HPV genotyping and potential progression markers in cervical intraepithelial neoplasia : Clinical and diagnostic impact

The aim of this thesis is to identify clinically useful early molecular markers to predict progression to carcinoma in women with preinvasive lesions of the cervix, for the purpose of improving care of these women and enabling more individualized treatment. In order to define the human papillomavirus (HPV) types in minor cytological abnormalities, 343 liquid-based cytology (LBC) samples with atypical squamous cells of uncertain significance (ASCUS) and with low-grade squamous intraepithelial lesions (LSIL) were genotyped using Linear Array. We found high-risk (HR-) HPV in 71% of LSIL and 49% of ASCUS cases (p 50 years) women had higher prevalence of HR-HPV and multiple infections (p=0.01). HPV16 was found in 23% and HPV18 in 10% (p<0.001) of HPV-positive women. To test the utility of HPV genotyping in post-surgical monitoring, we genotyped cones of 90 women and follow-up samples after conization, and evaluated cytological results from two consecutive visits. Margin status and presence of CIN3+ in the cone were poor predictors of treatment outcome (sensitivity <50%). Presence of any probable HR/HR-HPV (18 types) predicted all residual high-grade SIL/ cervical intraepithelial neoplasia (CIN) 2 or worse with 73% specificity. Consideration of only 13 HR-HPV types showed equal sensitivity, but higher specificity (86%, p<0.01). True persistent infection detected high-grade residual disease with 60% sensitivity and 95% specificity, resulting in a positive predictive value (PPV) of 43%. We also assessed the utility of p16 INK4a immunocytochemical detection of dysplastic cells in 118 samples from patients referred for further testing because of cytological abnormality. Intensity of p16 INK4a staining correlated well with CIN grade, particularly when diagnosis was based on simultaneous routine cytology (p<0.001, Rho 0.70). Immunostaining for p16 INK4a was feasible in clinical practice and helped to distinguish premalignant cells from reactive cells. To map local immune responses to HPV, we analyzed expression of several immune markers using real-time RT-PCR in cervical biopsies from 24 female volunteers who had been genotyped for HPV. No difference between the 11 HPV-positive and 13 HPV-negative women was found for mRNA expression of any of the immune markers. Surprisingly, levels of the B cell marker CD19 were elevated among women using hormonal contraception (p<0.05). HPV genotyping revealed age-dependent patterns of HPV infections in LSIL and ASCUS cases. We propose triage HPV testing of LSIL after 30 years of age. Genotyping after conization substantially increased PPV, but with loss in sensitivity. General HR-HPV testing will identify all recurrent or residual high-grade CIN. Demonstration of p16 INK4a accumulation in the cell nucleus is a simple way to enhance presence of dysplastic cells and to distinguish these from reactive atypia. HPV infection per se does not evoke local immune response as measured by semiquantitative RTPCR. Hormonal contraception may influence B cell activity in the cervix. Further studies are needed to identify potential progression markers. LIST OF PUBLICATIONS This thesis is based on the following papers: I. BRISMAR WENDEL S, Froberg M, Hjerpe A, Andersson S*, Johansson B*. (*Equal contribution) Age-specific prevalence of HPV genotypes in cervical cytology samples with equivocal or low-grade lesions. Submitted to British Journal of Cancer, February 2009. II. BRISMAR S, Johansson B, Borjesson M, Arbyn M, Andersson S. Follow-up after treatment of cervical intraepithelial neoplasia by HPVgenotyping. American Journal of Obstetrics and Gynecology 2009;201:x-ex-x-ex (e pub ahead of print). III. Norman I*, BRISMAR S*, Zhu J, Gaberi V, Hagmar B, Hjerpe A, Andersson S. (*Equal contribution) p16(INK4a) immunocytochemistry in liquid based cervical cytology: is it feasible for clinical use? International Journal of Oncology 2007 Dec; 31(6): 1339-43. IV. BRISMAR WENDEL, S, Kaldensjo T, Petersson P, Andersson S, Broliden K, Hirbod T. Slumbering mucosal immune response in the cervix of human papillomavirus DNA-positive and negative women. In manuscript.