Study Of Gene Chip And Immune Repertoire In Rapid Recurrence Early-stage Lung Adenocarcinoma

Background and objective:With the application of low-dose Computed Tomography(LDCT),great progress has been made in the diagnosis of early-stage lung cancer,including early-stage lung adenocarcinoma.More and more patients with early-stage lung adenocarcinoma can now receive surgical treatment,and the 5-year survival rate of these patients can reach 67% after surgical resection.But we have found in clinical practice that a small number of patients(about 15%)relapse rapidly within 1 year after surgery and their 5-year survival rate is only45.1%(well below 67%),which indicates that the prognosis is extremely poor once the disease relapse rapidly.More importantly,we also found that another part of patients did not relapse within 3 years after surgery,and their 5-year survival rate reached an astonishing94.5%.Therefore,we speculate that the early-stage lung adenocarcinoma may be a highly heterogeneous disease that includes different subtypes.If the two types of patients(rapid recurrence and late recurrence)are distinguished in advance,we can intervene as early as possible in patients with high risk of rapid recurrence to improve their clinical outcomes and avoid over-treatment in patients with late recurrence.It may be the most beneficial option for early-stage lung adenocarcinoma patients when there is no breakthrough about adjuvant therapy.There have been many studies on predictors and biomarkers of early-stage lung adenocarcinoma recurrence,including single nucleotide polymorphisms(SNP),DNA methylation,gene panels,protein expression,etc.,but they all cannot be used in clinical practice.And more importantly,the predictors or biomarkers cannot be used to distinguish patients with rapid recurrence.This study aims to find predictors that can distinguish patients with rapid recurrence of early-stage lung adenocarcinoma,and to understand the mechanism of rapid recurrence from a new perspective.Methods:1.Study on the relationship between rapid recurrence and clinicopathological characteristics: a univariate analysis of the clinicopathological features and the relapse free survival(RFS)was performed to obtain the clinicopathological characteristics that affect the patient's RFS.Subsequently,a multivariate analysis of the Cox regression model was performed to obtain the independent predictors of the early-stage lung adenocarcinoma recurrence.Then the patients who relapsed within 1 year(RFS?12 months)and those who did not relapse within 3 years(RFS>36 months)were respectively defined as rapid recurrence and late recurrence patients,and Pearson Chi-square test(Pearson Chi-square test)was used to compare the clinicopathological characteristics of the two groups of patients in order to obtain the statistically different indicators.2.Gene chip study on rapid recurrence early-stage lung adenocarcinoma: the RNA of paraffin-embedded tumor tissue from 20 early-stage lung adenocarcinoma patients(8 cases of rapid recurrence,12 cases of late recurrence)was extracted and then proceeded to gene expression profile chip detection.The gene chip data of each patient was imported into the software Transcriptome Analysis Console(TAC)3.0.0 to obtain the gene clustering heat map.The differentially expressed genes(DEG)of the two groups of patients were obtained through limma software package.GO(Gene Ontology)function enrichment analysis and KEGG(Kyoto Encyclopedia of Genes and Genomes)signal pathway analysis was performed on the DEGs so the significant differential functional enrichment were obtained.The protein-protein interaction(PPI)network analysis of DEGs was performed to obtain the key genes.After increasing the number of patients,immunohistochemistry(IHC)staining was performed on the key immune-related genes IL-1? and PTGS to analyze the effect of their protein expression levels on the RFS of early-stage lung adenocarcinoma.The chi-square test was also