| Background:Cervical cancer is one of the female malignant tumors with high morbidity and mortalit y in China.Persistent infections with the high-risk papillomavirus(HR-HPV)have been identified to be the main factor causing cervical cancer.However,most HPV infections are transient and show no clinical symptoms,and only HR-HPV persistent infections contribute to developing the progress of the cervical intraepithelial neoplasia(CIN)and even cervical cancer.And the process may occupy 8 to 12 years from the initial infection to cervical cancer at last.During this period,as long as clinically effective screening methods were used,it would nip the malignancy in the " the bud of precancerous lesions”.HR-HPV testing is a commonly primary screening in clinical practice.It can often improve the sensitivity,but is unable to distinguish transient infections from persistent infections,especially for child-bearing period female whose HPV infection rate is high but the clinically constructive significance is weak.Low specificity of the method can easily result in the waste of limited medical resources and over-diagnosis and over-treatment of patients.Although TCT can improve the specificity of cervical screening,low sensitivity and strong subjective dependence limits its application for triaging HR-HPV-positive patients.Therefore,there is an urgent need for triaging strategies that can distinguish transient infections from persistent infections and have the ability to help physicians complete the layered management of HR-HPV-positive patients.DNA methylation is an important form of epigenetic modification,which occurs in the early stages of malignant transformation.It is widely believed that abnormal DNA methylat io n contributes to carcinogenesis and malignant development,especially hypermethylation of tumor suppressor promoter,which is related to the severity of cervical lesions.The whole genome Cp G Island microarray is used for selecting six cervical cancer biomarkers includ ing ASTN1,DLX1,ITGA4,RXFP3,SOX17 and ZNF671 genes.Thus,the combined methylat io n detection of host genes forms a methylation screening panel,and it includes Cp G-rich sequences of these six genes which are frequently methylated in(pre)cervical cancer.Overseas studies have shown that the combined methylation detection based on ASTN1/DLX1/ITGA4/RXFP3/SOX17/ZNF671 genes showed the high specificity and sensitivity in cervical cancer screening.There is a large population of cervical lesions in China.However,no domestic studies have been conducted to compare the triaging effect of the combined methylation detection,TCT or the combination of the two methods for HR-HPVpositive patients.Purpose:In this study,we analyzed the different performance between the combined methylat io n detection of host genes and TCT in different grades of cervical lesions.And we determine the effect of the combined methylation detection of host genes,TCT and the combination of the two methods for triaging HR-HPV and diagnosing high-grade cervical lesions.Methods:According to the inclusion and exclusion criteria,residual cervical scrapes in LBC media from routine cytology screening were obtained from 131 women,who had abnormal cervical cancer screening results and thus underwent colposcopy and cervical biopsy in the Second Hospital of Jilin University from September 2019 to December 2019.The pathology results and clinical data were recorded accurately.Among the 132 samples,two cases were excluded due to the small sample size and could not be analyzed by the combined methylation detection of host genes.According to the pathological results,these samples were divided into 45 patients with cervicitis(CIN0 group),28 patients with CIN1,20 patients with CIN2,23 patients with CIN3,and 13 patients with cervical cancer(CC group).According to HR-HPV testing,these samples were divided into 99 patients with HPV16/18 infection(HPV16/18group)and 30 patients with other carcinogenic HR-HPV infection(non-HPV 16/18 group).According to age,these samples were divided into 94 patients aged 35 years or older(the older group)and 35 patients younger than 35 years(the younger group).The combined methylat io n detection of host genes was used to analyze the methylation status of different grades of cervical lesions,and statistical methods were used to analyze the relationship between methylation status and ages/HR-HPV types/pathological results.The triaging effect of the combined methylation detection,TCT or the combination of the two methods for HR-HPVpositive patients was compared,and p<0.05 was statistically significant.Results:1、Methylation rates of the combined methylation detection of host genes in CIN0,CIN1,CIN2,CIN3 and CC groups were 8.9%,7.1%,50.0%,91.3% and 100.0%,respectively,and the methylation rates of these five groups showed statistically significant differences.But pair comparison showed no statistically significant differences in methylation rates between CIN0 and CIN1 and between CIN3 and CC groups.And the methylation rate of CIN2 group was significantly higher than that of CIN1 group and lower than that of CIN3 group,both of which were statistically significant.In general,the methylation rates were significantly increased in CIN1-(8.2%),CIN2(50.0%)and CIN3+(94.4%),and the overall comparison and pair comparison all showed statistically significant differences.2 、 The methylation rate of the combined methylation detection of host genes in HPV16/18 group was slightly higher than that in non-HPV16/18 HR-HPV group,but the difference between