Improvement of DNA Extraction for Human Papillomavirus Genotyping from Formalin-Fixed Paraffin-Embedded Tissues

Mucosal human papillomaviruses (HPVs) play a crucial role in the development of cervical carcinoma, the third most common malignancy in women worldwide. These viruses are classified as high risk (HR) or low risk (LR), depending on their transforming ability. Formalinfixed paraffin-embedded (FFPE) tissues represent the most frequent form of tissue storage in pathology departments. These archival tissues represent a potentially useful resource for retrospective epidemiological studies. Several HPV studies have used FFPE specimens to correlate HPV genotypes with histological classification, to establish the geographical distribution of HPV genotypes, or to search for these viruses in other primary cancers. In these tissues, the detection of viruses by molecular biological tools based on molecular hybridization between DNA or RNA targets and specific probes is problematic because fixation causes damage to nucleic acids. Formalin fixation induces protein–protein and protein–nucleic acid crosslinking. As a result of protein–nucleic acid cross-linkages, it is difficult to separate DNA from histones and to obtain pure nucleic acids at extraction. The fixation of tissues also leads to the fragmentation of nucleic acids, such that polymerase chain reaction (PCR) methods that amplify a smaller portion of the viral genome are most effective. The aim of this study was to test whether HPV genotyping techniques used routinely in our diagnostic laboratory could be applied to FFPE tissues. For recovery of HPV DNA from FFPE tissues, we had to adapt extraction protocols to obtain viral DNA useful for PCR amplification. We tested different extraction protocols for HPV DNA, mainly to simplify this step, and we tested two genotyping methods. The first genotyping method involved sequencing of the L1 open reading frame. This method links PCR amplification of a conserved HPV L1 segment using GP5+/GP6 + consensus primers with automated sequencing of amplified PCR products. The second method used the Greiner Bio-one (GBO) PapilloCheck DNA microarray, which enables the detection and genotyping of 24 different HPV types (18 high risk and 6 low risk) from DNA preparations of human cervical cytology specimens.