第一部分 阿法替尼治疗非小细胞肺癌非常见基因突变疗效的研究

目的：表皮生长因子受体（epidermal growth factor receptor，EGFR）酪氨酸激酶抑制剂（tyrosine kinase inhibitors，TKIs）可改善EGFR突变型肺癌的预后；然而TKIs对非常见EGFR突变在真实世界的临床应用价值尚需更多证据支持，尤其是对于部分特定罕见突变类型的患者的疗效仍需进一步探索。方法：本研究为基于电子病历的回顾性研究，研究收集了在2017年1月至2021年1月期间接受阿法替尼治疗并携带EGFR非经典突变的IIIb/IV期肺腺癌（lung adenocarcinoma，LUAD）患者的疗效数据及耐药前后的分子检测结果。结果：共有42例经阿法替尼治疗的EGFR非常见突变患者入选，其中EGFR非常见突变主要包括G719X，L861Q和S768I，其他类型包括L747P、E709X及R776H等。总体人群的客观缓解率（objective response rate，ORR）为50.0%（20例患者中有10例）。中位治疗失败时间（time to treatment failure，TTF）为11.7个月（95%可信区间 = 8.5-18.3个月）。在42名患者中，Gly719Xaa（G719X）、Ser768Ile（S768I）和Leu861Gln（L861Q）突变患者的中位TTF分别为15.0、11.7和16.6个月。在20例其他类型突变患者中，总的TTF中位数为10.0个月，ORR为50.0%。阿法替尼在一系列特定罕见突变患者中表现出临床活性，包括EGFR L747P、A767_V769dup和L833V/H835L，均有一例患者TTF超过1年。共有16例阿法替尼治疗耐药后活检样本的分子检测结果可及，在一名EGFR L833V/H835L突变患者和一名EGFR S768I/L858R突变患者中检测到继发T790M突变。结论：阿法替尼对具有非常见突变NSCLC患者治疗有效，其耐药机制存在差异仍需要进一步研究。

第二部分 EGFR突变型肺腺癌免疫微环境的分析

目的：临床证据表明表皮生长因子受体（epidermal growth factor receptor，EGFR）突变的非小细胞肺癌（non-small cell lung cancer，NSCLC）患者较难从免疫治疗中获益，而肿瘤免疫微环境（tumor immune microenvironmen，TIME）是影响免疫治疗效果的重要因素。然而目前对于EGFR突变患者，尤其是不同突变亚型的TIME尚不清楚。方法：肺腺癌（lung adenocarcinoma，LUAD）的mRNA数据和突变数据以及临床数据来自癌症基因组图谱（The Cancer Genome Atlas，TCGA）数据库。采用肿瘤突变负荷（tumor mutation burden，TMB）、ESTIMATE、CIBERSORT和微环境细胞群计数器（microenvironment cell populations-counter，MCP-counter）分析EGFR突变患者的免疫景观。结果：共有585例TCGA数据库中的患者纳入分析。在这些患者中，98例具有EGFR突变。EGFR组的TMB（3.94 mut/Mb）低于KRAS突变组（6.09 mut/Mb，P<0.001）和整体肺腺癌组（6.58 mut/Mb，P<0.001）。EGFR组的活化免疫细胞数量较少，免疫抑制细胞得分更高（P<0.05）。进一步的组间TIME比较显示，TMB和肿瘤浸润淋巴细胞的差异仅在驱动突变和未知突变患者之间发现。同时EGFR 19del中的髓样树突状细胞（dendritic cell，DC）比L858R突变患者多，常见突变患者中的DC细胞比非常见突变患者多（P<0.05）。构建D值，D=细胞毒性T淋巴细胞（cytotoxic T lymphocytes，CTL）的MCP-counter评分/髓系DC细胞的MCP-counter评分。进一步分析显示较低的D值提示免疫抑制状态并与更低的免疫检查点相关基因mRNA表达相关。结论：EGFR突变NSCLC的TIME呈免疫抑制状态。在EGFR 19del、L858R和非常见突变中，髓样DC逐渐增加。CTL和DC在免疫微环境中起到重要作用，其潜在价值需要进一步研究。

