Tumor Evolution Trajectories And The Analysis On Immune Microenvironment In Early Stage Lung Adenocarcinoma

Background:Lung cancer is one of the most common malignancies.According to International Agency for Research on Cancer(IARC)and World Health Organization(WHO),the incidence and mortality of lung cancer ranked the second and the first place across worldwide in 2020,respectively.Lung adenocarcinoma(LUAD)is the major histopathological subtype,accounting for almost 60%of all lung cancer cases.In relevant studies,these lesions are also named malignant pulmonary nodules(MPNs).Tumor invasive status has a notable impact on prognosis for LUAD,especially in early-stage cases.Therefore,it is of great significance to explore the evolutionary process from pre-invasive to invasive components.By analyzing the phylogenetic relationship between pre-invasive and adjacent invasive components,we could understand the progression of early stage LUAD and improve the clinical prognosis of LUAD cases.Aims:To investigate the evolutionary trajectories during the invasiveness of early stage LUAD,we performed phylogenetic analysis and constructed the evolutionary routes between pre-invasive and adjacent invasive components,which aims to demonstrate the critical molecular events during the invasive progression and reveal the association between immune microenvironment and tumor evolution.We analyzed phylogenetics,driver mutations,and immune microenvironment to indicate the tumor progression and clinical prognosis of early stage LUAD.Methods:This study included four datasets.Patients enrolled in this study belonged to a cohort study(Chi CTR1900022521)from Jiangsu Cancer Hospital(JSCH).All included 53 cases were pathologically confirmed as early stage LUAD(50 T₁N₀M₀and3 T₁N₁M₀ stage cases).We performed genomic sequencing on 69 MPNs,5 metastatic lymph nodes,41 prior-operation,and 38 post-operation cf DNA(Circulating cell-free DNA)samples.A total of 61 MPNs were micro-dissected to separate pre-invasive and invasive components,and we finally got 113 MPN components,which including 52paired pre-invasive and invasive components.In the phase 1 study,80 specimens of the first 18 cases were subjected to wide panel-genomic sequencing(1021-gene panel)at the coverage depth of 1800×.In the phase 2 study,125 specimens of 35 cases were subjected to hotspot panel-genomic sequencing(425-gene panel)at the coverage depth of 1500×.In addition,we included the gene expression,somatic mutations and clinical prognosis data of 510 LUAD cases from the TCGA(The Cancer Genome Atlas)cohort,and we integrated the clinical and the Snapshot mutation data of 496 T₁N₀M₀stage LUAD patients from the BLCS(Boston Lung Cancer Study)cohort.Finally,we included Sc RNA-Seq(Single-cell RNA sequencing)data of 29 MPNs and WES(Whole exome sequencing)data from 27 of them.We showed the evolutionary trajectories among 52 paired pre-invasive and invasive components,identified critical molecular events,revealed the relationship between clinical prognosis and driver events in the BLCS cohort,and analyzed the association between driver events and immune microenvironment in both the TCGA and Sc RNA-Seq datasets.Results:This study demonstrated three evolution modes(EMs)among 52 paired pre-invasive and invasive MPN components.EM1(n=5)suggested none of the common driver events between paired components,but another two modes,EM2A(n=26)and EM2B(n=19),exhibited critical private alterations restricted to pre-invasive and invasive components,respectively.These results suggested that EM1 was similar to parallel evolution,which indicated the poor genomic correlation between the two MPN components.Truncal mutations of EM2 harbored driver events(i.e.,EGFR,TP53,and KRAS mutations),but significant different phylogenetic trees were also observed among MPNs in EM2,which indicated branch and linear evolutions of EM2A and EM2B,respectively.These results suggested the invasive progression of the stepwise process from pre-invasive to invasive components in EM2B.Furthermore,we performed analysis on critical truncal mutations in MPNs(EM2),and we found that these trunks harbored most of driver genes.Among all recurrent mutated driver genes,EGFR was found to be mutually exclusive from KRAS and STK11.The survival analysis indicated a better prognosis of EGFR-mutated patients than KRAS/STK11-mutated patients in the BLCS cohort.Analyses on variant allele frequency and d N/d S ratio both suggested that EGFR mutations were associated with high selective pressure in early stage LUAD,which may benefit the clinical prognosis.To investigate the potential origins of high selective pressure in EGFR-mutated LUAD,we analyzed the relationship between EGFR mutations and immune cell infiltration in the TCGA data,which indicated the intratumoral accumulation of B cells in EGFR-mutated MPNs.We further performed immunohistochemistry(IHC)assays on tissue samples to validate the microenvironmental B cell infiltration in the invasive components,which indicated that the infiltrated B cells correlated with the increase of selective pressure.In addition,we performed Sc RNA-Seq on 29 MPNs and conducted WES in 27 of them.These analyses identified the expression profile of a total of 70,896 cells,which including2,653 B cells.The results suggested that the accumulation of plasma B cells were associated the clonal EGFR mutations.Conclusions:Based on the micro-dissection and high-throughput sequencing,we identified three evolutionary trajectories during the acquisition of invasiveness in early stage LUAD.According to the phylogenetic analysis,EGFR mutations were mostly frequent truncal mutations,which was critical to selective pressure during tumor evolution.The analyses of immune microenvironment showed that intratumoral accumulation of infiltrated B cells,especially plasma cells,were associated with the increase of selective pressure.This study focused on the progression from pre-invasive to invasive components and investigated the genomic evolution during the invasiveness acquisition in early LUAD.Our findings extended the understanding of prognosis related evolutionary process and the relationship between driver mutations and immune microenvironment,which provided additional insights into the mechanisms of carcinogenesis and tumor progression.