| Objective1. To analysis HPV integration prevalence and genotypes’ distribution in cervical intraepithelial neoplasia and cervical cancer in Qingdao, furthermore analysis the preferential sites for common HPV integration and provide baseline information for Chinese women cervical abnormalities screening and prevention.2. To investigate the law of HPV 16 and HPV 58 integration status in cervical cancer tissues, the distribution of integrated sites in human genomes and the function of loci that virus integrated, discuss the interaction between integration status and clinical pathology.Methods We collect 149 paraffin-embedded tissue specimens of CIN and cervical cancer patients,who were given cervical cold knife ionization and radical hysterectomy in the Affiliated Hospital of Qingdao University Medical College between May 2008 and May 2013. HPV gene integration state and integration sites of all the samples were detected using whole-genome sequencing and high-throughput viral integration detection in Key Laboratory of Biotechnologies in BGI-Shenzhen.Results1. Mean age of the 149 cervical abnormality patients was 42.30±7.49, while patients who had HPV integrated was 41.34±6.84. Younger patients than 45 has a higher integration rate than that elder than 45.2. 93 samples were HPV-integrated, the integration rate of all samples was 62.42% and as the cervical lesions increase, the integration rate grow higher significantly(p<0.01). CIN3 and cervical cancer had obviously higher integration rates than other tissues. In 93 integrated samples, 83.87% of them was HR-HPV integrated and the integration rate also grow higher significantly(p<0.01).3. The most common genotypes of all the cervical lesions were HPV 16(42.28%),HPV58(20.13%),HPV 31(18.12%),HPV 35(16.11%)and HPV 33(8.05%); in 35 CIN 1 samples, the common genotypes were HPV 58(11.43%), 52(8.57%)and 56/ 66/68(each type was 5.71%); in 35 CIN 3 samples, the common genotypes were HPV 16/58(14.29%), 33/ 66(8.57%)and 52(5.71%);4 genotypes was detected in CIN 3, they were HPV 16(65.12%), 58(16.28%), 33(6.98%)and 31(2.33%); in 36 cervical cancer patients, the common types were HPV 16(80.56%),31(69.44%),35(66.67%),58(38.89%)and 51(22.22%).4. Multiple HPV integration were detected in the 149 samples, the multiple-integration rate of cervical cancer was significantly higher than CIN patients(p<0.01), among the CIN patients, the co-integration rates had no correlation with the severity of cervical lesions(p>0.05). Single HPV integration rate was higher than cervical cancer obviously(p<0.01), of all the CIN samples, single integration were more common than multiple integration, and integration rate grow higher while the CIN degree grows(p<0.05).5. Integration of HPV 16 in all the 149 samples was 42.28%, the integration sites was widely distributed in chr1 to chr X, the most common chromosome that had integration sites were chr1, chr2, chr4, chr X and chr5. Integration of HPV 58 in all the samples was20.13%, the integration sites was also widely distributed in chr1 to chr X, the most common chromosome that had integration sites were chr5, chr9, chr2, chr3 and chr4.HPV gene disruption happened in E6 to LCR, the frequency of HPV gene disruption was higher in L1 and E1 gene than in other regions of the viral genomes. Among 29 cervical cancer tissues that detected HPV 16 integration, 17 happened in the region, while 14 HPV58 integrated tissues, 1 sample had gene promoter integrated, of all the 94 sites, many of them had tumorigenesis and immune function.6. There was relation between cervical FIGO stage and HPV 16 integration(p<0.05),and so was HPV 58. HPV 16 integration rates grow higher when the tissue was squamous cell carcinoma or cervical stromal invasion ≥1/2 or the lymph vascular invasion; HPV 58 integration rate increases when patients’ age ≥50 years, poorly differentiated, squamous cell carcinoma, cervical stromal invasion ≥1/2, the lymph vascular invasion or the lymph node metastasis, although the former clinical factors didn’t have significantly correlation with HPV 16 and 58 integration, but they can promote that multiple virus integration was the risk factors for cervical stromal invasion ≥1/2 and the lymph vascular invasion.Conclusion1. HPV DNA prone to integrate in the younger patients below 45 years old, indicate that we should pay more attention on the strict follow-up for these women.2. HPV and HR-HPV integration not only detected in CIN tissues but in cervical cancer samples, the rate of integration grow higher while the cervical abnormalities increase,indicate that HPV integration play a key role in cervical cancer progression. Multiple integration rate was higher in cervical cancer but in CIN, the single integration rate was parallel with the severity, this may prompt in different stage of cervical abnormalities,characteristics of co-integration and single integration were different, HPV integration play different roles during the development of cervical cancer.3. The most common integration genotypes of HPV was HPV 16,HPV 58,HPV 31,HPV 35 and HPV 33; as the severity of cervical abnormalities increase, different integration rates were detected.4. HPV 16 and 58 genes can be widely integrated into the host chromosomes without any regularity. HPV fracture happened in every regions of its gene from E6 to LCR, the most common site was L1, then that was E1. Integration sites of HPV 16 and 58 showed a prone to near the tumorigenesis and immune related genes.5. In cervical cancer, HPV 16 and 58 integration rates grow while the FIGO stage increase;cervical stromal invasion ≥1/2 and the lymph vascular invasion were the risk factors for virus integration.
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