performed to compared the difference in protein expression of IL-1? and PTGS between patients with rapid and late recurrence.Finally,IHC staining on the tumor infiltrating lymphocytes(TILs)markers,including CD3,CD4,CD8 and CD20 was performed to compared the differences in the degree of TILs infiltration between the two groups.3.Immune repertoire study on rapid recurrence early-stage lung adenocarcinoma: the paraffin-embedded tumor tissue DNA from 39 patients(20 cases of rapid recurrence,19 cases of late recurrence)was extracted.The multiple PCR amplification technology was used to amplify the complementarity determining area 3(CDR3)of the variable region of the B cell receptor(BCR)heavy chain(IGH)and the T cell receptor(TCR)? chain(TR?),then sequencing of the amplified CDR3 region was performed through the Illumina Hiseq3000 platform.The heat map was used to show each patient's use frequency about each V gene segment(variable gene segment)and J gene segment(joining gene segment).The use frequency of V and J genes was compared between the two groups of patients in order to get the statistically different IGHV,IGHJ,TR?V,TR?J genes.The IGHV/J and TR?V/J gene rearrangement combinations of the two groups of patients was compared to obtained the statistically different combinations.The diversity index Clonality and Evenness of the BCR and TCR in the two groups of patients was calculated to evaluate the difference in clone diversity.The clone frequency of BCR and TCR was divided into 5 grades(rare,low,medium,high,and hyper)and the grades comparison was performed between the two groups to explore whether the clone distribution of each grade was different.4.The effect of APE1(Apurinic/Apyrimidinic Endonuclease 1)on the immune microenvironment of lung adenocarcinoma: the mouse Lewis cells were injected into APE1knock-out(KO)mice and wildtype(WT)mice,the immune cells were separated from the tumor and then labeled with the markers including CD8+T cells,macrophages,dendritic cells,and MDSC.The labeled cells were detected by flow cytometry.Results:1.Study on the relationship between rapid recurrence and clinicopathological characteristics: The results of univariate analysis showed that tumor size and histological type are the factors that affect the recurrence of early-stage lung adenocarcinoma;Cox multivariate analysis further showed that tumor diameters> 2cm and non-lepidic histological types are the independent risk factors that affect early-stage lung adenocarcinoma RFS,the Hazard Ratio(HR)are 2.688(95%CI: 1.211-5.920)and 2.040(95%CI: 1.006-4.135)respectively,P<0.05.The chi-square test results showed the histological type and differentiation degree were significantly different between rapid recurrence and late recurrence patients.The proportion of non-lepidic and poorly differentiation degree patients in the rapid recurrence group was significantly higher than that in the late recurrence group which was 95.5% vs 71.1% and 54.5%vs 23.9% respectively,P<0.05.2.Gene chip study on rapid recurrence early-stage lung adenocarcinoma: The gene clustering heat map showed that patients with rapid recurrence and late recurrence can basically be classified into two different categories at the genetic level.A total of 416 DEGs were obtained by Limma analysis.The DEGs were closely related to immune function through GO enrichment analysis: Among the top 10 functional categories,2 of the top 10categories(2/10)in the domain of cellular component,2 of the top 10(2/10)in the domain of molecular function,and 9 of the top 10(9/10)in the domain of biological process were functionally related to immunity.The results of KEGG analysis showed that 6 of the top 8pathways were functionally involved in immune regulation and inflammatory response.The PPI network analysis identified 10 key genes from the 416 DEGs,some of which were closely related to immunity or inflammation,such as IL-1? and PTGS2.Immunohistochemistry and Kaplan-Meier analysis showed that the early-stage