Detection Of Plasma RASSF1A Gene Hypermethylation And Its Preliminary Clinical Application

BackgroundsIn October 2003,Human Epigenome Consortium announced to initiate the Human Epigenome Project.DNA methylation is one of the most important epigenetic mechanisms and 5'-methylated cytosine is even called as the fifth kind of bases.5'-methylated cytosine,the most common base with covalent modification in vertebrate genome,is usually involved in CpG.The region rich of CpG dinucleotides,also called CpG islands,is widespread in genome and is usually located in promoter regions.Methylation of CpGs in promoter has been confirmed to be related with histone hypoacetylation,gene silencing and embryo development,inherited imprinting and x-chromatin inactivation.Recently,the study on tumorigenesis and development were more and more penetrating from genetics to epigenetics.It has been demonstrated that hypermethylation of TSG existed in early stage of cancer and this provide a new idea for searching novel cancer-specific biomarkers.Recent studies showed that more than 20 TSGs were associated with lung cancer,including p16,APC,RASSF1A and so on. Therefore,the detection of these TSGs promoter hypermethylation could play an important role in the diagnosis of early lung cancer.Ras-association domain family 1A gene,which is localized at 3p21.3,is a newly cloned putative TSG.Several studies presented that RASSF1A was involved in Ras signaling pathways,cell cycle and cell division althought we are far form understanding its true role(s). RASSF1A promoter hypermethylation leads to the loss of its function in certain lung cancer cell lines and its methylation was found to occur in 40%of lung cancer tissues.Nevertheless,all the studies mentioned above were aimed at diagnosed lung cancer patients,most of whom were at advanced stage. Therefore,in this study,a new real-time quantitative MSP(real-time QMSP)of detecting plasma RASSF1A promoter hypermethylation was developed to invest the methylation status of early lung cancer.Plasma RASSF1A promoter methylation of CT-detected thoracic solid tumor patients was detected by this novel assay to analysis its clinical application.ObjectiveTo develope a quantitative method for the quantification of RASSF1A gene methylation in plasma by real-time QMSP and to analyze plasma RASSF1A gene promoter methylation of CT-detected early stage lung cancer patients. Materials and MethodsLarge cell lung cancer cell line NCI-H460 was confirmed to be hypermethlated in RASSF 1A promoter by previous study.After extracted by phenol-chloroform method and quantified by spectrophotometric measurements,genomic DNA of NCI-H460 was added into 200μl plasma samples of healthy volunteers with decuple serial dilution.The simulated plasma samples of lung cancer patients which contained 1.5×10⁵,1.5×10⁴, 1.5×10³,1.5×10² and 15 copies genomic DNA of cancer cell per milliliter plasma were used as standard samples and its circulating DNA was extracted by magnetic bead protocol and modified by bisulfite.Specific primers and probe were designed to amplify RASSF1A promoter region by real-time QMSP.After optimization of annealing temperature and amplification system,a real-time QMSP method was developed.The specificity,sensitivity and repeatability of this assay were evaluated.The accuracy of this quantitative method was determined by the standard curve constructed by using simulated lung cancer patients plasma samples containing tumor cell genomic DNA(1.5×10⁵,1.5×10⁴,1.5×10³, 1.5×10² copies per ml).After further optimization,RASSF1A DNA methylation in plasma samples collected from 92 CT-detected thoracic solid tumor(diameter≤2cm)patients and 23 healthy volunteers were analyzed.Plasma DNA samples were purified by magnetic beads,then treated with sodium bisulfite,and promoter hypermethylation of RASSF1A gene was detected by real-time QMSR The concentration of methylated RASSF1A gene was calculated according to the standard equation C_RASSF1A=10(Ct-Interept/Slope)ResultsThe newly developed real-time QMSP assay allowed specific amplification of methylated RASSFIA promoter region and the 95 bp product could be detected sensitively by TaqMan probe with fluorescein FAM.This assay had been optimized for the best amplification while the annealing temperature was 59℃and the administered dose of Mg²⁺was 2μl.This assay allowed the detection of plasma samples of simulated lung cancer patients with concerntration of as low as 1.50×10² copies of methylated tumor genomic DNA per milliliter of plasma.The intra-assay and extra-assay CV caculated by Ct values were 1.26%and 1.89%, respectively.The difference of quantitative results between this novel assay and spectrophotometric assay was below 20%.Because of the same amplification efficiency,the three-step assay was then changed to a two-step for shorter detecting time.Genomic DNA of H460 monoclonal cell line(1 000μg/ml)was used as quality control and its normal amplification Ct value was 26.00～28.00.According to histopathological diagnosis,92 CT-detected thoracic solid tumor patients were composed of 58 early stage lung cancer patients and 31 benign lung disease patients and 3 uncertain cases.Plasma RASSF1A promoter hypermethylation was found in 43.1%(25/58)and 6.5%(2/31)of patients diagnosed with early stage lung cancer and benign lung disease, respectively.Hypermethylation was also detected in one of the three unconfirmed cases.Twenty-three healthy volunteers were all negative. The differences of RASSF1A gene promotor methylation frequency among early stage lung cancer,benign lung disease as well as healthy volunteers were significantly(p=0.000).But no significant difference was found between patients with benign lung disease and healthy controls (p=1.575).The concentrations of methylated RASSF1A gene in 25 early lung cancer patients ranged from 3.83×10² to 1.47×10⁵ copies/ml with a median of 2.90×10³ copies/ml in plasma.ConclusionsThis real-time QMSP assay allows quantitative detection of plasma RASSF1A gene hypermethylation with high sensitivity and specificity. Our data suggest that promoter hypermethylation of RASSF1A gene in plasma DNA is a promising molecular biomarker for the early diagnosis of lung canncer.