the two groups was not statistically significant.However,the methylat io n rate of the younger group was significantly lower than that of the older group,and the difference between the two groups showed statistical significance.3 、 Median scores of the combined methylation detection of host genes showed a significant increase trend in CIN1-(0),CIN2(0.45)and CIN3+(1.2).The overall comparison and pair comparison all showed statistically significant differences.4、The combined methylation detection of host genes enabled to reduce colposcopy referral rate to 38.8%,and had an excel ent performance in identifying CIN2+/CIN1-,CIN3+/CIN2-and CIN3+/CIN1-with the sensitivities of 78.6%,94.4% and 94.4%,respectively and the sensitivities of 91.8%,82.8% and 91.8%,respectively.Compared with the older group,the combined methylation detection showed lower sensitivity but higher specificity in the younger group.5、For the differential diagnosis of CIN2+/CIN1-and CIN3+/CIN1-,the sensitivities of the combined methylation detection of host genes were significantly higher than that of TCT(78.6% vs 44.6%;94.4% vs 61.1%),whereas the specificities of the combined methylat io n detection of host genes were slightly lower than that of TCT(both 91.8% vs 98.6%)and the differences were not statistically significant.For the differential diagnosis of CIN3+/CIN2-,there were statistically significant differences in sensitivity and specificity between the combined methylation detection and TCT(94.4% vs 61.1%;82.8% vs 95.7%).The Youden indices of the methylated combination detection for CIN2+/CIN1-,CIN3+/CIN2-and CIN3+/CIN1-were 70.4%,77.2% and 86.2%,respectively,whereas those of TCT were 43.2%,56.8% and 59.7%,respectively.Additionally,compared with the combined methylat io n detection,the combination of the combined methylation detection with TCT did not significantly improve the sensitivities and specificities for identifying all classifications,and the differences were not statistically significant.6、Methylation rates of the six independent host genes were statistically significant in the overall five groups,but pairwise comparison showed that the methylation rates of the six independent genes were no statistically significant differences between the CIN0 and CIN1 group and between the CIN3 and CC group.Furthermore,significant differences were found in methylation rates of only ZNF671 gene between CIN1 and CIN2 groups and between CIN2 and CIN3 groups.In terms of methylation analysis of a single gene,ITGA4 gene showed the highest specificities for detecting CIN2+/CIN1-,CIN3+/CIN2-and CIN3+/CIN1-of 97.3%,95.7% and 97.3% respectively,but lowest sensitivities of 35.7%,50.0% and 50.0%,respectively.Besides,ZNF671 gene showed the best performance for triaging HR-HPVpositive patients.And the sensitivities for identifying CIN2+/CIN1-,CIN3+/CIN2-and CIN3+/CIN1-were 78.6%,94.4% and 94.4%,respectively,and the specificities were 95.9%,86.0% and 95.9%,respectively.7、During the 1-year follow-up,one of the six combined methylation positive patients with histologic cervicitis and CIN1 was lost to follow-up,and the HR-HPV testing results of the remaining five patients were persistently positive,and one of them underwent cervical biopsy again with histologic progression to CIN2.Moreover,five patients with CIN2 who did not undergo surgical treatment were also followed up.Two of them had positive combined methylation results,and throughout the follow-up period,their HR-HPV tests still remained positive.The remaining three patients had negative combined methylation results,of which two patients turned to be HPV-negative.And the other one patient remained HR-HPV positive but had a decreased viral load,and her TCT results turned from ASC-H to NILM.Conclusions:1、In this study,the combined methylation detection of host genes was not related to HRHPV types,but was related to pathological results and ages.Methylation rates of the combined methylation detection were significantly increased in CIN1-,CIN2 and CIN3+,and significantly decreased in group under the age of 35.2 、 The combined methylation detection of host genes can significantly reduce the colposcopy referral rate without loss of sensitivity,which is especial y important for women in the reproductive age.And it had the best performance in the identification of CIN3+/CIN1-with sensitivity and specificity of 94.4% and 91.8%,respectively,following by CIN3+/CIN2-with sensitivity and specificity of 94.4% and 82.8%,respectively.3、The combined methylation detection of host genes was a better triage tool for HRHPV than TCT,but combining the combined methylation detection with TCT did not significantly improve diagnostic efficiency compared to the combined methylation detection.4、Methylation analysis of ZNF671 gene exerted the best triage effect than those of other five genes.Furthermore,methylation analysis of ZNF671 gene and the combined methylat io n detection of host genes performed equally well in detecting all classifications.The advantage of methylation analysis of ZNF671 gene lie in its simplicity and economy,while the advantage of the combined methylation detection lie in that its score may be related to the severity of cervical lesions.5、The combined methylation detection of host genes provided a novel idea for the management of CIN2,and it had the potential to recognize HR-HPV who were at risk for progression in a short period of time,but large sample data are still required to verify the conclusion. |
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