ObjectiveMethylation of the promoters of SHOX2 and RASSF1A are potentially informative biomarkers for the diagnosis of early lung adenocarcinoma (LUAD). Abnormal methylation of SHOX2 and RASSF1A promoters may promote the occurrence and facilitate the progression of LUAD.Materials and MethodsWe selected 54 patients with early LUAD and 31 patients with benign lung nodules as a NJDT cohort and evaluated their DNA methylation and mRNA sequencing levels. The DNA methylation sequencing, mRNA sequencing, and clinical data for patients with LUAD were obtained from The Cancer Genome Atlas, and served as a TCGA cohort. We evaluated the diagnostic potential of a SHOX2 and RASSF1A combined promoter methylation assay for detection of early LUAD in the NJDT cohort. Then we explored the promoter methylation levels of SHOX2 and RASSF1A and their gene expression between normal and tumor samples at different stages in both cohorts. Pathways enriched between tumor and normal samples of methylation-positive patients in the NJDT cohort were analyzed.ResultsIn the NJDT cohort, the sensitivity of the combined promoter methylation assay on tumor samples was 74.07%, the sensitivity on paired tumor and paracancerous samples was 77.78%, and the specificities in both contexts were 100%. The combined promoter methylation-positive patients had clinicopathologic features including older age, larger tumors, deeper invasion, and higher Ki-67 expression. In both cohorts, SHOX2 expression increased and RASSF1A expression decreased in tumor samples. The promoter methylation level of SHOX2 and RASSF1A was significantly higher in tumor samples at stage I-II than that in normal samples. The promoter methylation levels of these two genes were both negative associated with their expression in early tumor samples. In the NJDT cohort, methylation-positive patients of both individual SHOX2 and RASSF1A assays exhibited upregulation of folate acid metabolism and nucleotide metabolism in tumor samples. The SHOX2 methylation-positive and RASSF1A methylation-positive patients showed the downregulation of pathways related to cell proliferation and apoptosis and pathways involved in DNA repair, cell growth and cell adhesion, respectively.ConclusionThe combined promoter methylation assay for SHOX2 and RASSF1A can be used for screening and diagnosis of early LUAD, with good sensitivity and specificity. The promoter methylation levels of SHOX2 and RASSF1A were associated with their abnormal mRNA expression, and affected DNA instability, cell proliferation, apoptosis and tumor microenvironment in patients with LUAD.