| purposes:1.To detect the expression of high-risk HPV E6/E7 mRNA in cervical intraepithelial neoplasia and cervical cancer tissues by novel RNA scope technology,and to explore the diagnostic value of RNA in situ hybridization in cervical lesions.2.To analyze the consistency of high-risk HPV E6/E7 mRNA in situ hybridization detection and HPV DNA typing detection in cervical precancerous lesions,and its value in the diagnosis of cervical precancerous lesions.3.To analyze the relationship between high-risk HPV E6/E7 mRNA in situ hybridization and P16 protein expression in cervical lesions,and to explore the diversion effect of the two in cervical cancer screening.Material and Methods:1.Collecting cases:258 cases of cervical tissue from cervical biopsy or conical resection of the Department of Pathology of the General Hospital of PLA from January 2016 to December 2017,including pathological diagnosis of high-grade and low-grade squamous squamous intraepithelial lesions(SIL)/cervical squamous intraepithelial lesions(CIN?,CIN?,CIN?,and CIN?-? cases),and cases with inflammation diagnosed by cervical biopsy at the same time were selected as the control group.2.Review the section:and use the new LAST(lower anogenital squamous terminology project)two-stage classification method for re-diagnosis,including 33 cases of inflammation group,110 cases of LSIL group,and 115 cases of HSIL group.3.For all collected cases:1)All specimens were fixed in 10%neutral formalin,embedded in paraffin,serially sectioned,and the expression of p16 in different groups was detected by immunohistochemistry.2)High-risk HPV E6/E7 mRNA in situ hybridization assay and analysis of results.3)Review the HPV DNA test results of the clinical examination data of the enrolled patients.4.Statistical analysis:1)The SPSS22.0 statistical software package is used for processing,and the counting data is analyzed by chi-square test P<0.05 was considered statistically significant.2)ROC curve evaluation statistic calculation:The area under the ROC curve is between 1.0 and 0.5.In the case of AUC>0.5,the closer the AUC is to 1,the better the diagnostic effect.3)Kappa test:The calculated result is-1?1,and the closer the result is to 1,the better the consistency.Results:1.Age distribution and statistical results of high-risk HPV E6/E7 mRNA in situ hybridization assay:1)High-risk HPV E6/E7 mRNA in situ hybridization detection in all age groups and statistical results:in 258 cervical biopsy tissue samples,the minimum age of 19 years,the maximum age of 74 years,the average age of 40 years,the median age 38 years old.The average age of each group is,Inflammation group 42:33±10.08 years old,The LSIL group 40.15±11.36 years old and the HSIL group 40.06±10.52 years old.The age comparison between groups was analyzed by analysis of variance.There was no significant difference in age between the groups(p=0.5442).All cases were grouped according to?30 years old,31-40,41-50,51-60,61-70,?71 years old,the high-risk HPV E6/E7 mRNA positive rate was the highest in the?30 years age group,84.62%(44/52).2)HR HPV E6/E7 mRNA in situ hybridization results are expressed in different groups:In the 258 samples,the overall positive rate of HR HPV E6/E7 mRNA in situ hybridization was 79.46%(205/258);the expression in each group was,Inflammation group 45.45%(15/33)and LSIL group 70.91%(78/110),HSIL group 97.39%(112/115);there was a statistically significant difference between the groups(?2=50.960p<0.001).73%(11/15)of the positive expression of the inflammatory group was expressed in the superficial epithelium with a few granular signal points;44%(33/75)of the positive expression of LSIL group was mainly characterized by diffuse nuclear staining in the upper epidermis.The plaque-like positive signal was more common and concentrated mainly on the epidermis 1/2;83%(84/101)of the HSIL group were positively expressed as scattered or diffuse full-thickness signals.After chi-square test,HR HPV E6/E7 mRNA in situ hybridization was significantly different between the groups(?2=95.408p<0.001).As the degree of lesions increased,the positive expression of HR HPV E6/E7 mRNA gradually deepened from the superficial to the deep.2.Comparison of HR HPV E6/E7 mRNA in situ hybridization assay with HPV DNA assay results:1)Consistency of HR HPV E6/E7 mRNA in situ hybridization and HPV DNA detection:Kappa test showed that HR HPV E6/E7 mRNA in situ hybridization and HPV DNA had higher consistency in the detection of HSIL,kappa=0.796.2)Comparison of HR HPV E6/E7 mRNA in situ hybridization and HPV DNA detection in cervical squamous intraepithelial lesions:ROC curves were used to verify the sensitivity and specificity of cervical squamous intraepithelial lesions.According to the test results,the areas under the ROC curve of HR HPV DNA detection for identifying inflammation and LSIL,LSIL and HSIL were 0.584(95%CI464-0.705,p=0.147)and 0.500(95%CI0.424-0.577,p=0.990),respectively;HR HPV E6/E7 mRNA in situ hybridization to identify inflammation and LSIL,LSIL and HSIL under the ROC curve were0.726(95%CI00.640-0.812,p<0.001)and 0.716(95%CI00.647-0.784,p<0.001).3.Relationship between HR HPV E6/E7 mRNA in situ hybridization assay and p16 protein expression.1)Consistency of HR HPV E6/E7 mRNA in situ hybridization and p16 protein expression:Kappa test showed that HR HPV E6/E7 mRNA in situ hybridization and p16 detection for the diagnosis of precancerous lesions,kappa=0.550.2)Comparison of HR HPV E6/E7 mRNA in situ hybridization and p16 protein detection for the sensitivity of cervical squamous intraepithelial lesions:Statistical analysis and analysis of HR HPV E6/E7 mRNA in situ hybridization and p16 protein expression in the LSIL group and the expression of the HSIL group and the ROC curve were used to verify the sensitivity and specificity of the two methods for cervical squamous intraepithelial lesions.HR-HPV E6/E7 mRNA in situ hybridization identified the area under the ROC curve of LSIL and HSIL as0.718(95%CI00.650-0.786,p<0.001),and the area under the ROC curve of p16 protein detection to identify LSIL and HSIL was0.835(95%CI00.778-0.892,p<0.001).Conclusions:1.The positive rate of HR-HPV E6/E7 mRNA in situ hybridization increases as the level of cervical lesions rises.It is confirmed that the results of HR-HPV E6/E7 mRNA are valuable for diagnosis and differentiation of cervical lesions,and can provide more evidence for the diversion of cervical precancerous lesions..2.The positive expression of HR-HPV E6/E7 mRNA in situ hybridization showed different expression patterns from superficial to deep layer with the severity of cervical precancerous lesions.HR-HPV E6/E7 mRNA in situ hybridization can reflect the state of HR HPV virus in the host.3.HR-HPV E6/E7 mRNA in situ hybridization and HPV DNA detection have the highest rate of agreement in HSIL.HPV DNA detection has higher sensitivity to inflammation and LSIL.HR-HPV E6/E7 mRNA in situ hybridization detection is more efficient for LSIL and HSIL.4.Compared with p16 protein detection,HR HPV E6/E7 mRNA in situ hybridization can reflect the integration status of HR HPV virus and host cells earlier,while p16 protein detection has better diagnostic value for identifying LSIL and HSIL.To focus and follow-up patients who express inconsistent results through the two methods can be quite helpful for improving screening and have early warning. |
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