| Background:Cervical cancer is one of the major malignant tumors threating women’s health.Different from other malignant tumors,cervical cancer has a clear etiology,that is,persistent infection of high-risk human papillomavirus(HPV),mainly HPV16 and 18 infection.Most HPV infections are transient,and the progression from viral infection to cervical cancer need to go through the cervical intraepithelial neoplasia(CIN)stage,which provides the possibility of detecting early precancerous lesions and preventing the occurrence of cervical cancer.The treatment of cervical lesions is mainly based on the pathological results of cervical biopsy under colposcope,but there are differences between biopsy pathological results and postoperative pathological results due to subjective limitations of the examiner and the difficulty in assessing the lesions in the cervical canal.In addition,there is a possibility of spontaneous regression in some patients with high grade squamous intraepithelial lesions(HSIL)of the cervix,and there is still a controversy on whether surgical treatment is necessary for these patients.Young patients choose conization due to the panic over the progression of lesions,which leads to the risk of adverse pregnancy outcomes.Therefore,it is of great clinical significance to explore a tool that can improve the accuracy of cervical biopsy under colposcopy and identify the risk of cervical disease progression.Abnormal DNA methylation is closely related to the occurrence and development of tumors.Studies have shown that DNA methylation detection has a broad application prospect in the early diagnosis of tumors.Foreign scholars screened six cervical cancer markers(ASTN1,DLX1,ITGA4,RXFP3,SOX17,ZNF671)with high sensitivity and specificity through genome-wide CpG island microarray and methylation-specific PCR verification.It has been proved that this methylation test has high sensitivity and specificity in cervical cancer screening.However,there is still a lack of clear research on the application of this methylation test in improving the accuracy of cervical biopsy pathological diagnosis and predicting the risk of cervical disease progression.Objective:The methylation degree of related genes(ASTN1,DLX1,ITGA4,RXFP3,SOX17 and ZNF671)in cervical exfoliated cells was detected to explore the potential of improving the accuracy of cervical biopsy pathological diagnosis and predicting the risk of cervical disease progression.Methods:We compared the coincidence rate,under-diagnosis rate and over-diagnosis rate of 144 patients between colposcopy-directed biopsy pathology and cervical tissue resection pathology in Qilu Hospital of Shandong University.Related genes in cervical exfoliated cells were detected by methylation-specific PCR,and We compared the positive rate of methylation in patients with under-diagnosis and over-diagnosis of colposcopy-directed biopsy.They were divided into young group(≤35 years old,n=56)and old group(>35 years old,n=88)according to age.According to the postoperative pathological results,they were divided into cervical carcinoma(CC)group(n=41),CIN3(n=34),CIN2(n=33),CIN1(n=24),and cervicitis(CIN0,n=12).According to HPV results,they were divided into HPV positive group(including 85 cases of HPV16/18 positive group,36 cases of non-HPV16/18 positive group,10 cases of HC2 positive group,3 cases of E6E7 positive group,n=134),HPV negative group(n=7)and HPV untested group(n=3).According to TCT results,they were divided into cytological low-risk group(including 21 cases of NILM,26 cases of ASCUS,20 cases of LSIL,n=67),cytological high-risk group(including 15 cases of ASC-H,31 cases of HSIL,n=46)and others(such as untested TCT,n=31).We compared the methylation positive rates of relevant genes in 144 patients under different clinical factors,and analyzed the diagnostic efficacy of methylation for CIN2+and CIN3+.Test standard was 0.05(α=0.05),when P<0.05 considered that the difference was statistically significant.Results:1.Based on the analysis of postoperative pathology,overall rates for under-,correctly-,and over-diagnosed cases were 17.3%(22/127),55.9%(71/127),and 26.8%(34/127),respectively.The pathological correctly-diagnosed rates of different pathological grades of colposcopy biopsy was respectively:CIN1(7/10,70.0%),CIN2(18/39,46.2%),CIN3(21/49,42.9%),CC(25/29,86.2%),the difference was statistically significant(P<0.05).There was no significant difference in the coincidence rate of colposcopy biopsy and postoperative pathology between the young group and the old group(56.8%vs 54.7%)(P>0.05).The