的研究尚无统一定论。本研究通过观察MM患者骨髓CD4+和CD8+T细胞及骨髓瘤细胞中PD-1、PD-L1/2的表达水平,分析3种负性共刺激分子表达特点与临床特征的关系,以期为免疫检查点抑制剂治疗MM提供一定的试验依据及方向。
1 资料与方法
1.1 一般资料
选取2019年6月—2020年12月中国人民解放军联勤保障部队第九四〇医院收治的22例初诊多发性骨髓瘤(newly diagnosed multiple myeloma, NDMM)患者及同期18例健康对照者临床资料;符合《中国多发性骨髓瘤诊治指南》(2017年)[6]诊断标准的患者为NDMM组;排除临床资料不完整的MM患者、使用Daratumumab单抗化疗的MM患者、冒烟型骨髓瘤患者和孤立性浆细胞瘤患者;排除孕期或哺乳期、合并有自身免疫性疾病、其他肿瘤及传染性疾病的患者。本研究已通过中国人民解放军联勤保障部队第九四〇医院伦理委员会批准。
1.2 研究方法
收集NDMM和对照组骨髓标本,利用流式细胞术分选有核细胞中CD4+、CD8+T细胞及骨髓瘤细胞,检测细胞中PD-1、PD-L1/2的表达水平,并用统计学方法分析患者PD-1、PD-L1/2的表达量与临床检验数据的相关性。患者的一般临床数据包括性别、年龄、免疫分型、Durie-Salmon(DS)分期亚型、国际分期体系(ISS分期)和mSMART 3.0危险分层。
1.3 主要试剂及仪器
所用荧光单克隆抗体:FITC标记的CD38单抗、APC标记的CD138单抗、PE标记的CD273(PD-L2)单抗、CD274(PD-L1)单抗、CD279单抗(PD-1)均购自美国Biolegend公司;所用荧光单克隆抗体:FITC标记的CD4单抗、PerCP-cy5.5标记的CD8单抗均购自美国Bioscience或BioLegend公司;流式细胞仪为德国美天旎生物技术有限公司的MACSQuantifyTM。
1.4 标本采集与处理
收集MM患者及对照组骨髓标本1 ml,加入乙二胺四乙酸(EDTA)抗凝管,置于4℃冰箱保存备用。流式细胞术检测T细胞亚群和骨髓瘤细胞中PD-1、PD-L1及PD-L2的表达水平。
将1 ml骨髓加入缓冲液离心洗涤后弃上清液;取已加入CD4和CD8单抗的3支流式管中和已加入CD38和CD138单抗的3支流式管,两组分别加入PD-1、PD-L1及PD-L2单抗;在6支流式管中加入制备好的骨髓100 μl并混匀,避光孵育10 min;溶血、洗涤及离心后加入500 μl缓冲液上机检测。采用FSC/SSC双参数设门,获取1×104个以上有核细胞进行检测,分析CD4+、CD8+T细胞、骨髓瘤细胞占有核细胞比例及其PD-1、PD-L1与PD-L2的表达水平。
1.5 统计学方法
采用SPSS26.0与GraphPad Prism 8.0统计软件进行统计分析。计量资料若呈正态分布以(x±s)表示,若呈偏态分布以M(P25-P75)表示;计量资料组间对比若符合正态分布且方差齐则采用独立样本t检验,若方差不齐则采用近似t检验;计量资料组间对比若不符合正态分布则采用Mann-Whitney U检验;P < 0.05为差异有统计学意义。
2 结果
2.1 临床特征
22例NDMM患者中,男12例(54.55%),女10例(45.45%),平均年龄(62±11)岁,其中≥65岁患者10例(45.45%), < 65岁12例(54.55%)。免疫分型:IgG型10例(45.45%)、IgA型4例(18.18%)、κ型4例(18.18%)、λ型4例(18.19%)。DS分期:Ⅰ期2例(9.10%)、Ⅱ期2例(9.10%)、Ⅲ期18例(81.80%);DS亚型:A型9例(40.91%)、B型13例(59.09%)。ISS分期:Ⅰ期3例(13.63%)、Ⅱ期7例(31.82%)、Ⅲ期12例(54.55%)。根据美国梅奥医院(Mayo Clinic)2018年发布的多发性骨髓瘤mSMART 3.0危险分层分为标危组15例(68.18%)、高危组7例(31.82%)。
2.2 NDMM和对照组CD4+、CD8+T细胞比例及CD4+/CD8+T细胞比值
NDMM组CD8+T细胞水平较对照组明显升高,差异有统计学意义(P=0.004);NDMM组CD4+/CD8+T细胞比值较对照组明显降低,差异有统计学意义(P=0.005);NDMM组CD4+T细胞水平与对照组比较,差异无统计学意义(P=0.572),见表 1。
表 1 NDMM和对照组T细胞亚群水平及CD4+/CD8+T细胞比值水平Table 1 Proportion of T cell subsets and CD4+/CD8+ ratio in NDMM and control groups2.3 NDMM和对照组CD4+、CD8+T细胞中PD-1、PD-L1和PD-L2的表达
