Impact of HPV Vaccination on the Distribution of HPV in High Grade Lesions of the Cervix in France

The prevalence of the different HPV in high-grade lesions (CIN2/3) will be determined using the INNOLiPA HPV extra v2 genotyping kit (Fujirebio). Only samples with a histologically proven high-grade cervical lesion (grade 2 or 3 intraepithelial neoplasia) are concerned. The patient year of birth, the year of the lesion diagnosis and the histological diagnosis will be recorded Participating laboratories will select eligible cervical samples (biopsies or surgical specimen) according to the inclusion criteria defined in the protocol. Only cervical samples with sufficient biological material will be selected. The selected tissue blocks will be sent to the Papillomavirus National Reference Center (NRC) by mail (pre-paid envelope provided) in accordance with the rules for the transport of biological samples. After receipt by the NRC, the tissue blocks will be anonymized. After HPV genotyping, the blocks will be returned to the laboratory. Transport costs are covered by the Papillomavirus NRC.

Samples have to be fixed in 4% buffered formaldehyde (maximum 24 hours) and then included in paraffin for histopathological evaluation. On each of the selected histological pieces, the papillomavirus NRC will make 5μm thick sections using a microtome. The number of sections to be made depends on the size of the histological specimen. A minimum surface area equivalent to 5 sections of 5μm of a 125 mm2 piece is required for genotyping.

Tissue sections are grouped in a labelled 2 mL screw tube. To avoid any contamination between samples, the microtome blade will be carefully cleaned with xylene and then 95% ethanol between each block.

After DNA extraction using the QiaAmp DNA mini Kit (Qiagen), HPV genotype analysis will be performed using molecular techniques for the detection of human papillomavirus DNA. The investigators will use the INNO-LiPA HPV Genotyping Extra II (Fujirebio) kit. This is a Polymerase Chain Reaction (PCR) technique using degenerate primers followed by reverse hybridization on strips on which the specific probes of 32 alpha HPV genotypes are immobilized. This kit allows the detection of 13 high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 52, 56, 58, 59, 68), 6 HPVs that are likely to be at risk (HPV 26, 53, 66, 70, 73, 82) and 14 low-risk or unclassified HPVs (HPV6, 11, 40, 42, 43, 44, 54, 61, 62, 67, 81, 83, 89). In addition, the samples will be analyzed by PCR luminex according to the technique published by Schmitt et al, J Clin Microbiol 2010. In addition to alpha HPV, this technique allows the detection of sixty beta and gamma HPVs.

Characteristics of the subjects to be included A description of patients included in the study (women born between 1972 and 1982, on the one hand, and women born between 1983 and 1993, on the other) will include age at time of sampling, year of sampling, stage of lesion CIN2 or 3, and histological type.