Recent increase in incidence of HPV-related Oropharyngeal Squamous Cell Carcinoma (OPSCC) highlights the need for effective tools to evaluate cancer HPV status, also considering the improved survival and response to treatment for HPV-positive OPSCC. To this end, it is essential to have a simple and reliable method to detect high-risk (HR)-HPV types also in formalin-fixed paraffin-embedded (FFPE) tissues. Although there is no agreement regarding the most appropriate method for HPV testing on FFPE materials, the PCR-based INNO-LiPA genotyping assay is currently considered one of the most reliable assays due to the small length of the amplification fragment. The Xpert® HPV assay is a qualitative real-time PCR assay, validated to detect HR-HPV in cervical cytology, which also gives a partial genotyping result. This method is very fast and simple to perform, and could be also applicable in low resource settings. The aim of this study was to investigate the performance of the Xpert HPV assay on FFPE OPSCC samples, compared to INNO-LiPA HPV genotyping assay and p16ink4a immunostaining.
A series of 32 FFPE OPSCC samples, already analyzed by the INNO-LiPA HPV genotyping Extra assay (Fujirebio), was evaluated by Xpert HPV assay (Cepheid). Moreover, samples were immunostained for p16 (Roche Diagnostics), considered a biomarker of HPV transforming activity and, by many authors, a surrogate marker for HPV infection. Deparaffinization was carried out with xylene, followed by absolute ethanol washing, and tissue lysis was performed overnight in ATL buffer with Proteinase K (Qiagen). After 1 hour incubation at 90°C, half of the crude lysate was analyzed by the Xpert HPV assay.
All the samples gave valid results with the Xpert, whereas two samples had been found invalid by the INNO-LiPA. These two samples were negative to both Xpert and p16 evaluations. Among the remaining 30 cases, 28 were concordant for HPV status, with a very high raw agreement and K value (93.3%; K=0.83). HPV genotyping results for the 21 cases positive by both methods, were also concordant. Regarding the two discordant cases, it is worth noting that one, Xpert positive/INNO-LiPA negative, was also p16 positive, whereas the other, Xpert negative/INNO-LiPA positive, was p16 negative. Comparing p16 and HPV results with the two methods, we found that the concordance rate with p16 staining was higher for Xpert than for INNO-LiPA (K= 0.92 vs K=0.73).
The Xpert HPV assay, a fast and easy method for HPV detection and partial genotyping, shows a very high concordance with INNO-LiPA genotyping assay and p16 findings, and appears to be reliable when used on FFPE OPSCC samples