[Correlation between Immune Microenvironment Features and EGFR Mutation Status 
in Lung Adenocarcinoma].

The morbidity and mortality rates of lung cancer remain high worldwide, and lung adenocarcinoma is one of the most important tissue subtypes of lung cancer. Epidermal growth factor receptor (EGFR) mutation is an important driver gene mutation for lung adenocarcinoma. In recent years, immune checkpoint inhibitors (ICIs), such as programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) inhibitors, have achieved remarkable efficacy in some lung cancer patients. Patients with EGFR mutations enjoyed limited benefits from immunotherapy according to recent studies. This study aimed to explore the relationship between EGFR mutation status and the spatial distribution as well as infiltration number of various immune cells in patients with EGFR mutant lung adenocarcinoma.This study included 62 lung adenocarcinoma patients who underwent surgery. Through multi-point sampling of surgically removed tumor tissues in different areas, 223 tumor tissue samples were finally obtained. We aquired EGFR mutations status including variant allele frequency (VAF) and mutation subtype in each tumor tissue by genetic test. Afterwards, hematoxylin-eosin (HE) staining, immunohistochemical staining and multiplex fluorescence immunohistochemistry staining have been performed, therefore the infiltration of various immune cells and the distribution of tertiary lymphoid structure (TLS) in tumor tissues were obtained by calculating the immunohistochemical score.Compared with EGFR wild-type patients, patients with EGFR-mutant lung adenocarcinoma had more infiltration of CD68+ macrophages and major histocompatibility complex (MHC) class II antigen-presenting cells and higher spatial distribution heterogeneity of MHC class II antigen presenting cells in tumor tissues, while CD56+ natural killer cells and CD8+ T cells had lower infiltration. Tumor tissues with higher EGFR VAF were associated with lower cell infiltration such as CD3+ T cells, CD20+ B cells, CD56+ natural killer cells, CD68+ macrophages, CD8+ T cells, and only CD3+ T cells showed a lower spatial distribution heterogeneity. For the two common subtypes of EGFR mutations in Chinese population, tumor tissues with EGFR exon 19 deletion mutations have lower immune cell infiltration but higher spatial distribution heterogeneity of CD3+ T cells, CD56+ natural killer cells, CD68+ macrophages, and CD8+ T cells than that in EGFR exon 21 L858R mutant tumor tissues. Prognostic analysis found that patients with EGFR mutations with high degree of CD3+ T cells, CD20+ B cell infiltration and larger numbers of TLS formation and high spatial distribution heterogeneity of CD8+ T cell had longer disease-free survival.EGFR-mutated lung adenocarcinoma had a unique "non-inflammatory" tumor microenvironment with low infiltration of immune cells, and there was also heterogeneity in the tumor microenvironment among the tumors with different mutation subtypes and mutation abundance. These differences were not only reflected in the number but also the spatial distribution of immune cell infiltration. Hence, further studies on the immune microenvironment of EGFR-mutant lung adenocarcinoma were of great significance for improving the efficacy of immunotherapy in EGFR-mutant lung adenocarcinoma patients in the future.【中文题目：肺腺癌中免疫微环境特征与 EGFR突变状态的相关性研究】 【中文摘要：背景与目的 在全球范围内肺癌发病率和死亡率一直居高不下，而肺腺癌是其中最主要的一个组织亚型，表皮生长因子受体（epidermal growth factor receptor, EGFR）突变是肺腺癌的一个重要驱动基因突变。近年来以程序性死亡受体1（programmed cell death 1, PD-1）及程序性死亡配体1（programmed cell death ligand 1, PD-L1）抑制剂为代表的免疫检查点抑制剂（immune checkpoint inhibitors, ICIs）治疗在部分肺癌患者中疗效显著。有研究表明EGFR突变的患者对免疫治疗的获益程度有限，本研究旨在讨论肺腺癌患者中EGFR突变状态与各类免疫细胞浸润数量、空间分布的关系。方法 本研究纳入62例接受手术的肺腺癌患者。通过对手术切除的肿瘤组织进行不同区域多点采样，最终获取了223份肿瘤组织样本。通过基因检测获取了每份肿瘤组织的EGFR突变情况，包括突变丰度（variant allele frequency, VAF）以及突变亚型等信息。对肿瘤组织进行苏木精-伊红（hematoxylin-eosin, HE）染色、免疫组化染色、多重荧光免疫组化染色，并通过计算免疫组化评分获取各类免疫细胞在肿瘤组织中的浸润情况以及三级淋巴结构（tertiary lymphoid structure, TLS）的分布情况。结果 与野生型患者相比，EGFR突变肺腺癌患者肿瘤组织中CD68+巨噬细胞以及组织相容性复合物（major histocompatibility complex, MHC）II类抗原呈递细胞浸润更多，且MHC II类抗原呈递细胞空间分布的异质性较大，而CD56+自然杀伤细胞以及CD8+ T细胞等浸润更低。EGFR VAF高的肿瘤组织与更低的CD3+ T细胞、CD20+ B细胞、CD56+自然杀伤细胞、CD68+巨噬细胞、CD8+ T细胞等细胞浸润相关，其中只有CD3+ T细胞分布的空间异质性较小。对于中国人群常见的两种EGFR突变亚型，EGFR外显子19缺失突变相较于EGFR外显子21 L858R突变肿瘤组织中有更低的免疫细胞浸润，但CD3+ T细胞、CD56+自然杀伤细胞、CD68+巨噬细胞以及CD8+ T细胞等免疫细胞分布的空间异质性更高。预后分析发现，CD3+ T细胞、CD20+ B细胞浸润程度高、TLS形成数量多以及CD8+ T细胞高分布异质性的EGFR突变患者拥有更长的无病生存期。结论 EGFR突变肺腺癌具有独特的低免疫细胞浸润的“非炎性”肿瘤微环境，不同突变亚型以及VAF的肿瘤微环境之间也表现出异质性，这些差异不仅体现在免疫细胞的浸润数量上，也体现在免疫细胞的空间分布情况。因此对EGFR突变肺腺癌的免疫微环境进行进一步更深入的研究对未来提高EGFR突变肺腺癌患者的免疫治疗疗效具有重大意义。 】 【中文关键词：肺肿瘤；EGFR突变；免疫微环境；肿瘤异质性；免疫治疗】.