The Investigation Of HAS1 Gene Methylation In Screening Application Of Cervical Cancer

Cervical cancer is one of the common malignancies that threaten women’s health.Studies have found that persistent infection with high-risk human papillomavirus(hr HPV)is the main cause of the occurrence of cervical cancer and precancerous lesions.While the cervical cancer was considered preventable because of its clear etiology.The Thinprep cytologic test(TCT)and HPV testing are both method for screening cervical cancer,although both tests have their own limitations.TCT has high specificity and low sensitivity,meaning that its precision is related to the diagnostic level of pathologists.Achieving high-quality TCT in developing countries is difficult,especially in the remote areas.Available data suggests that more than 30% number of the high-grade cervical lesions or cervical cancer may not be detected by even high-quality cytology.Another method is HPV genotyping test,although,its low specificity leads to higher colposcopy referral rates.One time of specific hr HPV genotyping test cannot distinguish between temporary and persistent infection,and the temporary infection can be detected in 80% of women and heal on their own.Therefore,it is necessary to find an appropriate,objective and reproducible screening strategy.Methylation in the promoter regions of human oncogenes will lead to the decreasing expression of tumor suppressor genes,and then the downstream products of corresponding gene will lose their normal biological functions.This process will lead to precancerous lesions and malignancies.Studies have identified over 100 human gene promoters methylation was associated to cervical cancer.In the screening or managing the cervical cancer and precancerous lesions,gene methylation testing is a valuable testing method.It also can be used to triage people with abnormal screening results,assist in the selection of individualized treatment regimens,follow after post-treatment,and assess the prognosis of the disease.A recent study found a significant correlation between the methylation level of the hyaluronate synthase 1(HAS1)gene and cervical disease progression.In this study,we quantified the methylation level of HAS1 gene in cervical exfoliated cells from different lesion degree patients by Quantitative methylation specific Polymerase chain reaction(QMSP)method.We also analyzed its efficacy in diagnosing HSIL+,and compared it with TCT and hr HPV(Since cervical cancer is caused by persistent hr HPV infection,this paper only compares the diagnostic efficacy of the hr HPV test)to explore the application value of HAS1 gene methylation detection in cervical cancer screening.Women who attended our hospital for cervical cancer screening from February 2022 to September 2022 were selected,and their cervical exfoliated cells were collected and stored in a refrigerator at 4°C after TCT and HPV testing.The cells were used to extract DNA for methylation testing.Among them,if the result of TCT or HPV is positive,the owners were referred for colposcopy according to colposcopy norms.Furthermore,according to white acetate and iodine tests,cervical tissues with suspected lesions should be asked for biopsy and pathological diagnosis.The cases were divided into four groups according to pathological results: cervical inflammation group(34 cases),Low grade squamous intraepithelial lesion group,(LSIL,43 cases),High grade squamous intraepithelial lesion group(HSIL,53 cases),and Squamous cell carcinoma of the cervix group(SCC,15 cases).Further,inflammation and LSIL merged into LSIL-group(77cases)and HSIL and SCC group merged into HSIL+ group(68 cases).In this study,we studied the HAS1 gene methylation level from LSIL-group and HSIL+ group by QMSP method.And we used the Receiver operating characteristic(ROC)curve to understand the efficacy of HAS1 gene methylation in the diagnosis of HSIL+.Then,the optimal cut-off value for diagnosis was selected,and the quantitative data of HAS1 gene methylation were converted into qualitative data.Finally,the diagnostic performance of HPV and TCT detection was compared.For the diagnosis of HSIL+,the AUC of HAS1 gene methylation test was higher than that of hr HPV,and the difference was statistically significant(0.703 vs 0.549,P < 0.05).Furthermore,the AUC of HAS1 gene methylation test was no statistical difference compared with TCT test(0.703 vs 0.592,P > 0.05).The sensitivity of the hr HPV test was the highest among the three tests at 93.60%,while the sensitivity of HAS1 gene methylation test was similar to that of the TCT test(69.10% vs69.12%,P > 0.05.The specificity of the HAS1 gene methylation test was higher than that of hr HPV and TCT,71.40%,49.25%,and 14.29%,respectively.Comparing two by two,all of the differences were statistically significant(P<0.05).These three methods were similar in terms of negative predictive values(0.644,0.786,0.724)and positive predictive values(0.547,0.496,0.681),all P> 0.05.The methylation level of HAS1 gene in HPV16/18 positive patients was 26.31(0.06,862.83)which was significantly higher than that of other hr HPV positive patients,0.12(0.01,227.52),P<0.05.Among the patients with the’ ASCUS’ or ’Higher’ TCT results,the HSIL+’s sensitivity of HAS1 gene methylation test was lower than that of hr HPV test(66.00% vs93.60% P<0.05),but the specificity of HAS1 gene methylation test was significantly higher than that of hr HPV test(76.90% vs.28.2% P<0.001).Moreover,they had similar AUC(0.714 vs 0.609 P>0.05),negative predictive value(0.652 vs 0.786 P>0.05)and positive predictive values(0.775 vs 0.611 P>0.05).For hr HPV-positive patients,the AUC,sensitivity,specificity,negative predictive value and positive predictive value of HAS1 gene methylation test for the diagnosis of HSIL+ were 0.695(95% CI0.603-0.786),69.2%,69.7%,0.697,0.692,respectively.The AUC,sensitivity,specificity,negative predictive value and positive predictive value of TCT for the diagnosis of HSIL+ were 0.626(95% CI 0.530-0.722),67.7%,57.6%,0.644 and 0.611,respectively.Comparing two by two,all of the differences were not statistically significant(all P > 0.05).In conclusion,the methylation level of HAS1 gene increases with the increasing severity of cervical lesions.The methylation level of patients with HPV 16/18 infection is significantly higher than that of patients with other high-risk positive types.These two signs suggest that HAS1 gene methylation is involved in the development of cervical precancer and cervical cancer.HAS1 gene methylation test has some application value in cervical cancer screening and can be used for triage of patients with abnormal TCT and/or hr HPV primary screening.