correctly-diagnosed rate of colposcopy biopsy pathology and postoperative pathology was higher in HPV16/18 positive group than in non-HPV16/18 group(62.3%vs 38.7%),and the difference was statistically significant(P<0.05)2.For patients with colposcopy-directed biopsy pathology results of CIN2 and CIN3,compared with postoperative pathology,the positive rate of methylation in patients with over-diagnosis was significantly lower than that in patients with under-diagnosis(0%vs 66.7%,33.3%vs 92.3%),with statistically significant differences(P<0.05).3.For the total sample,the methylation positive rate in CC,HSIL and LSIL-groups was 97.6%,50.7%and 8.3%,respectively.With the increase of pathological grade,the positive rate of methylation increased significantly(P<0.05),and the difference between each two groups was statistically significant(P<0.005).The positive rate of methylation in HPV16/18 positive group was 63.5%higher than that in non-HPV16/18 positive group(30.1%),and the difference was statistically significant(P<0.05).The positive rate of methylation in high-risk cytology group was higher than that in low-risk cytology group(69.6%vs 32.8%),the difference was statistically significant(P<0.05).For CIN3+patients,the methylation positive rate in the young group(77.8%)was lower than that in the old group(95.8%),and the difference was statistically significant(P<0.05).4.Methylation test showed good differentiation ability for CIN3+/CIN2-,CIN3+/CIN1and CIN2+/CIN1-with high sensitivity and specificity(89.3%、85.5%,89.3%、91.7%,69.7%,91.7%).Methylation test had higher sensitivity and specificity for CIN3+/CIN2-,CIN3+/CIN1-and CIN2+/CIN1-in the elderly group(95.8%、87.5%,95.8%、95.0%,73.5%、95.0%).5.The coincidence rate of biopsy pathology under colposcopy and postoperative pathology CIN3+(that is,the sensitivity of colposcopic biopsy to diagnose postoperative pathology as CIN3+)was 89.4%,and the specificity was 69.4%.The sensitivity and specificity of methylation for the diagnosis of postoperative pathological CIN3+were 90.9%and 85.5%respectively.Colposcopy-directed biopsy combined with methylation test had a sensitivity of 97.0%and specificity of 64.5%for the diagnosis of postoperative pathological CIN3+.The sensitivity of CIN3+detected by colposcopy-directed biopsy combined with methylation(97.0%)was higher than that by colposcopic cervical biopsy(89.4%),and the difference was not statistically significant(P>0.05).The specificity of methylation was higher than that colposcopy-directed biopsy,and the difference was statistically significant(P<0.05).6.Analysis of HPV-positive patients with biopsy results of CIN1+showed that the sensitivity of host polygene methylation test to CIN3+was 88.9%,although slightly lower than cytology test and HPV16/18 typing test combined cytology test(92.6%,100%),the specificity was much higher than cytology test and HPV16/18 typing test combined cytology test(89.4%vs 22.5%,10.6%).7.For a single gene,the methylation positive rate of each gene showed an upward trend with the aggravation of pathological grade(P<0.001).The specificity and sensitivity of ZNF671 for CIN3+detection were 85.5%and 84.0%respectively,and The Youden index is 0.668.There were no significant differences in sensitivity(84.0%vs 89.3%)and specificity(85.5%vs 85.5%)between ZNF671 single gene and host polygene methylation combination for detection of CIN3+(P<0.05).Conclusions1.The coincidence rate of cervical biopsy pathology guided by colposcopy and postoperative pathology is not related to age,but is related to HPV16/18 typing.2.The positive rate of methylation in patients with biopsy pathology for HSIL is significantly lower in patients with over-diagnosis than in patients with under-diagnosis.3.The positive rate of methylation increases with the severity of pathology.Methylation test has a high sensitivity and specificity in predicting postoperative pathological CIN3+,especially in older women.4.For patients with cervical biopsy under colposcopy,combined methylation detection can improve the sensitivity of predicting postoperative CIN3+to a certain extent and significantly improve the specificity.5.For a single gene,the specificity and sensitivity of ZNF671 to detect CIN3+are consistent with the host polygene methylation combination,and it has the advantage of simple detection and low cost.6.Host polygene methylation is a potential biomarker for predicting the risk of progression of precancerous lesions,but it needs to be confirmed by more prospective observational studies. |
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