Active human papillomavirus involvement in Barrett's dysplasia and oesophageal adenocarcinoma is characterized by wild‐type p53 and aberrations of the retinoblastoma protein pathway

Abstract

We have previously demonstrated that transcriptionally active high-risk HPV (hr-HPV) is strongly incriminated in Barrett's dysplasia (BD) and oesophageal adenocarcinoma (OAC) using mainly fresh frozen tissue. This study aimed to identify biomarkers of active HPV infection in Barrett's metaplasia, (BM)/BD/OAC by immunohistochemical staining (IHC) of formalin-fixed paraffin embedded (FFPE) tissue for aberrations of p53 and the retinoblastoma (pRb) pathway, which are targets for the viral oncoproteins, E6/E7, respectively. Prospectively, BM (n = 81)/BD (n = 72)/OAC (n = 65) FFPE specimens were subjected to IHC staining for pRb, p16^INK4A , cyclin D₁ , p53 and RNA in-situ hybridization for E6/E7 transcripts. HPV DNA was determined via PCR in fresh frozen specimens. Viral load measurement (real-time PCR) and Next Generation Sequencing of TP53 was performed. Of 218 patients, 56 were HPV DNA positive [HPV16 (n = 42), 18 (n = 13), 6 (n = 1)]. Viral load was low. Transcriptionally active HPV (DNA⁺ /RNA⁺ ) was only found in the dysplastic and adenocarcinoma group (n = 21). The majority of HPV DNA⁺ /RNA⁺ BD/OAC were characterized by p 16highINK4A (14/21, 66.7%), pRb_low (15/21, 71.4%) and p53_low (20/21, 95%) and was significantly different to controls [combination of HPV DNA^- /RNA^- (n = 94) and HPV DNA⁺ /RNA^- cohorts (n = 22)]. p53_low had the strongest association with DNA⁺ /RNA⁺ oesophageal lesions (OR = 23.5, 95% CI = 2.94-187.8, p = 0.0029). Seventeen HPV DNA⁺ /RNA⁺ BD/OAC identified as p53_low, were sequenced and all but one exhibited wild-type status. pRb_low /p53_low provided the best balance of strength of association (OR = 8.0, 95% CI = 2.6-25.0, p = 0.0003) and sensitivity (71.4%)/specificity (71.6%) for DNA⁺ /RNA⁺ BD/OAC. Active HPV involvement in BD/OAC is characterized by wild-type p53 and aberrations of the retinoblastoma protein pathway.