第三部分 TGFBR2突变作为非小细胞肺癌免疫检查点抑制剂治疗耐药预测因子的研究

目的：肿瘤对免疫检查点抑制剂（immune checkpoint inhibitors，ICIs）治疗的耐药及超进展阻碍了ICIs的临床应用。研究提示转化生长因子-β（transforming growth factor beta，TGF-β）受体途径与免疫功能障碍密切相关，可能与ICIs治疗耐药有关。本研究旨在探讨TGF-β在非小细胞肺癌（non-small cell lung cancer，NSCLC）ICIs治疗耐药中的作用。方法：从POPLAR/ OAK（n = 853）、MSKCC（n = 1662） 及Van Allen（n = 57）三项队列中获取接受ICIs治疗或化疗患者数据，并将基于TCGA（n=3210）队列数据进行分析。结果：与TGF-β受体2（TGF-β receptor，TGFBR2）野生型相比，TGFBR2突变型NSCLC具有更高表达的细胞程序性死亡配体1（programmed cell death ligand 1，PD-L1/CD274），淋巴细胞激活基因3（lymphocyte-activation gene 3，LAG3）、具有Ig和ITIM结构域的T细胞免疫受体（T cell immunoreceptor with Ig and ITIM domains，TIGIT）、细胞毒性T淋巴细胞相关蛋白4（cytotoxic T-lymphocyte-associated protein 4，CTLA-4）、细胞程序性死亡受体1（programmed cell death 1，PDCD1）和细胞程序性死亡1配体2（programmed cell death 1 ligand 2，PDCD1LG2）（P<0.05）。在POPLAR/OAK队列中发现，在接受ICIs治疗组中，TGFBR2突变型患者较TGFBR2野生型患者的无进展生存期（progression-free survival，PFS）【P = 0.004；风险比（hazard ratio，HR），2.83；95%可信区间（confidence interval，CI），1.34–6.00】和总生存期（overall survival，OS）（P = 0.0006；HR，3.46；95% CI，1.63–7.35）更短；而在接受化疗组中无类似发现。在合并的MSKCC和Van Allen队列中，同样发现在接受ICIs的NSCLC中，突变型TGFBR2患者较野生型TGFBR2患者的OS较差（P = 0.007；HR，2.53；95%CI，1.25–5.12）。多因素cox回归提示TGBFR2突变与生存在POPLAR/OAK队列（p = 0.02；HR，2.53；95%CI，1.17–5.45）和合并队列（p = 0.008；HR，2.63；95%CI，1.29–5.35）中均显著相关。我们进一步评估了多种肿瘤中TGFBR2突变与免疫治疗的OS之间的关联，分析发现仅在NSCLC中TGFBR2突变与免疫治疗的OS仍显著相关（P = 0.02；HR，2.47；95%CI，1.16–5.26），但在其他类型的肿瘤中两者则不显著。结论：TGFBR2突变可预测NSCLC对ICIs治疗的耐药性。ICIs在TGFBR2突变患者中的临床应用应谨慎。

Part I Afatinib treatment response in advanced lung adenocarcinomas harboring uncommon mutations

Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have improved the prognosis of EGFR mutant lung cancer; however, the clinical application value of TKIs for nonclassical EGFR mutation is unclear, especially for patients with rare uncommon mutations. Methods: A retrospective study based on electronic medical records was conducted to collect data on the effectiveness of afatinib in patients with stage IIIB/IV lung adenocarcinoma (LUAD) bearing uncommon mutations between January 2017 and January 2021. Results: In total, 42 patients with uncommon EGFR mutations treated with afatinib were included in the analysis. The objective response rate (ORR) was 50.0% (10 of 20 patients). The median time to treatment failure (TTF) was 11.7 months (95% confidence interval = 8.5–18.3 months). Of the 42 patients, the median TTF was 15.0, 11.7, and 16.6 months in patients with Gly719Xaa (G719X), Ser768Ile (S768I), and Leu861Gln (L861Q) mutations, respectively. In patients with the rare uncommon mutation, the median TTF was 10.0 months, and the ORR was 50.0%. Afatinib demonstrated clinical activity across a set type of specific rare uncommon mutations, including EGFR L747P, A767_V769dup, and L833V/H835L, with a case having a TTF of more than 1 year. Molecular profiling reports of 16 afatinib-resistant biopsy samples were available, and the secondary T790M mutation was detected in one patient with L833V/ H835L mutation and one harboring S768I/L858R mutation. Conclusions: Our findings suggested that afatinib was effective in patients with uncommon mutations. Mechanisms of afatinib resistance vary and need further investigation.

