Validation of Transcriptionally Active HPV in Salivary Mucoepidermoid Carcinomas
Zhiyong Ren, Rong Li, Tatyana Isayeva, Shuting Bai, Nasser Said-Al-Naief, Margaret Brandwein-Gensler. University of Alabama at Birmingham, Birmingham, AL; University of the Pacific, San Francisco, CA
Background: Mucoepidermoid carcinoma (MEC) is a common salivary malignancy. We have recently demonstrated expression of HPV16 and HPV18 specific E6/E7 transcripts in 30.2%, and 20.5% of MEC, respectively, by nested RT-PCR. Here we validate these findings by demonstrating HR HPV E6 protein in MEC by immunofluorescence (IF). In addition, we compared p16 expression with HPV status in these tumors.
Design: We studied 84 MEC with known HPV status. E6 protein was determined by mouse monoclonal HPV16/18 E6 (C1P5) primary antibody and goat anti-mouse secondary antibody conjugated to Alexa 488. Immunohistochemistry stains were performed for p16, and expression was categorized for staining intensity (scale 0-4) and percent staining distribution, for the nuclei and cytoplasm of the squamous and mucinous tumor elements. All slides were examined blinded to HPV status by two pathologists.
Results: E6 protein was demonstrated in 18/84 MEC (21%), with either nuclear and/or cytoplasmic staining in both mucinous and squamoid tumor elements, as shown in figure 1 (arrows).
All E6+ MEC were HPV16/18+ by RT-PCR. Fourteen additional tumors were negative by IF and positive by RT-PCR (sensitivity 55%, specificity 100%). For p16 staining, 15/84 MEC (18%) demonstrated strong (+4) nuclear and cytoplasmic p16 expression in ≥ 50% of solid MEC components; 53/84 MEC (64%) demonstrated strong (+4) nuclear and cytoplasmic p16 expression in ≥ 50% of the mucinous glandular components. No correlation, however, was seen between p16 expression and HPV status.
Conclusions: Here we validate our previous RT-PCR data demonstrating transcriptionally active HPV16/18 in MEC, by detecting E6 viral oncoprotein in tumor cells. As expected, the sensitivity of IF is lower than RT-PCR, but the specificity is high. These findings support the concept that HPV 16/18 promotes the carcinogenesis of MEC. Interestingly, we found no relationship between transcriptionally active HPV status and p16INK4a expression in MEC, in contrast to the paradigm of HPV-mediated tonsillar cancers. Lack of p16 overexpression in transcriptionally active HPV16/18 MEC may be attributed promoter hypermethylation, or deletions and point mutations in p16INK4a.
Category: Head & Neck
Monday, March 19, 2012 1:30 PM
Platform Session: Section F, Monday Afternoon