Blood FOLR3 methylation dysregulations and heterogeneity in non-small lung cancer highlight its strong associations with lung squamous carcinoma

Background Non-small cell lung cancer (NSCLC) accounts for the vast majority of lung cancers. Early detection is crucial to reduce lung cancer-related mortality. Aberrant DNA methylation occurs early during carcinogenesis and can be detected in blood. It is essential to investigate the dysregulated blood methylation markers for early diagnosis of NSCLC. Methods NSCLC-associated methylation gene folate receptor gamma ( FOLR3 ) was selected from an Illumina 850K array analysis of peripheral blood samples. Mass spectrometry was used for validation in two independent case–control studies (validation I: n = 2548; validation II: n = 3866). Patients with lung squamous carcinoma (LUSC) or lung adenocarcinoma (LUAD), normal controls (NCs) and benign pulmonary nodule (BPN) cases were included. FOLR3 methylations were compared among different populations. Their associations with NSCLC clinical features were investigated. Receiver operating characteristic analyses, Kruskal–Wallis test, Wilcoxon test, logistics regression analysis and nomogram analysis were performed. Results Two CpG sites (CpG_1 and CpG_2) of FOLR3 was significantly lower methylated in NSCLC patients than NCs in the discovery round. In the two validations, both LUSC and LUAD patients presented significant FOLR3 hypomethylations. LUSC patients were highlighted to have significantly lower methylation levels of CpG_1 and CpG_2 than BPN cases and LUAD patients. Both in the two validations, CpG_1 methylation and CpG_2 methylation could discriminate LUSC from NCs well, with areas under the curve (AUCs) of 0.818 and 0.832 in validation I, and 0.789 and 0.780 in validation II. They could also differentiate LUAD from NCs, but with lower efficiency. CpG_1 and CpG_2 methylations could also discriminate LUSC from BPNs well individually in the two validations. With the combined dataset of two validations, the independent associations of age, gender, and FOLR3 methylation with LUSC and LUAD risk were shown and the age-gender-CpG_1 signature could discriminate LUSC and LUAD from NCs and BPNs, with higher efficiency for LUSC. Conclusions Blood-based FOLR3 hypomethylation was shown in LUSC and LUAD. FOLR3 methylation heterogeneity between LUSC and LUAD highlighted its stronger associations with LUSC. FOLR3 methylation and the age-gender-CpG_1 signature might be novel diagnostic markers for the early detection of NSCLC, especially for LUSC.