Part II Exploration of the Tumor-Suppressive Immune Microenvironment by Integrated Analysis in EGFR-Mutant Lung Adenocarcinoma

Background: Clinical evidence has shown that few non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations can benefit from immunotherapy. The tumor immune microenvironment (TIME) is a significant factor affecting the efficacy of immunotherapy. However, the TIME transformational process in EGFR-mutation patients was unknown. Methods: The mRNA expression and mutation data and lung adenocarcinoma (LUAD) clinical data were obtained from The Cancer Genome Atlas (TCGA) database. Profiles describing the immune landscape of patients with EGFR mutations were characterized by differences in tumor mutation burden (TMB), ESTIMATE, CIBERSORT, and microenvironment cell populations-counter (MCP-counter). Results: In total, the TCGA data for 585 patients were analyzed. Among these patients, 98 had EGFR mutations. The TMB was lower in the EGFR group (3.94 mut/Mb) than in the KRAS mutation group (6.09 mut/Mb, P<0.001) and the entire LUAD (6.58 mut/Mb, P<0.001). The EGFR group had a lower population of activated immune cells and an even higher score of immunosuppressive cells. A further inter-group comparison showed that differences in the TMB and tumor-infiltrating lymphocytes were only found between patients with oncogenic mutations and unknown mutation. Meanwhile, there were more myeloid dendritic cells (DCs) in EGFR 19del than in L858R-mutation patients and in common mutation patents than in uncommon mutation patients (P<0.05). Additionally, we established a D score, where D = MCP-counter score for cytotoxic T lymphocytes (CTLs)/MCP-counter score for myeloid DCs. Further analysis revealed that lower D scores indicated immune suppression and were negatively related to several immunotherapy biomarkers. Conclusions: The TIME of EGFR mutant NSCLC was immunosuppressive. Myeloid DCs gradually increased in EGFR 19del, L858R, and uncommon mutations. The potential role of CTLs and DCs in the TIME of patients requires further investigation.

Part III TGFBR2 mutation predicts resistance to immune checkpoint inhibitors in patients with non-small cell lung cancer

Background: Resistance or even hyper-progression to immune checkpoint inhibitors (ICIs) manifesting as accelerated disease progression or death has impeded the clinical use of ICIs. The transforming growth factor beta (TGF-β) receptor pathway has been identified in contributing to immune dysfunction, which might be associated with resistance to ICIs. We aimed to explore the role of TGF-βin the resistance to ICIs in non-small cell lung cancer (NSCLC) in this study. Methods: Public cohorts with patients treated with ICIs or chemotherapy including POPLAR/ OAK (n = 853), MSKCC (n = 1662) and Van Allen (n = 57) and TCGA (n = 3210) cohorts were obtained and analyzed. Results: The expression of immune-checkpoint related genes, including programmed death-ligand 1 (CD274), lymphocyte-activation gene 3 (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death 1 (PDCD1), and programmed cell death 1 ligand 2 (PDCD1LG2) were significantly upregulated in transforming growth factor beta TGF-β receptor 2 (TGFβR2)-mutated patients than those with wild-type TGFBR2 (p<0.05). In the POPLAR/OAK cohort, TGFBR2-mutated patients showed shorter progression-free survival (PFS) [ P = 0.004; hazard ratio (HR), 2.83; 95% confidence interval (CI), 1.34–6.00] and overall survival (OS) ( P = 0.0006; HR, 3.46; 95% CI, 1.63–7.35) than those with wild-type TGFBR2 when treated with ICIs but not chemotherapy.In the merged MSKCC and Van Allen cohorts, a similar result was observed that the OS was inferior in patients with mutated TGFBR2 compared with those with wild-type TGFBR2 (P = 0.007; HR, 2.53; 95% CI, 1.25–5.12). The association between TGBFR2 mutation and survival remained significant in multivariable cox regression in both POPLAR/OAK cohort (P = 0.02; HR, 2.53; 95% CI, 1.17–5.45) and merged cohort (P = 0.008; HR, 2.63; 95% CI, 1.29–5.35). We further evaluated the association between TGFBR2 mutations and OS of immunotherapy in multiple types of tumors. The association between TGFBR2 mutations and OS remained significant in NSCLC (P = 0.02; HR, 2.47; 95% CI, 1.16–5.26), but not in other type of tumors. Conclusions: We identified that TGFBR2 mutation predicted the resistance to ICIs in NSCLCs. The clinical delivery of ICIs should be cautious in those patients.