HUMAN PAPILLOMAVIRUS GENOTYPING IN OROPHARYNGEAL SQUAMOUS CELL CARCINOMA WITHOUT DNA EXTRACTION THROUGH EASYPAP DIRECT HPV DNA TEST

Objective. Human Papillomavirus (HPV)-induced oropharyngeal squamous cell carcinoma (OPSCC) represents an increasing tumor entity with typical clinical and histopathological features, associated with a rising incidence and improved prognosis (1). Detection of HPV-DNA is the most widely used method to diagnose HPV infection in clinical samples. Commercially available diagnostic assays show variable sensitivity and specificity and no international standards have been proposed yet (2). The main purpose of this preliminary study was to evaluate the reliability of EasyPap Direct HPV DNA Test (Ulisse BioMed) on formalin-fixed paraffin-embedded samples (FFPE), despite this test has been designed to work in vaginal mucus, where preliminary data showed a sensitivity of 97%. The test is real-time PCR based, it does not require DNA extraction and it can perform high-risk HPV genotyping. Kit performances have been compared with a commercial kit which requires DNA extraction. Materials and Methods. This pilot study included 38 cases of OPSCC that were surgically treated in our institute. Tumor samples were fixed in buffered formalin, embedded in paraffin and stored at room temperature for 1-13 years (mean storage period 4 ± 4 years). In order to confirm histology and lesion grade, all samples were reviewed by hematoxylin-eosin staining. DNA extraction has been performed starting from FFPE samples through QIAamp® DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) and HPV has been typed through Real Time PCR HPV sign® Pyro Mark System Q96 IDTM (CE-IVD Diatech Pharmacogentetics Iesi Italy). We found 16 cases positive for HPV16, 1 case positive for HPV35 and 21 HPV negative samples. In order to perform HPV detection through EasyPap Direct HPV DNA test, samples underwent deparaffinization with xylene and rehydration with ethanol. Genotyping data were compared to those obtained with Qiagen kit. Results. EasyPap Direct HPV DNA test identified 16 HPVpositive cases and 22 HPV negative cases. Genotyping data showed that 15 samples were HPV16 and 1 sample was HPV35 positive. Concordance analysis showed an agreement- coefficient Kappa Cohen K=0.95 IC95% [0.84-1.00] (excellent agreement, Fleiss classification). The sensibility and specificity of the EasyPap HPV DNA test was 94.1% (95%, confidence interval 80.1%-98.8%) and 100% (95%, confidence interval 88.6%-100%), respectively. The positive predictive value was 100% (95%, confidence interval 88.6%-100%) and the negative predictive value was 95.5% (95% confidence interval, 81.9%-99.3%). Conclusions. Hitherto it was well known that DNA extraction contributes to increase the sensitivity of PCR-based HPV test starting from FFPE tissue samples (3). Despite small sample size has been analyzed, preliminary data obtained through EasyPap Direct HPV DNA test successfully showed that high sensitivity and specificity can be achieved without DNA extraction, with a remarkable decrease of assay cost and time. These results lead us to perform large scale EasyPap testing.