The utility of HPV typing and relative quantification of HPV-16 transcripts for monitoring HPV vaccine efficacy and improving colposcopy triage of women with abnormal cervical cytology.

Abstract

Human papillomaviruses (HPV) can cause benign or malignant disease with the majority of infections without symptoms. The viral origin of cervical cancer is now proven with HPV proteins E6 and E7 defining part of the molecular basis of oncogenesis in vitro. Integration and/or malignant transformation of cervical cells in vivo are expected to be accompanied by the over-expression of HPV genes for E6 and E7 oncoproteins and a reduction of both expression for the L1 capsid protein and viral DNA. Cervical intraepithelial neoplasia (CIN) has been associated with particular HPV types that can be distinguished by DNA sequence differences. The research work studied two important aspects of HPV and its role in cervical disease. First, the distribution of HPV types and the epidemiology of HPV infection in a population of Saskatchewan women referred to a colposcopy clinic. Second, the potential of HPV-16 transcripts and HPV viral load for the detection of CIN.

The most commonly identified genotype in patients with CIN grade 2 or worse was HPV-16 (46.7%) followed by HPV-31 (14.7%) and then HPV-18 (3.9%). The risk of CIN associated with HPV-18 infection, odds ratio 0.8 (95% CI, 0.4 to 1.7) is significantly lower than either the odds ratio of 6.3 for HPV-16 (95% CI, 3.6 to 11.0) or 4.3 for HPV-31 (95% CI, 1.8 to 12.6). Thus in Saskatchewan, the prevalence of HPV-31 is high whereas HPV-18 is associated with less clinical disease. Consequently, the efficacy the new cervical cancer vaccine, which target only HPV-16 and HPV-18, may be diminished in Saskatchewan’s population.

Analysis of variance (P = 0.2) indicated no significant correlation between grade of CIN and HPV viral load. The presence of E6 RNA (P = 0.0002) and relative quantification of HPV-16 E6 transcripts (P < 0.0001) displayed the most significant median difference among the various grades of CIN when standardized to HPV viral load and human RNA and DNA levels. There was no correlation with L1 transcripts and cervical disease. Likelihood ratios indicate that the combination of Pap smear cervical cytology screening test with E6 relative quantification, on populations with higher cervical disease prevalence, would find more true positives than simply an additional Pap test. Using molecular testing for triage, HPV genotype information identifies 96% of women with CIN grade 2 or worse while eliminating 44% of women with CIN grade 1 or better. Information from the relative quantification of HPV-16 E6 transcripts identified 31.0% (n=13) of HPV-16 positive women with CIN grade 1 or better while retaining 92.4% of women with CIN grade 2 or worse. This work shows that there is diagnostic utility in relative quantification of HPV transcripts and that it benefits from standardization for variables such as the amount of HPV DNA and the total cellular nucleic acids. Relative quantification of HPV-16 E6 and HPV genotyping can be used to reduce medical procedures for women. HPV molecular tests could be useful in a cascade of diagnostic testing designed to refer women with cervical abnormalities for colposcopy, or treatment, while reducing the number of women needing triage.