Diagnostic Performance Of HPV E6/E7 MRNA Assay For Detection Of Cervical High-grade Intraepithelial Neoplasia And Cancer Among Women With ASCUS

Cervical cancer act as the most common gynecological malignancy in the world and have high mortality and morbidity among all female cancers.However,since the1950 s,cervical cancer has been screened by cytology test,which has resulted in a significant decrease in the incidence and mortality of cervical cancer in the population.At present,the cervical exfoliative cytology results reporting system has adopted the TBS classification method,and then conducted further treatment for different reported results.However,up to now,there is still controversy over the treatment of patients with atypical squamous cells of undetermined significance(ASCUS).Currently the following three methods unanimously approved by experts and scholars:1.cervical cytology again 6-12 months later;2.further colposcopy and cervical biopsy if necessary;3.Using High risk-human papillomavirus(HR-HPV)DNA testing to triage ASCUS.After big data analysis,for ASCUS patients,further HR-HPV DNA test can not only reduce the referral for colposcopy and avoid the waste of medical resources,but also reduce the chances of missing high-grade cervical lesion.However,with further research,many scholars found that using HR-HPV DNA test to triage ASCUS patients resulted in such a problem of lowspecificity and low positive predictive value,and similarly increased the unnecessary colposcopy.Therefore,exploring a test index that can accurately shunt ASCUS patients is necessary and meaningful to manage ASCUS patients.A number of studies have shown that the expression of HR-HPV E6/E7 gene is a necessary step for cervical persistent HPV infection and carcinogenesis.During the carcinogenesis procedure of cervical epithelial cells,HPV integrates its E6/E7 DNA into the genome of cervical epithelial cells,so that cervical epithelial cells acquire oncogene E6/E7 which is activated and transcribed to E6/E7 mRNA under the stimulation of external carcinogens,and then E6/E7 protein is produced,which binds to the tumor suppressor protein P53 and the retinoblastoma protein(PRb)respectively,resulting in the inactivation of the latter two proteins,eventually resulting in cervical carcinogenesis.Therefore,the detection of HR-HPV E6/E7 mRNA not only represented the persistent infection of HPV,but also to some extent reflected the expression of HR-HPV E6/E7 oncoprotein and the activity of cervical lesions.Therefore,detecting the expression of HR-HPV E6/E7 mRNA in ASCUS patients is expected to make the further treatment problems of ASCUS patients be resolved.ObjectiveIn this study,we investigate the risk factors affecting pathological biopsy results of high-grade cervical intraepithelial neoplasia and cervical cancer,including cervical intraepithelial neoplasia(CIN)grade and above lesions,namely CIN ?+ in patients with ASCUS,by analyzing the age,number of pregnancies and deliveries,age at first childbirth,qualitative results of HR-HPV DNA,category loads of HR-HPV DNA,qualitative results of HR-HPV E6/E7 mRNA and category expression level of HR-HPV E6/E7 mRNA of ASCUS patients.Again,we evaluate the value of HR-HPV E6/E7 mRNA quantitative assay in triage of ASCUS Pap smears,so as to provide an objective basis for guiding the clinical treatment.Methods1.A total of 160 effective residual sample solution were consecutively collected in the period from December 2015 to July 2017 from patients who underwent a regular liquid-based cytology(LBC)test using the ThinPrep system diagnosed as ASCUS and performed colposcopy and underwent HR-HPV DNA testby hybridization capture ?(HC ?)method at the Department of Cytopathology in the third Affiliated Hospital of Zhengzhou University.The clinical factors such as patient age,number of pregnancies and deliveries,age at first childbirth,result of HR-HPV DNA detection and cervical biopsy pathology results of all patients were recorded in detail.2.Residual LBC specimens were used for the detection of E6/E7 mRNA by branched DNA technology according to the manufacturer's instructions.Statistical analysisStatistical analysis was carried out using SPSS 21.0 software.Univariate and multivariate analysis were used to evaluate the association between clinical factors and CIN ?+.The differences of HR-HPV E6/E7 mRNA expression levels among different grades of cervical lesions were evaluated by Mann-Whitney U test.Receiver operating characteristic(ROC)curve was used to assess the optimal diagnosis of E6/E7 mRNA quantitative assay.Test level taken bilateral ? = 0.05.Results1.A total of 160 patients with ASCUS underwent pathology diagnosis.Cervical inflammation was diagnosed in 69(43.125%).CIN ? was diagnosed in 60(37.50%),CIN ? in 22(13.75%)and CIN ? in 8(5.00%).There was one case of cervical squamous cell carcinoma(0.625%).Among them,31 cases of cervical biopsy were CIN ? +(19.38%).2.The clinical factors(qualitative results of HR-HPV DNA,qualitative results of HR-HPV E6/E7 mRNA and high expression level of HR-HPV E6/E7 mRNA)were associated with CIN ?+(all P < 0.05).There was no significant correlation between the clinical factors(patient age,the number of pregnancies and deliveries,age at first childbirth and category loads of HR-HPV DNA)and incidence of CIN?+ in women with ASCUS.Multivariable analysis was performed by using Logistic regression analysis of the likelihood ratio method to screen variables,which found that only the expression level of HR-HPV E6/E7 mRNA was associated with CIN ?+(OR = 8.971,95% CI = 2.572–31.289,P = 0.001).3.The median loads of HR-HPV E6/E7 mRNA in CIN ?+ tissues were11775.97 copies/ml.Cervical inflammation and CIN ? was 537.07 copies / ml,with statistical significance(P <0.05).4.The sensitivity of HR-HPV E6/E7 mRNA classification analysis,HR-HPV E6/E7 mRNA qualitative analysis,and HR-HPV DNA qualitative analysis to diagnose CIN II+ were 90.32%,88.46%,100%,and specificity were 49.61%,40.30%,15.05%.Using the ROC curve to further analyze the HR-HPV E6/E7 mRNA load analysis,the best critical point for diagnosis of CIN II+ in ASCUS patients was558.26 copies/ml,and then,the sensitivity was 90.32% and the specificity was51.16%.The area under the curve of the HR-HPV E6/E7 mRNA load analysis and the above three methods were further compared.The area under the curve of the HR-HPV E6/E7 mRNA load analysis was the highest,with 0.749(95%CI=0.658-0.840).Conclusion1.ASCUS patients with pathological diversity,located in inflammation,CIN lesions at all levels and invasive cervical cancer.2.HR-HPV E6/E7 mRNA high load is an important factor affecting cervical pathology results of CIN ? + in ASCUS patients.3.HR-HPV E6/E7 mRNA assay can accurately diagnose ASCUS patients with CIN ? + lesions and is expected to replace the HR-HPV DNA detection to triage ASCUS patients.