Improved human papillomavirus DNA typing methods and biology of cervival cancer

University dissertation from Stockholm : Karolinska Institutet, Department of Molecular Medicine and Surgery

Abstract: Cervical cancer is the second most common cancer among women worldwide. Persistent HPV has been identified as a main risk factor in cervical cancer development. Oncoproteins E6 and E7 of highrisk HPV interact with cellular proteins, subverting cell cycle checkpoint, inducing carcinogenesis. However, only a small proportion of HPV-Minfected women will develop cervical cancer. Other factors may be involved in carcinogenesis, such as other sexually transmitted infections (STIs), genetic predisposition and impaired immune response. Early diagnosis and treatment of cervical precancerous lesions can prevent cervical cancer progression. Widespread use of the Pap smear has decreased the incidence of cervical cancer by 70% in developed countries. HPV DNA testing may further improve the efficiency of cervical cancer screening. In addition, HPV induced cellular protein-biomarkers may act as diagnostic markers in predicting disease progression. HPV vaccines, currently in development, may eventually prevent HPV infection and further decrease the incidence of cervical cancer. Accurate detection and genotyping of HPV is important for the clinics. By using multiple sequencing primers method, we were able to genotype a panel of 65 cervical and 17 oropharyngeal samples for the most clinically important HPV genotypes (HPV 6, 11, 16, 18, 31, 33, and 45) using Pyrosequencing technique. By introducing the sequence pattern recognition, we are able to identify multiple coinfections of HPV types. We also showed that this method is suitable for amplicons with non-specific amplification products and samples with low amplification yields (Paper I). Furthermore, we developed a rapid assay for twelve most common oncogenic HPV genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) that use a single base, acting as a sentinel to identify the presence of particular HPV genotypes in the specimen. 244 HPV positive cervical specimens were genotyped with this sentinel-base method. The results showed about 25% of them had multiple co-infections of HPV. Thus this sentinel-base method is capable of rapidly genotyping samples containing single or multiple HPV co-infections (Paper II). Other STIs may act as cofactors for cervical carcinogenesis. Co-infection of AAV and HPV can occur in the cervix. AAV-2 Rep78 gene inhibits HPV-induced cell transformation in vitro. To investigate the role of AAV in cervical cancer development, we detected AAV-2 DNA in a retrospective, populationbased nested case-control study. 104 cases and 104 controls were matched by age and time of sampling. Our data showed a link between AAV and cervical cancer. This has to be confirmed in a larger population study base would be required and experiments to show causality (Paper III). Immunological factors play an important role in cervical cancer development. Two genetic variations in chemokine receptors CCR2 and CCR5 were investigated for their association with HPV infection in cervical neoplasia. We analyzed 50 CIS or CIN cases and 50 matched normal controls, and 100 cervical cancer cases and 100 matched healthy controls. All the cases and their responding controls were matched by age and date of sampling. We found that those with the A32/A32 genotype of the CCR5 have a risk of 4.58 (Cl 0.40-52.64, pvalue=0.045), whereas those with CCR2-641 polymorphism did not confer any increased risk for HPV infection. However, there is no evidence of association between these two chemokine receptor gene polymorphisms and the development of cervical neoplasia (Paper IV). During carcinogenesis, oncoproteins E6 and E7 of high-risk HPVs interact with host cellular proteins, particularly through p 1 CINK4A-cyclin D 1 -CDK4/6-pRb-E2F and p 14ARF-MDM2-p53 pathway, resulting in over proliferation and genomic instability of the host cell, further leading to tumorigenesis. We employed immunohistochemistry to investigate the expression of host cellular proteins (biomarkers) p16INK4A, p14ARF, p53 and PCNA on paraffin sections of serial consecutive biopsies from cervical cancer patients. Our results suggested that increased p16INK4A and p14ARF , and decreased or stable p53 expression, are associated with disease progression. The disease developed more quickly among samples staining positive for p16INK4A or p14 ARF, and those with altered p53 expression. Thus, these biomarkers can act as prediction markers for cervical cancer progression (Paper V).

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