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.

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