| BackgroundThymic epithelial tumors（TETs）are rare neoplasms arising from the epithelial cells of the thymus with an incidence of 0.13 per 100,000 person/year.TETs are divided into thymomas（A,A/B,B1,B2,B3 subtypes）and thymic carcinomas（TCs）by 2015 World Health Organization（WHO）classification.Masaoka Staging System and WHO classification at diagnosis were reported to be the main prognostic factors for recurrence and survival.Most type A and AB thymomas have low malignant potential,whereas type B1,B2,and B3 thymomas are more aggressive,with B3 thymoma having the greatest tendency for intrathoracic spread.On the contrary,thymic carcinoma is a highly aggressive tumor with frequent lymphatic and hematogenous metastasis.Most MasaokaⅠ-Ⅱthymic epithelial tumors have better prognosis,whereas MasaokaⅢ-Ⅳthymic epithelial tumors have worse prognosis.However,its prognostic significance in guiding further treatment is controversial.Therefore,we need to find better biomarkers for prognosis in thymic epithelial tumors.Epigenetic alterations such as DNA methylation,histone modification,and loss of genome imprinting play crucial roles in the formation and progression of cancer.Increased methylation of tumour suppressor genes is an early event in many tumours,suggesting that altered DNA methylation patterns could be one of the first detectable neoplastic changes associated with tumorigenesis.Over the past decade,many researchers have demonstrated the presence of aberrant DNA methylation in various types of tumor.However,few studies have investigated DNA methylation in TETs.It is therefore necessary to identify DNA methylation biomarkers that could be used for detection and prognosis in TETs.In this present study,in order to evaluate the potential of DNA methylation markers in the prognosis of TETs,we compared various methylation profiles of thymoma tissues and thymic carcinomas tissues by analyzing CpG markers.We managed to identify a methylation marker panel for prognosis in TETs.ObjectiveUsing the resources of public databases such as the TCGA database,the DNA methylation sites for the prognosis of thymic epithelial tumors were screened by bioinformatics analysis and validated in the thymic epithelial tumor follow-up cohort in our hospital.In order to evaluate the potential of DNA methylation markers in the prognosis of TETs,we compared various methylation profiles of thymoma tissues and thymic carcinomas tissues by analyzing CpG markers.Methods1.Screening for DNA methylation sites affecting OS in patients with thymic epithelial tumors via TCGA database.Obtain thymic epithelial tumor clinical data,whole genome DNA methylation sequencing data,transcriptome sequencing data through TCGA database,and then screen out differential DNA methylation sites,and then screen out the promoter region by gene enrichment method and adjust Differential DNA methylation sites of mRNA expression,and then differentially analyzed DNA methylation sites and overall survival（OS）were analyzed by univariate and multivariate Cox retrospective analysis to screen out.2.The clinical data of thymic epithelial tumors in our hospital from 2007 to 2017 were followed up.DNA methylation sequencing and real-time PCR were performed in paraffin-embedded tumor tissues to verify the OS of candidate DNA methylation sites.3.Screening for DNA methylation sites affecting RFS in patients with thymic epithelial tumors via TCGA database.Obtain thymic epithelial tumor clinical data,whole genome DNA methylation sequencing data,transcriptome sequencing data through TCGA database,and then screen out differential DNA methylation sites,and then screen out the promoter region by gene enrichment method and adjust Differential DNA methylation sites of mRNA expression,and then differentially analyzed DNA methylation sites and RFS were anal yzed by univariate and multivariate Cox retrospective analysis to screen out.4.The clinical data of thymic epithelial tumors in our hospital from 2007 to 2017 were followed up.DNA methylation sequencing and real-time PCR were performed in paraffin-embedded tumor tissues to verify the RFS of candidate DNA methylation sites.Result1.Through the TCGA database,we screened 12122 differential DNA methylation sites from 392,653 DNA methylation sites,and 3,600 of the 12,122 differential DNA methylation sites were located in the gene promoter region,of which only 542 DNA methylation sites regulate gene expression,and by univariate and multivariate Cox regression analysis,we screened four DNA methylation sites that most significantly affect OS as death risk prediction marker.cg05784862（KSR1）（HR=0.014 95%CI:0.000-1.297,P=0.065）、cg07154254（ELF3）(HR=1.091×10-6 95%CI:0.000-0.098,P=0.018)、cg02543462（ILRN）(HR=9.744×10-4 95%CI:0.000-0.669,P=0.037)和cg06288355（RAG1）（HR=62.037 95%CI:0.934-4122.5,P=0.054）.2.DNA methylation sequencing of candidate DNA methylation sites in 100 thymic epithelial tumor tissue samples from our hospital also confirmed its ability to predict OS of thymic epithelial tumors and construct a risk prognostic model.Through the area analysis under the ROC curve,the DNA methylation site prognosis model was more accurate than the Masaoka stage in predicting the 5-year survival rate(AUC:1.000 vs 0.742,P=2.7×10-6).3.Through the TCGA database,we screened 12122 differential DNA methylation sites from 392,653 DNA methylation sites,and 3,600 of the 12,122 differential DNA methylation sites were located in the gene promoter region,of which only 542 DNA methylation sites regulate gene expression,and by univariate and multivariate Cox regression analysis,we screened four DNA methylation sites that most significantly affect OS as death risk prediction marker.cg20068620（MAPK4）（HR=0.02 95%CI:0.000-1.287,P=0.045）、cg18770944（USP51）（HR=1.041×10-6 95%CI:0.000-0.098,P=0.012）和cg16272791（GPC3）（HR=62.037 95%CI:0.924-4132.5,P=0.034）.4.DNA methylation sequencing of candidate DNA methylation sites in 100 thymic epithelial tumor tissue samples from our hospital also confirmed its ability to predict OS of thymic epithelial tumors and construct a risk prognostic model..Through the area analysis under the ROC curve,the DNA methylation site prognosis model was more accurate than the Masaoka stage in predicting the 5-year recurrence rate(AUC:0.870 vs 0.642,P=1.7×10-6)and 10-year recurrence rate(AUC:0.890 vs 0.742,P=2.4×10-6).Conclusion1.Masaoka clinical stage is an independent prognostic factor for thymic epithelial tumors.2.The methylation levels of cg05784862（KSR1）,cg07154254（ELF3）,cg02543462（ILRN）and cg06288355（RAG1）sites in thymic epithelial tumors are associated with the OS of thymic epithelial tumors and can be used as a biomarker.3.The methylation levels of cg20068620（MAPK4）,cg18770944（USP51）,cg16272791（GPC3）sites in thymic epithelial tumors are associated with the OS of thymic epithelial tumors and can be used as a biomarker.4.The prognostic model of DNA methylation in thymic epithelial tumors is more predictive than Masaoka’s clinical staging. |
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