High-risk HPV infections are known to cause cervical and anal precursor lesions varying from productive infections that are likely to regress, to advanced transforming infections that might lead to cancer. In order to differentiate between these two types of lesions, different molecular markers that focus on human gene expression patterns or viral gene expression patterns are used. This study aims to provide insights in the value of markers that detect: (1) changes in human gene expression, hyper methylation of tumour suppressor genes in cervical smears, and in (2) viral gene expression patterns, immunohistochemical (IHC) staining with HPV E4 and p16INK4a in cervical and anal biopsies.
Women who underwent LEEP treatment for the suspicion of a high-grade CIN lesion (CIN2+) and had a cervical smear suitable for methylation testing were selected from a follow-up study conducted in Delft, The Netherlands and Barcelona Spain (N=199). Cervical smears were tested for hyper methylation of the tumour suppressor genes CADM1, MAL and miR124-2. Methylation results were compared to biopsy diagnoses. Cervical biopsies were stained with primary antibodies HPV panE4 and p16INK4a and immunostainings were scored to identify productive lesions (HPV E4+) and transforming lesions (HPV E4-, p16≥2/3 of the epithelium). In addition, the same IHC staining’s were performed on a selection of 67 anal biopsies collected in New York, USA. Results were compared with previously generated research.
In both cervical and anal lesions, the combination of HPV E4/p16 immunostaining assists in distinguishing between different stages of lesion development and disease severity. E4 expression is associated with low-grade disease and the use in combination with p16 might allow us to divide the cervical and anal intraepithelial neoplasia grade 2 (-IN2) group into E4 expressing lesions which are more low-grade-like and E4 negative lesions which are more high-grade-like. Hyper methylation detected in a cervical smear correlates to the worst lesion found on biopsy, with >70% of CIN3+ lesions positive for hyper methylation of at least one of the tested tumour suppressor genes. The relation of immunohistochemical markers tested on biopsies and methylation markers tested on smears to identify advanced transforming high-grade lesions is important for the identification of patients suitable for treatment.