Mutigene Methylations For Early Diagnosis Of Lung Cancer In Yunnan, China

Background and Objective:Lung cancer (LC) is a complex disease. Aberrant DNA methylation is considered to be an early event during process of tumorigenesis through mediating the interaction between genetic and environmental factors. Currently, most LC patients diagnosed in hospital are already in middle or late stage. Thus, it is urgently needed to improve technologies and identify biomarkers for risk prediction and/or diagnosis of LC. Liquid biopsies are non-invasive alternatives to traditional methods for cancer detection and monitoration. In many areas of Yunnan Province, such as Xuanwei City, rates of LC are among the highest nation-wide. Here, we aimed to conduct researches on clinical samples of LC patients from these areas, and analysis of multigene methylations for early diagnosis of LC by using cfDNA in plasma as liquid biopsy.Methods:Twenty-three tumor tissue samples and 42 blood samples separately collected from LC patients, and 10 blood samples from healthy controls were used for the present study. Cell DNA or cfDNA in plasma was extracted and bisulfite converted with kits. Aberrant methylation in DNA was then detected by nested methylation-specific PCR (nMSP). According to the previous reports,8 LC-related candidate genes including p16, DLEC1, CDH1, DAPK, RUNX3, APC. WIF1 and MGMT were first tested on the tumor tissue samples. A panel of genes was then selected for the follow-up examination on cfDNA. In addition, the Bisulfite Sequencing PCR (BSP) technique was used on some of the samples to confirm the methylation results. Clinical information was collected meanwhile on all patients and analyzed with the methylation data together. For the 9 patients having both tumor tissue and blood samples collected, comparison of the methylation status was also preformed between them.Results:In tumor tissue samples,8 genes except MGMT had a relatively high sensitivity ranging from 39%-74%. Using a cutoff of present of at least any one methylated gene as a criterion,96%samples were positive; and using that of at least any two methylated genes,91%samples were detected out. Wherein, a panel of genes including p16, DLEC1, CDH1. DAPK and RUNX3 were with higher positive ratios (≥48%), which were then used for the testing on cfDNA in plasma. The results showed that their methylation rates were 14%-76%. Moreover, using cutoff of at least any one and two methylated genes, separately 95%and 71%samples were screened out for methylations. With regard to the healthy controls, no sample could be detected positively. In addition, the methylation status was basically consistent between the matched tumor tissue samples and blood samples for the 9 patients. When analysis was performed by combining the clinical data from all LC patients, the methylation status of CDH1 gene were related to LC pathological classification, clinical stage and metastasis, and W1F1 gene was associated with the pathological classification of LC. Finally, the methylation status of CDH1, RUNX3 and W1F1 genes in tumor or plasma DNA were found to be likely affected by age, gender or smoking status.Main conclusions:(1) LC tumorigensis in Yunnan area are related to aberrant changes in DNA methylation of multigene; (2) A panel of genes including p16, DLEC1, CDH1, DAPK and RUNX3 has relatively higher sensitivity and specificity for detection of LC related methylation using plasma cfDNA, which could be used as potential biomarkers for early diagnosis of LC in Yunnan; (3) The methylation status of CDH1 gene is correlates well the clinicopathologic characteristics of LC, and could be a potential prognostic biomarker for LC.