The Development Of A Novel Method For The Quantification Of Plasma APC Gene Promoter Methylation And Its Preliminary Clinical Application In Diagnosis Of Early Lung Cancer

BackgroundsLung cancer,which has a high morbidity,is a major public health problem in the world and remains a leading cause of cancer-related deaths.The low survival rate is attributable to the lack of method to early diagnosis of lung cancer.Thus,the development of efficient diagnostic methods to enable the early detection is imperative.Recently,much research suggests that the activation of oncogene after the inactivation of TSG induced by DNA methylation play an important role in tumorigeness.DNA methylation that lead to the inactivation and low expression of TSG could make the cell to malignant transformation and play a leading role in some types of carcinoma,this change may be detected in early stage of cancer.It can be detected in lung cancer tissues that the methylation in the promoter of p16,APC,RASSF1A and DAPK.Circulating DNA,also called cell-free DNA,is a kind of extracellular DNA present in the blood(plasma or serum),cerebrospinal fluid and synovial fluid.Cell-free DNA concentrations increase in the circulating blood of cancer patients and mainly be responsible for the tumor cell death.The adenomatous polyposis coli(APC)gene is located at chromosome 5q21.It was found that the hypermethylation of APC promoter in the tissue of lung cancer and may be useful in lung cancer diagnostic.So the methylation of APC was detected in plasma may have clinical application in early diagnosis of lung cancer.ObjectiveTo develop a real-time quantilative methylation-specific PCR(real-time QMSP)method of plasma APC gene promoter methylation,to quantify the plasma APC gene promoter methylation of lung solid tumor patients and study its application in the early diagnosis for lung cancer patients.Materials and MethodsThe methylated status of APC gene promoter 1A in lung cancer cell line NCI-H460 was confirmed by methylation specific PCR(MSP)and BSP.After extraction by phenol-chloroform method and quantification by spectrophotometric measurements,genomic DNA with methylated APC gene of lung cancer cell line NCI-H460 was added into 200μl plasma samples of healthy volunteer with serial dilution.Circulating DNA samples were extracted from simulated lung cancer patient plasma samples with different concentration(1.5×10⁵,1.5×10⁴,1.5×10³, 1.5×10²,15 copies/ml plasma)by magnetic bead protocol,and modified by bisulfite sodium together with CB DNA transferred by methyl transferase(positive control)and CB DNA(negative control).According to Prime Priemer 5.0,specific primers and probe were designed to do APC promoter region by real-time QMSP.The specificity,sensitivity, repeatability and accuracy of this assay were evaluated.Blood samples were collected from 92 CT-detected thoracic solid tumor(tumor diameter≤2cm)patients and 23 healthy volunteers.Plasma DNA samples were purified with magnetic beads,then treated with sodium bisulfite,and promoter hypermethylations of APC gene were detected by real-time QMSP.ResultsThe standard curve of this real-time QMSP was constructed by the samples with different concentration(1.5×10⁵,1.5×10⁴,1.5×10³, 1.5×10²copies/ml)and C_APC=10^Ct-Intercept/Slope)was used to calculate the quantitation of methylated APC DNA.Real-time QMSP could detect 1.5×10² copies of methylated APC DNA in one milliliter simulated lung cancer patient plasma.The CVs of intra-assay and extra-assay Ct value were 0.91%and 2.43%,respectively.All 92 patients,including 58 lung cancer cases,31 benign lung diseases and 3 unconfirm cases were confirmed by histopathology.14 cases were detected positively in 58 lung cancer patients(14/58,24.1%),but not in 31 benign lung diseases,3 unconfirm cases and 23 health controls.ConclusionsThis real-time QMSP assay allows quantitative detection of plasma APC gene hypermethylation with high sensitivity and specificity.Our data suggests that promoter hypermethylation of APC gene of plasma DNA is a promising molecular biomarker for the early diagnosis of lung cancer.