NDMM组CD4+T细胞中PD-1和PD-L2的表达水平较对照组明显升高,差异有统计学意义(P=0.001;P=0.001);NDMM组中CD4+T细胞PD-L1的表达水平与对照组相比差异无统计学意义(P=0.326);NDMM组中CD8+T细胞PD-1、PD-L1及PD-L2的表达与对照组间相比差异无统计学意义(P=0.423;P=0.211;P=0.348),见图 1、表 2。
图 1 流式细胞术检测T细胞亚群和骨髓瘤细胞中PD-1、PD-L1及PD-L2的表达Figure 1 Expression of PD-1, PD-L1 and PD-L2 in T cell subsets and myeloma cells detected by flow cytometryA: P2 was defined as nucleated cells which circled by FSC and SSC, and CD4+T lymphocytes and CD8+T lymphocytes were selected by P2; B: P2 was defined as nucleated cells which circled by FSC and SSC, and CD38+CD138+ myeloma cells were selected by P2; C: The expression of PD-1, PD-L1 and PD-L2.表 2 NDMM与对照组CD4+T和CD8+T细胞中PD-1、PD-L1和PD-L2的表达Table 2 Expression of PD-1, PD-L1 and PD-L2 in CD4+T and CD8+T cells in NDMM and control groups2.4 NDMM骨髓瘤细胞中PD-1、PD-L1和PD-L2的表达
NDMM组骨髓瘤细胞中PD-1的表达水平为(37.28±20.97)%,PD-L1的表达水平为(95.33±6.56)%,PD-L2的表达水平为44.7(33.20~48.12)%。
2.5 CD4+T、CD8+T细胞和骨髓瘤细胞中PD-1、PD-L1及PD-L2表达与NDMM患者临床特征的关系
PD-1、PD-L1及PD-L2水平与NDMM患者性别、年龄、免疫分型、DS分期和亚型、ISS分期及mSMART 3.0分层差异无统计学意义(均P > 0.05),见表 3~5。
表 3 CD4+T细胞中PD-1、PD-L1及PD-L2的表达与NDMM患者临床特征的关系Table 3 Correlation of PD-1, PD-L1 and PD-L2 expression in CD4+T cells with clinical features of NDMM patients表 4 CD8+T细胞中PD-1、PD-L1及PD-L2的表达与NDMM患者临床特征的关系Table 4 Correlation of PD-1, PD-L1 and PD-L2 expression in CD8+T cells with clinical features of NDMM patients表 5 骨髓瘤细胞中PD-1、PD-L1及PD-L2的表达与NDMM患者临床特征的关系Table 5 Correlation of PD-1, PD-L1 and PD-L2 expression in myeloma cells with clinical features of NDMM patients3 讨论
MM的发生发展与机体免疫功能密切相关。当骨髓瘤细胞特异性抗原被淋巴细胞识别并攻击时,一方面活化T细胞使其表达PD-1并产生γ链细胞因子,与白介素-21及干扰素-γ共同诱导骨髓瘤细胞及免疫细胞表达PD-L1,另一方面活化B细胞与树突细胞使其分泌粒细胞-巨噬细胞集落刺激因子(granulocyte macrophage colony-stimulating factor, GM-CSF),与白介素-4诱导PD-L2的表达[7-8],PD-1与PD-L1/2的结合使T细胞衰老并功能衰竭,导致骨髓瘤细胞迁移和增殖[1-2]。这种抑制并未破坏T细胞的功能,阻断PD-1/PD-L1/2通路可激活和恢复T细胞功能并对抗肿瘤进展[1]。
