Page 120 - Strategies for non-invasive managementof high-grade cervical intraepithelial neoplasia - prognostic biomarkers and immunotherapy Margot Maria Koeneman

Chapter 6
HPV typing
Annotations of the affected epithelium were made on the HE slide, after which DNA isolation of the formaline fixed paraffin-embedded tissue was performed via Maxwell 16 (Promega). A PCR GP5+/6+ was run with HPV universal primers. HPV positivity was assessed via agarose gel electrophoresis. Positive HPV cases were subtyped using an Enzym Immuno Assay. Possible outcomes are positivity for HPV16, HPV18 or cocktail hrHPV (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66).
Outcome measures
Disease regression was defined as follows. For patients who completed the follow-up schedule, disease regression was defined PAP 1/2 cytology or ≤CIN 1 histology at the 24-month follow-up and no diagnosis of CIN 2 or higher before the 24-month follow-up visit. For patients who did not complete the 24-month follow-up schedule, those with PAP 1/2 cytology at the last follow-up visit were included in the regression group, and all other patients were included in the persistence group.
Potential prognostic factors were selected based on expert opinion and published studies reporting factors that influence the natural course of CIN, taking into account the availability of these factors in the studied population. The following potential predictors were assessed: (1) HPV16/18 negative lesion in baseline biopsy, (2) weak p16 staining in baseline biopsy, (3) weak KI67 staining in baseline biopsy, (4) lower age at CIN2 diagnosis, (5) not smoking at time of CIN2 diagnosis, (6) last PAP smear result before CIN2 diagnosis < PAP 3 (i.e. PAP 1 or PAP 2), (7), no more than one CIN2 lesion in case of multiple baseline biopsies, (8) no use of oral contraception at time of diagnosis and (9) nulliparity at time of diagnosis.
Statistical analysis
Statistical analysis was performed using IBM SPSS Statistics for windows (version 24.0. Armonk, NY: IBM Corp). Baseline characteristics were summarized using mean (standard deviation (SD), range) for numerical variables, and number of patients (%) for categorical variables. The influence of potential prognostic factors on spontaneous regression was first assessed using univariable logistic regression. Additionally, multivariable logistic regression analysis was performed, correcting for age and HPV 16/18 status. For each prognostic variable, the correction was performed in two separate analyses, as correction for both potential confounders was considered inappropriate given the small sample size. Outcomes were presented as odds ratios (OR) with 95% confidence intervals (CI). In case of missing data, these cases were omitted from the analysis (complete case analysis, CCA). A two-sided p-value smaller than or equal to 0.05 was considered statistically significant.
Results
Population
81 cases of CIN2 were identified and included in the study. After revision, 23 cases were excluded (8 cases were considered CIN1 or CIN1-2 and 15 cases were considered CIN3). Of the remaining 58 CIN2 lesions, 56 were positive for hrHPV and were included in the current study. Patient characteristics can be found in table 1.
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