Executive summary DNA methylation as a tumor biomarkerEpigenetic abnormalities are present in all human cancers and are currently considered to be the hallmark of cancer initiation and progression. Currently, the best-studied epigenetic event in the mammalian genome is DNA methylation, which is a chemical covalent modification of cytosine. The virtually universal presence of DNA hypermethylation in all types of cancer makes it an ideal candidate tumor biomarker. DNA methylation is chemically stable, it can be readily amplifiable and is easy to detect. In lung cancer, aberrant DNA methylation can be detected in tissues, in plasma/serum, in sputum, in bronchial lavage and saliva.Circulating tumor DNA Cell-free DNA maintains the genetic and epigenetic profile of its primary tumor source. A plethora of loci have been reported to be methylated in the plasma/serum of non-small-cell lung cancer (NSCLC) patients, suggesting novel potential diagnostic and prognostic targets such as APC, CDH1, MGMT, DCC, RASSF1A and others. DNA methylation as a tumor biomarker in lung cancerA variety of epigenetic biomarkers have been evaluated so far in lung cancer diagnostics. The diagnostic value of RASSF1A methylation and KRAS mutations in bronchial washings reported a 29% efficiency of detecting malignancy in false-negative or ambiguous cytology outcomes. SHOX2 quantified methylation showed promising results in bronchial aspirates, with a sensitivity of 78% and a specificity of 96% while SHOX2 sensitivity and specificity in plasma was 60 and 90%, respectively. Promoter methylation of APC, RASSF1A, p16 and CDH13 associated with early recurrence in stage 1 NSCLC. Promoter methylation of BRMS1 correlates with smoking history and poor survival in NSCLC.

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Background:Breast-cancer metastasis suppressor 1 (BRMS1) gene encodes for a predominantly nuclear protein that differentially regulates the expression of multiple genes, leading to suppression of metastasis without blocking orthotropic tumour growth. The aim of the present study was to evaluate for the first time the prognostic significance of BRMS1 promoter methylation in cell-free DNA (cfDNA) circulating in plasma of non-small cell lung cancer (NSCLC) patients. Towards this goal, we examined the methylation status of BRMS1 promoter in NSCLC tissues, matched adjacent non-cancerous tissues and corresponding cfDNA as well as in an independent cohort of patients with advanced NSCLC and healthy individuals.Methods:Methylation of BRMS1 promoter was examined in 57 NSCLC tumours and adjacent non-cancerous tissues, in cfDNA isolated from 48 corresponding plasma samples, in cfDNA isolated from plasma of 74 patients with advanced NSCLC and 24 healthy individuals.Results:The BRMS1 promoter was highly methylated both in operable NSCLC primary tissues (59.6%) and in corresponding cfDNA (47.9%) but not in cfDNA from healthy individuals (0%), while it was also highly methylated in cfDNA from advanced NSCLC patients (63.5%). In operable NSCLC, Kaplan–Meier estimates were significantly different in favour of patients with non-methylated BRMS1 promoter in cfDNA, concerning both disease-free interval (DFI) (P=0.048) and overall survival (OS) (P=0.007). In advanced NSCLC, OS was significantly different in favour of patients with non-methylated BRMS1 promoter in their cfDNA (P=0.003). Multivariate analysis confirmed that BRMS1 promoter methylation has a statistical significant influence both on operable NSCLC patients' DFI time and OS and on advanced NSCLC patients' PFS and OS.Conclusions:Methylation of BRMS1 promoter in cfDNA isolated from plasma of NSCLC patients provides important prognostic information and merits to be further evaluated as a circulating tumour biomarker.

Our results show that SOX17 promoter is highly methylated in primary tumors and in corresponding plasma samples both in operable and advanced NSCLC. In the advanced setting, SOX17 promoter methylation in plasma ctDNA has a statistical significant influence on NSCLC patient's survival time. Detection of SOX17 promoter methylation in plasma provides prognostic information and merits to be further evaluated as a circulating tumor biomarker in patients with operable and advanced NSCLC.