| Cervical cancer is one of the most common malignant tumor in the word. Its incidence showed a steady rise in recent years. The malignancy often happened in the less-developed regions, and it is a main problem for the health of women in the developing countries. According to the statistics, about 174.000 women are died of cervical cancer every year worldwide. Lots of studies have shown that HPV infection is a necessary but not sufficient condition for the development of cervical cancer, and HPV 16 and 18 are the main subtypes that cause this diseases. Recently, it has been risen people's attention of the relation between the epigenetic factors of the virus and cervical cancer. There has been several researchers to study its founction in the process of malignancy, and the most studies were focused on the change of HPV 16 and HPV 18 gene methylation. As for HPV type 16, the target regions which were most frequent studied were L2, L1, LCR and E5/E6/E7. But it still not clear for the changing and the pathogenic mechanism of the HPV methylation, so the conclution of these studies are controversial. However, most of the researchers think that the methylation of L1 gene were related to the progression of cervical cancer, and suggested that L1 gene methylation has potential as a surrogate marker of viral integration and neoplastic progression.In 2011, the HPV mRNA detection mathod of Aptima(?) HPV Assay has been approved by FDA for clinical application. However, in the current study, most of the studies were compared the detection value between HPV E6/E7 mRNA and HPV DNA, but reraly to study the performance of HPV E6/E7 mRNA test compared with HPV DNA methylation test in the women who were positive with HPV screening. In the present study, we using MS-HRM and Quantivirus (?) HPV E6/E7 RNA 3.0 assay (b-DNA) to measure the HPV16 L1 DNA methylation and E6/E7 mRNA levels of various cervical lesions in the same residue of liquid-based cervical cytological (LBC) samples with HPV16-positive, respectively, to assess the performance of the two assays for detecting cervical precancer and cancer.Methods1. Materials:A total of 947 specimens (median age 35 years, range 17-76 years)were consecutively collected in the period from February 2012 to May 2013 from patients who underwent a regular liquid-based cytology (LBC) test using the ThinPrep system at the Department of Cytopathology in the 3rd Affiliated Hospital of Zhengzhou University. In order to identify HPV genotyping, all DNA were purified from the residual material of LBC samples and tested using PCR-Reverse dot blot hybridization technique. Only those women that were diagnosed with HPV type 16 only were enrolled in this study. Among the 947 participants,114 women (median age 37 years, range 25-76 years) with the results of cytology, histology and positive HPV DNA test for HPV type 16 only were included in this study, including 17 samples with chronic inflammation or negtive for cervical lesions served as the control group, CIN1 (25), CIN2 (29), CIN3 (32), SCC (11). This study was approved by the institutional Ethics Committee of the bioscience, Zhengzhou University. All patients signed an informed consent form before inclusion.2. Methods:(2.1) The target sequence of HPV16 L1 gene was searched from the Genebank and the specific primers design by primer Premier 5.0 for the methylation-independent PCR that targets the sequence. The 100% and 0% DNA methylation standards were synthesized according to the genetic base sequence of the target gene. The two standards were precisely quantified using quantitative real-time PCR (qPCR). Then, we mixed the two DNA standards in 0%,1%,10%,25%,50%,75% and 100% methylated to unmethylated template ratios, which served as the DNA methylation standards for MS-HRM. (2.2) The correct amount of DNA were isolated and purified from all the 114 residual LBC samples, then the HPV16 L1 DNA methylation levels of these DNA and the 100% methylated standard were measured by MS-HRM after bisulfite-converted. (2.3) The E6/E7 mRNA levels of these residue of liquid-based cervical cytological (LBC) samples were detected by Quantivirus(?)HPV E6/E7 RNA 3.0 assay (b-DNA).3. SPSS 17.0 software was used for the statistical analyses. Spearman rank correlations were used to evaluate the associations between HPV 16 L1 DNA methylation and E6/E7 mRNA levels and severity of cervical lesions. To analyze the difference at different ranges of DNA methylation and E6/E7 mRNA levels of different histological grades, the non-parametric 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. The overall positive rates of the HPV16 L1 DNA methylation were 6.3%,62.5%, 81.3%,92.3%, and 100.0% in control samples, CIN1, CIN2, CIN3, and SCC, respectively. The HPV16 L1 methylation levels increased with the histological diagnostic grades (rs,=0.661,P<0.001).CIN2+(CIN2,CIN3 and SCC) showed significantly higher methylation levels when compared with CIN2-(P<0.001).2. Among all of the 114 samples,81 samples were successfully detected for the HPV E6/E7 mRNA levels and the results were compared to histological diagnoses. The E6/E7 mRNA levels showed a statistically significant increase corresponding with the grades of histological follow-up diagnosis (rs=0.477, P<0.001). The E6/E7 mRNA positive rates of control samples, CIN1,CIN2,CIN3,and SCC were 50.0%,81.3%,93.8%,100.0% and 100.0%. respectively. Furthermore, the E6/E7 mRNA expression levels of CIN2+ were significantly higher than that of CIN2-(P<0.001).3. The overall positive rate of E6/E7 mRNA (85.2%) was higher than HPV 16 L1 methylation (72.8%,?2=4.221, P<0.05). E6/E7 mRNA levels increased with the grades of HPV16 L1 methylation (rs=0.313,P<0.05).4. When defining disease of histology-confirmed CIN2+ as the target condition, there were no statistical differences in the sensitivity. PPVs, and NPVs of HPV16 L1 DNA methylation, E6/E7 mRNA and the detection strategy of mRNA testing combining withHPV16 L1 methylation testing methods (all P>0.05). The specificity of HPV16 L1 methylation and combined detection methods were higher than E6/E7 mRNA testing method (all P<0.05). The specificity of combined detection method was higher than HPV16 L1 methylation testing, but there was no statistical difference.Conclusions1. The HPV16 L1 methylation and E6/E7 mRNA levels both increased with disease severity.2. We supposed that assessment of HPV16 L1 methylation testing combined with HPV E6/E7 mRNA testing is a promising method for the triage of women with a positive HPV DNA test for HPV type 16 only.3. When compared with HPV E6/E7 mRNA testing method, HPV 16 L1 methylation test was more advantage in reducing the false positive rate. The combined detection methods should has better clinical application prospects in the triage of the women with positive of HPV and cytological detection because of its higher specificity compared with separate detection of the two assays. |
PDF Full Text Request