The System Evaluation Of RASSF1A,RAR?,and CDH1 Promoter Hypermethylation With Oral Cancer Risk

Background and Objective:Oral cancer is one of the most frequent tumor of head and neck tumor,in which oral squamous cell carcinoma(OSCC)accounts for approximately90%of all oral malignancies.As the sixth most common cancer worldwide,OSCC has a high mortality and low cure rate.Although advancements have been made in the prevention and treatment of oral cancer,the five-year survival rate of OSCC still remained approximately 50%.Oral tumor is a heterogeneous group of tumors,in which it has several different histopathological and molecular features.Recently,genetic and epigenetic alterations are often detected in the development of oral cancer.Gene promoter hypermethylation could lead to the silencing of cancer related genes without changes of genes sequence.RASSF1A played a key role in the cell cycle control,microtubule stabilization,cellular adhesion and motility as well as apoptosis.Hypermethylation of RASSF1A gene was reported in many different cancers and was described as a good prognostic indicator.Several studies have been also performed to evaluate the relationship between RASSF1A promoter hypermethylation and oral cancer risk.However,the results remained inconsistent.Moreover,the results of other studies indicated that CDH1 and RAR?promoter hypermethylation frequencies were very high in oral cancer patients.Thus,these tumor suppressor genes which occurred aberrant methylation might be good biomarkers for the early detection and early treatment of oral cancer,however,the results are still inconsistent.In order to strengthen the clinical screening and laboratory diagnosis of oral cancer?improve the clinical treatment?postoperative prognosis status and update the current diagnosis and treatment of oral cancer for the clinical,To clarify the effect of RASSF1A,Retinoic acid receptor beta(RAR?),and E-cadherin(CDH1)promoter hypermethylation on the risk of oral cancer,we perform this system evaluation.Methods:1 Search strategy for included studiesTwo researchers independently retrieved PubMed,Web of Science,Embase,and Chinese National Knowledge Infrastructure(CNKI)databases to identify eligible articles.The retrieval time of literature research was started from the earliest time of database collection.The literature research was up to 15 April 2017.The following keywords or medical subject headings(Me SH)words:�oral cancer�,�oral tumor�,�oral carcinoma�,�oral squamous cell carcinoma�,�OSCC�,�Salivary Gland Carcinoma�,�Buccal Carcinoma�,�Salivary Adenoid Cystic Carcinoma�,�RASSF1A�,�CDH1�,�E-cadherin�,�RAR?�,�methylation�,and�hypermethylation�were used to search eligible articles.In addition,references of included articles were reviewed for additional eligible studies.The search language includes both Chinese and English.2 Study selection criteria(1)Studies assessing the associations of RASSF1A,RAR?,and CDH1 hypermethylation with oral cancer risk.Case-control or cohort studies that contained data of hypermethylation frequency both in control and case group.(2)This literature included was based on the research literature of the population,and the literature of animal experiments was not included in this study.(3)If studies didn't meet the following criteria,they would be removed:no or incomplete relevant data about RASSF1A,RAR?,and CDH1 methylation data;duplicate data(If duplicate data was showed in different studies,the most complete and latest data were selected);Meta-analysis and review article;and low quality studies.(4)All relevant articles were evaluated and selected by two investigators.If discrepancies occurred in the process of studies selection,the third researcher would help to resolve this problem through discussion with the two reviewers.3 Data extraction and methodology quality assessmentTwo reviewers independently extracted the data of promoter methylation of RASSF1A,RAR?and CDH1,and hypermethylation frequency of oral cancer and peripheral oral cancer.The following information were extracted from included articles:first author's name,publication year,race,frequency of gene methylation,frequency of gene unmethylated,disease type,detection method of genes methylation,and country of studied population.The methodological quality,including selection