lung adenocarcinoma patients with lower IL-1? expression had a worse RFS(21.5 months vs 55.0 months,P<0.05).Chi-square test further showed the proportion of patients with low IL-1? expression is more in the rapid recurrence group(31.8% vs 8.1%,P<0.05).The results of PTGS2 were similar to IL-1?,that is,patients with lower PTGS2 expression had a worse RFS(19.0 months vs 77.0 months,P<0.05),and the proportion of patients with low PTGS2 expression is more in the rapid recurrence group(68.2% vs 10.8%,P<0.05).The IHC results showed that the proportion of patients with low TILs infiltration was higher in the rapid recurrence group: CD3(81.8% vs54.1%),CD4(86.4 vs 55.4%),CD8(90.9% vs 62.6%),CD20(95.5% vs71.6%),P<0.05.3.Immune repertoire study on rapid recurrence early-stage lung adenocarcinoma: ?the use frequency of V and J genes: for BCR,there were 4 different genes between the two groups of patients,namely IGHV1-69 D,IGHV3-NL1,IGHV4-59 and IGHJ2,which were all used more frequently in the rapid recurrence group;for TCR,there were 6 differential genes: TR?V6-9 was used more frequently in the rapid recurrence group,while TR?V3-2,TR?V6-1,TR?V6-4,TR?J1-4,TR?J2-6 were used more frequently in the late recurrence group.?V/J gene rearrangement: for BCR,the two groups of patients had 8 different rearrangement combinations,among which IGHV3-43/IGHJ6,IGHV3-NL1/IGHJ3,IGHV3-69-1/IGHJ5,IGHV1-18/IGHJ3 appeared more frequently in rapid recurrence group while IGHV3-23D/IGHJ6,IGHV3-11/IGHJ6,IGHV3-23/IGHJ6,IGHV3-11/IGHJ2 appeared more frequently in the late recurrence group(IGHV3-11/IGHJ2 only appeared in the late recurrence group);for TCR,there were 23 different combinations in the two groups,among which TRBV14/TRBJ2-4,TRBV7-8/TRBJ1-4,TRBV3-1/TRBJ1-3,TRBV5-1/TRBJ2-4,TRBV13/TRBJ2-7,TRBV5-1/TRBJ1-4,TRBV4-1/TRBJ2-1,TRBV5-6/TRBJ1-1,TRBV4-3/TRBJ2-2,TRBV9/TRBJ2-1,TRBV6-1/TRBJ1-3,TRBV6-9/TRBJ2-7,TRBV3-2/TRBJ2-7 had a higher frequency in the rapid recurrence group(TRBV6-9/TRBJ2-7,TRBV3-2/TRBJ2-7 only appeared in the rapid recurrence group)while TRBV28/TRBJ1-4,TRBV10-3/TRBJ2-3,TRBV7-9/TRBJ1-1,TRBV2/TRBJ2-2,TRBV6-6/TRBJ2-4,TRBV4-1/TRBJ2-2,TRBV12-5/TRBJ1-1,TRBV6-3/TRBJ2-5,TRBV10-2/TRBJ1-4,TRBV16/TRBJ1-1 had a higher frequency in the late recurrence group(TRBV16/TRBJ1-1 only appeared in the late recurrence group).?Clonal diversity indexes Clonality and Evenness: for BCR,there was no statistical difference in Clonality and Evenness between the two groups;for TCR,patients with rapid recurrence had higher Clonality and lower Evenness,the diversity of their TCR clones was lower.?Distribution of cloning frequency of BCR and TCR: there was no significant difference in the frequency distribution of both BCR and TCR between the two groups.4.The effect of APE1 on the immune microenvironment of lung adenocarcinoma: APE1 KO mice had significantly fewer tumor-infiltrating functional CD8+ T cells and dendritic cells than WT mice,while tumor-infiltrating MDSCs are significantly more than WT mice.Conclusion:1.The tumor size(>2cm)and histological type(non-lepidic)are independent factors influencing early-stage lung adenocarcinoma recurrence;the histological type and differentiation degree have a certain indicating role in distinguishing patients with rapid and late recurrence.2.There are obvious differences between rapid and late recurrence patients in immune-related molecules,immune function-related enrichment pathways,and tumor lymphocyte infiltration.3.BCR and TCR specific V or J gene frequency and V/J gene rearrangement have indicating significance for distinguishing patients with rapid and late recurrence;the decreased clonal diversity of TCR may be the important reason for rapid recurrence of early-stage lung adenocarcinoma.4.APE1 can affect the proportion of infiltrating immune cells in mouse lung adenocarcinoma transplanted tumors and interfere with tumor immune function.This may be an important reason for APE1 to affect early-stage lung adenocarcinoma recurrence.