MM多处于免疫功能失调状态[9]。目前有关MM免疫功能的研究有限且说法不一,但几乎所有结果都提示存在CD4+/CD8+比值失调[9-11]。国内外大多数研究致力于MM外周血水平的免疫功能,多表现为CD4+T细胞减少[12],部分表现为CD8+T细胞升高[12-13];骨髓水平免疫亚群的研究相对较少,国内有关MM骨髓免疫状态的研究非常有限,相当部分研究小组认为主要为CD4+T细胞减少[10-12];也有人观察到以CD8+T细胞升高为主[14]。本研究观察到NDMM骨髓CD4+/CD8+T细胞比值降低,分析细胞亚群发现表现为CD8+T细胞增多,CD4+T细胞无差异。前者可能与骨髓CD8+T细胞抑制造血干细胞动员的功能减退以及CD8+T细胞在肿瘤细胞抗原作用下活化形成发挥特异性抗肿瘤免疫应答的细胞毒性T淋巴细胞(cytotoxic lymphocyte, CTL)有关,但这种活化的CTL无法分泌穿孔素、白介素-2和干扰素-γ等细胞因子发挥细胞毒作用,多表现为衰老及功能受限[13];CD4+T细胞无差异可能与肿瘤微环境中对其免疫抑制作用相对较小有关。
PD-1/PD-L1/2通路在MM免疫功能失调中发挥重要作用抑。首先,本研究观察到T淋巴细胞PD-1的表达水平表现为CD4+T细胞上PD-1的高表达(PD-1highCD4+T),CD8+T细胞PD-1表达与对照组无差异,当PD-1highCD4+T细胞与骨髓瘤细胞表达的免疫检查点分子配体结合会抑制CD4+T细胞功能;其次,本研究还观察到骨髓瘤细胞高表达PD-L1(PD-L1highTumer),CD4+与CD8+T细胞PD-L1表达与对照组差异虽无统计学意义,但从现有数据来看有升高趋势,且不论在骨髓瘤细胞还是T淋巴细胞PD-L1表达率多高于84.00%,当PD-L1highTumer细胞分别与PD-1highCD4+T细胞以及增多的CD8+T细胞相互作用也会抑制CD4+与CD8+T细胞功能;随后,本研究又观察了PD-L2在T淋巴细胞和骨髓瘤细胞的表达,发现CD4+T细胞PD-L2的表达升高(PD-L2highCD4+T),CD8+T细胞表达较CD4+T细胞少约10%,PD-L2highCD4+T细胞与PD-L1highTumer细胞相互作用抑制CD4+T细胞功能;以上细胞之间负性共刺激分子的相互作用使T淋巴细胞(尤其是CD4+T细胞)功能失调、功能缺陷甚至失活,最终使骨髓瘤细胞发生免疫逃逸,促进MM耐药及进展。
本研究进一步研究了MM患者机体免疫功能缺陷与临床特征的关系。通过分别对CD4+、CD8+T细胞及骨髓瘤细胞PD-1、PD-L1和PD-L2的表达与性别、年龄、DS分期及亚型、免疫表型、ISS分期及mSMART 3.0危险分层进行统计学分析,发现以上差异均无统计学意义,与Crescenzi等结果一致[15-16]。有研究认为PD-L1水平与个体免疫系统的状况相关[16]。现有的研究无法解释这种现象,考虑到本研究涉及的临床特征较少,期待后期大样本、多中心以及多种临床特征的研究能给我们带来答案。
综上,MM患者多伴有免疫功能失调,可能与表达PD-1及PD-L1/2的T淋巴细胞和肿瘤细胞之间的相互作用有关,阻断此通路或可对抗骨髓瘤细胞增殖潜力及耐药性。期待进一步的研究可以具体阐明T细胞亚群及功能(包括增殖和细胞毒性),提供细胞免疫抑制的整体概况,也希望后续基于MM治疗方案的多中心大样本的PD-1/PD-L1/2通路研究能给我们带来新思路。
Competing interests: The authors declare that they have no competing interests.利益冲突声明:所有作者均声明不存在利益冲突。作者贡献:孙文佳:数据收集及分析、论文撰写岳君秋:文章撰写指导王满香:文章审阅及修改 -
表 1 EGFR罕见突变与EGFR野生型、EGFR经典突变NSCLC患者临床病理特征比较
Table 1 Clinicopathological features comparation of patients with NSCLC and uncommon EGFR mutations, wildtype EGFR or common EGFR mutation
表 2 各EGFR罕见突变与EGFR野生型NSCLC患者临床病理特征比较
Table 2 Clinicopathological features comparison of patients with NSCLC and uncommon EGFR mutations or wildtype EGFR
表 3 23例晚期EGFR罕见突变NSCLC患者治疗及随访
Table 3 Treatment and follow-up of 23 patients with advanced NSCLC and uncommon EGFR mutations
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