of case and control(4 stars),comparability of the groups(2 stars),and ascertainment of exposure(3 stars),were evaluated with the Newcastle�Ottawa Scale table.If a study got36 stars,the study was considered as high quality and included for this Meta-analysis,otherwise it would be removed.4 Statistical MethodsSTATA software(version 12.0,Stata Corporation,College Station,Texas,USA)was used to conduct all statistical analysis.The associations of RASSF1A,RAR?,and CDH1 methylation with oral cancer risk were evaluated with OR and 95%CI.All P value were two-sided in which P<0.05 was considered as statistically significant.Heterogeneity among studies were detected by chi squared test based on Q-statistic test.If P value was<0.05 or I~2 value was>50%,which indicated a significant heterogeneity,a random-effects model was used;otherwise,a fixed-effect model would be applied.In addition,Z-test was conducted to determine the strength of pooled OR.In order to assess the publication bias,egger's test and begg's test were performed to detect between-study publication bias,in which P<0.05 indicated a significant publication bias.Moreover,a sensitivity analysis was conducted to further detect the stability of overall OR by sequential deletion of each study.In the Meta-analysis of CDH1promoter hypermethylation,Meta-regression was also performed to explore the source of heterogeneity due to significant heterogeneity.Results:1 Literature screening results160 potentially relevant articles were identified in the initial searching.After removing repeat articles,75 relevant records were reviewed and selected.Through reading titles and abstracts,55 articles were remained,of which 20 irrelevant literatures were excluded.In addition,the full-text of remained 55 articles were read,in which 32 studies didn't contained relevant effective data and were eliminated.Finally,23 literatures with 29studies were included in the final Meta-analysis,in which 12 studies were about RASSF1A promoter methylation,4 studies were about RAR?promoter methylation,and 13 studies were about CDH1 promoter methylation.Moreover,all studies obtained a score of36,which indicated high methodological quantity of included studies.2 RASSF1A promoter hyper methylation and oral cancerriskThe RASSF1A methylation data of 12 case-control studies was pooled together and the OR was calculated to assess the association between RASSF1A promoter hypermethylation and oral cancer risk and peripheral oral cancer.On the basis of the results,the overall pooled OR clarified that RASSF1A promoter hypermethylation was significantly associated with oral cancer risk(OR=11.8,95%CI=6.14-22.66).According to the results of Q-statistic test,no heterogeneity among studies was found(P=0.337,I~2=12.0%).Then,subgroup analysis based on oral cancer subtype was performed and significant association was detected in OSCC and SGC(Salivary Gland Carcinoma)(OSCC,OR=6.78,95%CI=3.20-14.37;SGC,OR=18.51,95%CI=3.58-95.79).3 RAR?,and CDH1 promoter hypermethylation and oral cancer riskThe overall pooled OR clarified that RAR?and CDH1 promoter hypermethylation were significantly associated with oral cancer risk and peripheral oral cancer.(RAR?,OR=20.35,95%CI=5.64-73.39;CDH1,OR=13.46,95%CI=5.31-34.17).In the analysis for CDH1 methylation,significant heterogeneity among studies was found(I~2=73.1%,P=0.000).In order to explore the source of heterogeneity,we performed a Meta-regression,in which the results indicated that ethnicity might be the mainly source of heterogeneity(P=0.028,95%CI=0.275-3.976).In the stratified analysis based on ethnicity,CDH1 promoter hypermethylation might significantly increase the oral cancer risk in Asians,but not in Caucasians(Asians,OR=21.79,95%CI=8.66-54.82;Caucasians,OR=2.57,95%CI=0.71-9.31).However,only three studies for CDH1methylation in Caucasians were included to calculate the pooled OR,its conclusions need to be further studied..4 RASSF1A promoter hypermethylation and tongue tumorriskWe found that RASSF1A promoter hypermethylation exerted higher frequency in the tongue tumor than other site tumor in mouth(RASSF1A,tongue tumor vs other site tumor in mouth,unmethylation vs methylation,OR=0.65,95%CI=0.44-0.98).Conclusion:RASSF1A,RAR?,and CDH1 promoter hypermethylation might significantly increase the risk of oral cancer.