Today the HPV status of oropharyngeal carcinomas is a prognostic marker impacting treatment.
The Xpert®HPV test (Cepheid) detects HR-HPV 16, 18-45 and 3 groups of other HPV types (P3 : 31-33-35-52-58, P4 : 51-59 and P5 : 39-56-66-68) by PCR including the detection of a reference gene, confirming the presence of an adequate number of cells. To date only one publication used this technique on FFPE tissue sections (1).
The aim of the study was to evaluate the Xpert HPV test on FFPE tumor samples by comparing it to the INNO-LIPA PCR Version Extra II (Fujirebio) genotyping and p16 immunohistochemistry (IHC) as reference methods.
The Xpert HPV-test on FFPE was validated on six 4 µm FFPE tissue sections of ten cervical biopsies (5 CIN2/3 and 5 within normal limits) and 5 anal biopsies with AIN 2/3 compared to p16 staining.
Tissue sections were deparaffinised, followed by a simple lysis (ATL lysis buffer, Qiagen) with proteinase K for 4 hours, heated for 1 hour at 90°C then diluted in 1 ml ultra-filtered water and processed.
Four of the positive samples were tested at different lysate dilutions: 1/2, 1/4, 1/8, 1/16 and 1/32.
We then performed Xpert HPV tests on 28 FFPE tumor samples of head and neck carcinomas formerly tested by INNO-LIPA PCR (13 HR-HPV positive and 15 negative) and compared them to the IHC expression of p16. Only an intense diffuse staining of > 80% of tumor cells was considered positive, patchy staining was considered negative
All high grade cervical and anal neoplasias were Xpert HPV and p16 positive.
For the 4 diluted CIN2/3 lysates, HR-HPV was still detected at a dilution of 1/32 and two 4µm sections showed to be sufficient.
Of the 28 head and neck tumor samples, 13 were INNO-LIPA HR-HPV positive, 15 were negative. The overall concordance was 85.7% with a negative concordance of 93.3% and a positive concordance of 76.9%.
One Inno-Lipa negative sample was Xpert HPV as well as p16 positive. Among 13 INNO-LIPA positive samples, 3 were Xpert HPV negative, p16 staining was negative favoring a non viral carcinogenesis. These cases could reflect a possible latent HPV infection in the viscinity of the tumors, detected by the INNO-LIPA PCR.
In all samples tested the overall concordance between Xpert HPV and p16 IHC was 100%.
Xpert HPV testing is feasible without DNA extraction even on very small tissue samples and may be considered as a valuable method for the detection of HR-HPV in FFPE tissues.
1. Guerendiain et al., FFPE material is amenable to HPV detection by the Xpert HPV assay, J. of Cli. Virology, 2017.