| ObjectiveIn this study, the b-DNA and immunohistochemistry techniques were used to test the expression levels of HPV E6/E7 mRNA and HPV L1, respectively. The aim was to investigate the relation between the levels of HPV E6/E7 mRNA and L1 protien, and to assess the performance of the two assays for detecting cervical precancer and cancer, and to look for new cervical lesion detection method that could monitor the progress and recurrence of cervical lesions effectively. Methods1. Materials: The residue of liquid-based cervical cytological(LBC) samples of patients who underwent LBC test using ThinPrep system from Feb. 2014 to Feb. 2016 at the Department of Cytopathology in The third people's Hospital of Henan Province. The hybrid capture(HC2) assay was used to test the HR?-HPV DNA, and then the histologic results were tracked of these patients. Finally, 127 samples(median age 37 years, range 17-75 years) that were diagnosed with positive HPV DNA and with both the results of cytology and histology test were included in this study, in which 24 samples with negative cytological results(NILM) and histological diagnosis of normal or chronic inflammation were performed as the control. The residue of these patient's LBC samples and the paraffin-embedded specimen were collected to conduct HPV E6/E7 mRNA and HPV L1 protein testing, respectively.2. Methods:(2.1) The HPV E6/E7 mRNA levels of these residue of LBC samples were detected by b-DNA assay.(2.2) The HPV L1 protein expression levels were detected by immunohistochemistry technique.3. SPSS 17.0 software was used for the statistical analyses. Association analysis were used to evaluate the associations between HPV E6/E7 mRNA and HPV L1 protein expression levels and severity of cervical lesions. The McNemar was used to compare the positive rates of E6/E7 mRNA and HPV L1 protein. To analyze the difference at different ranges of E6/E7 mRNA and HPV L1 protein expression levels of different cytological and histological grades, the chi-square test were employed. The sensitivity, specificity, predictive values of diffierent methods were compared by chi-square test. Kappa statistic was used to assess the agreement between tests. P<0.05 was considered to be significant. Results1. All of the cases were detected for the HPV E6/E7 mRNA and HPV L1 protein expression levels and the results were compared to histological diagnoses. The E6/E7 mRNA and HPV L1 protein positive rates of control samples, CIN1, CIN2, CIN3, and SCC were 37.5%, 53.6%, 85.2%, 94.1%, 100.0% and 16.7%, 60.7%, 29.6%, 11.8%, 0%, respectively. Both of the E6/E7 mRNA and HPV L1 protein expression levels showed statistically significant assiociation corresponding with the grades of histological diagnosis(?2 were 35.801 and 27.320, respectively, all P<0.001), the correlation coefficient were 0.469 and 0.421, respectively(all P<0.001). Furthermore, the E6/E7 m RNA expression levels of CIN1-(include control and CIN1) were significantly lower than that of CIN2+(include CIN2,CIN3 and SCC, ?2=32.924, P<0.001), the HPV L1 expression levels of CIN1- were higher than that of CIN2+(?2=9.494, P<0.05).2. All of the cases were detected for the HPV E6/E7 mRNA HPV L1 protein expression levels and the results were compared to cytological diagnoses. The E6/E7 mRNA and HPV L1 protein positive rates of NILM, ASCUS, LSIL, ASC-H/LSIL-H, AGC, HSIL and SCC were 37.5%, 63.3%, 68.8%, 84.6%, 100%, 96.6%, 100.0%?16.7%, 43.3%, 68.8%, 15.4%, 14.3%, 6.9%, 0%, respectively. Both of the E6/E7 mRNA and HPV L1 protein expression levels showed statistically significant assiociation corresponding with the grades of cytological diagnoses(?2 were 31.680 and 30.553, respectively, all P<0.001), the correlation coefficient were 0.477 and 0.440, respectively(all P<0.001). Moreover, the E6/E7 mRNA expression levels of LSIL-(include NILM, ASCUS and LSIL) were significantly lower than that of ASC-H+(include ASC-H, LSIL-H, AGC, HSIL and SCC, ?2=24.403,P<0.001), the HPV L1 expression levels of LSIL- were significantly higher than that of ASC-H+(?2=15.930,P<0.001).3. The overall positive rate of HPV E6/E7 mRNA was significantly higher than that of HPV L1(26.0%, ?2=42.451, P<0.001). The agreement rate between the two assays was 64.57%(82/127). There was no association between E6/E7 mRNA and HPV L1 expression levels(?2=0.979,P>0.05).4. When take the histological diagnosis as the gold standard, there were no statistical differences in the sensitivity and NPV of E6/E7 mRNA, HPV L1 protein and the detection strategy of mRNA testing combining with HPV L1 protein testing methods(all P>0.05) in detecting CIN2+. The specificity of the combined testing methods were higher than HPV E6/E7 mRNA testing method(?2=7.278, P<0.016). The PPV of combined detection method was higher than HPV L1 protein testing(?2=6.518,P<0.016), but there was no statistical differences when comparing between the other groups(all P>0.016). Conclusions1. The HPV E6/E7 mRNA were increased with disease severity, but HPV L1 protein expression levels were decreased with disease severity.2. HPV E6/E7 mRNA testing combined with HPV L1 protein testing has higher specificity compared with separate detection of the two assays. The conbined testing method should haves better clinical application prospects in the triage of the women with positive of HPV. |
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