Background: In China, lung cancer remains the cancer with the highest incidence and mortality rate. Among early-stage lung adenocarcinomas (LUAD), the micropapillary (MPP) component is prevalent and typically exhibits high aggressiveness, significantly correlating with early metastasis, lymphatic infiltration, and reduced five-year survival rates. Therefore, the study is to explore the similarities and differences between MPP and non-micropapillary (non-MPP) components in malignant pulmonary nodules characterized by GGOs in early-stage LUAD, identify unique mutational features of the MPP component and analyze the relationship between the ZNF469 gene, a member of the zinc-finger protein family, and the prognosis of early-stage LUAD, as well as its correlation with immune infiltration.
Methods: A total of 31 malignant pulmonary nodules of LUAD were collected and dissected into paired MPP and non-MPP components using microdissection. Whole-exome sequencing (WES) was performed on the components of early-stage malignant pulmonary nodules. Mutational signatures analysis was conducted using R packages such as maftools, Nonnegative Matrix Factorization (NMF), and Sigminer to unveil the genomic mutational characteristics unique to MPP components in invasive LUAD compared to other tumor tissues. Furthermore, we explored the expression of the ZNF469 gene in LUAD using The Cancer Genome Atlas (TCGA) database to investigate its potential association with the prognosis. We also investigated gene interaction networks and signaling pathways related to ZNF469 in LUAD using the GeneMANIA database and conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Lastly, we analyzed the correlation between ZNF469 gene expression and levels of immune cell infiltration in LUAD using the TIMER and TISIDB databases.
Results: MPP components exhibited a higher number of genomic variations, particularly the 13th COSMIC (Catalogue of Somatic Mutations in Cancer) mutational signature characterized by the activity of the cytidine deaminase APOBEC family, which was unique to MPP components compared to non-MPP components in tumor tissues. This suggests the potential involvement of APOBEC in the progression of MPP components in early-stage LUAD. Additionally, MPP samples with high similarity to APOBEC signature displayed a higher tumor mutational burden (TMB), indicating that these patients may be more likely to benefit from immunotherapy. The expression of ZNF469 was significantly upregulated in LUAD compared to normal tissue, and was associated with poor prognosis in LUAD patients (P<0.05). Gene interaction network analysis and GO/KEGG enrichment analysis revealed that COL6A1, COL1A1, COL1A2, TGFB2, MMP2, COL8A2 and C2CD4C interacted with ZNF469 and were mainly involved in encoding collagen proteins and participating in the constitution of extracellular matrix. ZNF469 expression was positively correlated with immune cell infiltration in LUAD (P<0.05).
Conclusions: The study has unveiled distinctive mutational signatures in the MPP components of early-stage invasive LUAD in the Asian population. Furthermore, we have identified that the elevated expression of mutated ZNF469 impacts the prognosis and immune infiltration in LUAD, suggesting its potential as a diagnostic and prognostic biomarker in LUAD.
【中文题目：“磨玻璃影”特征的早期肺腺癌中MPP成分的突变特征分析及ZNF469基因的探索】 【中文摘要：背景与目的 目前，肺癌依然是我国发病率和死亡率最高的恶性肿瘤。而在早期肺腺癌（lung adenocarcinoma, LUAD）中，微乳头（micropapillary, MPP）成分尤其常见，且通常表现出高侵袭性，其与早期转移、淋巴浸润的风险以及患者的5年生存率显著相关。本研究旨在探究以磨玻璃影（ground-glass opacities, GGOs）为特征的早期 LUAD中MPP成分和非MPP成分的异同，寻找MPP成分所特有的突变特征，并分析锌指蛋白家族的ZNF469基因与早期LUAD预后以及免疫浸润的关系。方法 收集31例LUAD恶性肺结节，采用显微解剖法将其分为成对的MPP和非MPP成分。对早期恶性肺结节组分进行全外显子组测序（whole-exome sequencing, WES），利用maftools、非负矩阵分解（Nonnegative Matrix Factorization, NMF）法、Sigminer等方法进行突变特征分析，以揭示侵袭性LUAD中MPP组分相比于其他肿瘤组织所特有的基因组突变特征。利用癌症基因组图谱（The Cancer Genome Atlas, TCGA）的LUAD数据库中ZNF469的表达情况，探讨其与肺癌预后的关系；利用GeneMANIA数据库以及基因本体（Gene Ontology, GO）、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析探索LUAD中与ZNF469相关基因的互作网络及信号通路；利用TIMER和TISIDB数据库分析ZNF469表达与LUAD中免疫细胞浸润水平的相关性。结果 MPP成分具有较多的基因组变异，相比于非MPP成分的肿瘤组织，癌症体细胞突变目录（Catalogue of Somatic Mutations in Cancer, COSMIC）的13号突变特征（胞苷脱氨酶家族，APOBEC）是MPP成分所特有的，这提示其参与了MPP成分对LUAD早期侵袭过程的促进作用；并且APOBEC特征高的MPP样本具有更高的肿瘤突变负荷（tumor mutational burden, TMB），提示这些患者更能从免疫治疗中获益。LUAD中突变ZNF469的表达高于正常组织，与LUAD患者的不良预后有关。基因互作网络分析以及GO和KEGG富集分析发现，COL6A1、COL1A1、COL1A2、TGFB2、MMP2、COL8A2、C2CD4C等与ZNF469具有相互作用，且主要与编码胶原蛋白、参与细胞外基质构成有关。ZNF469表达与肿瘤的免疫浸润呈正相关。结论 本研究揭示了中国人群侵袭性LUAD中MPP成分的特有突变特征，并发现突变ZNF469的高表达影响LUAD预后与免疫浸润，推测ZNF469可作为LUAD潜在的诊断及预后生物标志物。 】 【中文关键词：肺肿瘤；磨玻璃影；微乳头成分；突变特征分析；ZNF469基因】.
Keywords: Ground-glass opacities; Lung neoplasms; Micropapillary; Mutational signatures analysis; ZNF469 gene.