Salivary Mucoepidermoid Carcinoma: Demonstration of Transcriptionally Active Human Papillomavirus 16/18

Abstract

Herein we test the following hypotheses: (1) High-risk Human Papillomavirus (HR-HPV) may be involved in the etiology of mucoepidermoid carcinoma (MEC), and (2) The detection rate of HR-HPV in MEC has been increasing over time. Ninety-eight archival MEC specimens from three institutions spanning three decades were studied for HPV16/18 E6/E7 transcripts. RNA was extracted from formalin-fixed paraffin embedded specimens and HPV16/18 E6/E7 expression assessed by nested reverse transcription polymerase chain reaction (RT-PCR). A subset of MEC were also studied for MECT1-MAML2 fusion transcripts by nested RT-PCR and amplicon sequencing. The HPV expression data was validated by immunofluorescence (IF) with monoclonal HPV16/18 E6 antibody, PCR with the GP5+/6+ consensus primers, and sequencing of RT-PCR amplicons. HPV genome was localized by in-situ hybridization with the Ventana Inform HPVIII Family 16 probe. P16^INK4a overexpression and aberrant p53 expression were assessed by immunohistochemistry. HPV16 E6/E7 transcripts were demonstrated in (29/98) 30 % of MEC by RT-PCR. HPV18 E6/E7 transcripts were demonstrated in 13/98 (13 %) of MEC by RT-PCR. Seven of 98 tumors (7 %) demonstrated both HPV16/18. No significant association was found between HPV status and gender, age, and tumor site. All 13 HPV18+ MEC were diagnosed between 2001 and 2010, whereas 45 MEC diagnosed from 1977 to 2000 were negative for HPV18 (p = 0.002). By contrast, there was no significant difference with respect to HPV16 detection and date of diagnosis. All MEC that were positive for E6 protein were also HPV16/18 positive by RT-PCR. Sequencing a subset of RT-PCR amplicons confirmed HPV type- and region-specific sequences. PCR using GP5+/6+ consensus primers demonstrated HPV status concordance in 9 of 10 cases. DNA degradation was present in the last case; the RT-PCR amplicons were sequenced from this case which confirmed the presence of HPV type- and region-specific sequences. Strong (+4/+4) and diffuse (>50 %) nuclear and cytoplasmic p16 expression was seen in 64 % of MEC in the glandular regions, and 18 % of MEC in the solid, squamoid regions. No correlation was seen between p16 expression and HPV status. Twenty-nine MEC (22 HPV+ and 7 HPV-negative) were selected for further evaluation for p53 expression. Strong aberrant nuclear p53 expression was present in only 2/22 HPV + MEC (9 %, both Grade 3); no HPV-negative MEC demonstrated aberrant p53 expression. MECT1-MAML2 fusion transcripts were demonstrated in 23/37 (62 %) MEC. No significant association was found between the presence of the MECT1-MAML2 fusion transcripts and tumor grade, HPV status, gender, era of diagnosis (2000 and earlier vs. 2001–2010) or tumor site. We demonstrate for the first time that transcriptionally active HPV16/18 is common to MEC. These findings were validated by demonstrating concordant results by separate PCR with consensus primers, and/or confirming the presence of HPV type- and region-specific sequences in the RT-PCR amplicons. We also visualized E6 viral oncoprotein and HPV genome within tumor cells. HR-HPV is thus potentially implicated in the pathogenesis of MEC. The frequency of HPV18 detection is significantly increased in MEC diagnosed after 2001, whereas we found no differences in the HPV16 detection rates per era of diagnosis.