Afrogheh, Amir H2026-06-172026-06-172018https://hdl.handle.net/10566/24504Purpose: High risk human papillomavirus (HR-HPV) is well established as a causative agent of squamous cell carcinoma (SCC) of the orophaynx. HR-HPV has also been reported in periocular cancers and precancers, but controversy exists about its overall incidence and clinicopathologic profile. The purpose of this study is to evaluate the role of HR-HPV infection in periocular cancers and precancers, using multiple methods of detection. Design: Retrospective observational case series with laboratory investigations. Methods: Sequential surgical samples of 87 carcinomas (invasive SCC, SCC in situ and sebaceous carcinoma) from three different periocular sites (conjunctiva, lacrimal sac and the eyelid) diagnosed over a 15-year period (2000-2015) were selected for evaluation. Unstained paraffin sections of 87 cases of periocular carcinomas were analyzed with immunohistochemistry (IHC) for p16 as a screening test. p16 positive conjunctival- and lacrimal sac SCC were further evaluated for HR-HPV using DNA in situ hybridization (DNA ISH), and a subset was also analyzed by DNA Polymerase Chain Reaction (DNA PCR). p16 positive periocular sebaceous carcinomas (SC) were analyzed with PCR, and a subset of 18 cases was further studied with a novel method of mRNA ISH, an advanced technique with an enhanced sensitivity and specificity. Relevant patient clinical information was obtained from review of the electronic medical records. Results: Of 43 ocular surface squamous cell neoplasia (OSSN), 30% (n=13; 8 SCC in situ and 5 invasive SCC), were positive for HR-HPV. The HPV-positive OSSN occurred in 8 men and 5 women with a mean age of 60 years (range: 39 to 94 years). HPV type16 was detected in all conjunctival cases evaluated by DNA PCR. All 5 conjunctival invasive SCC (I-SCC) showed a non-keratinizing (n=4) or partially keratinizing (n=1) histomorphology, and were managed by simple excision. In contrast, the HPV-negative conjunctival I-SCC were predominantly keratinizing (11 keratinizing, 2 non-keratinizing). Of 9 lacrimal sac I-SCC (LSSCC), 66.7% (n=6/9) were positive for HR-HPV by p16 and DNA ISH; HPV subtypes were HPV-16 (n=5) and HPV-58 (n=1). In addition, two p16-positive cases with negative DNA ISH results were HR-HPV positive (HPV-16 and HPV-33) when evaluated by DNA PCR, suggesting that the rate of HRHPV positivity among the LSSCC may be as high as 89% (n=8/9). The combined group of HR-HPV positive LSSCC occurred in 4 men and 4 women with a mean age of 60 years (range: 34 to 71 years). Seven of the 8 HPV-positive LSSCC (87.5%) had a non-keratinizing or partially keratinizing histomorphology while 1 case (12.5%) was predominantly keratinizing. Strong immunohistochemical p16 positivity was present in 29 of 35 cases of periocular SC (82.9%). The selected 18 p16-positive cases tested were negative for HR-HPV using mRNA ISH. DNA PCR was unequivocal with adequate DNA isolated in 24 cases, 23 of which were negative for HR-HPV. One case was positive for HPV type 16, which was found to be a false positive as determined by mRNA ISH negativity. Conclusion: The presence of HR-HPV in 30% of OSSN and at least 66.7% of LSSCC confirms HR HPV as an important etiologic agent at these sites. No evidence was found for HR-HPV as an etiologic agent in the development of periocular SC using DNA PCR, and mRNA ISH to maximize sensitivity and specificity. p16 overexpression is common in periocular SC but unrelated to HR-HPV status. Immunohistochemical testing for p16, however, can be a valuable adjunct for identifying pagetoid intraepithelial spread of disease and small invasive cellular clusters. Although p16 may be used as a surrogate marker for HR-HPV status in conjuctival- and lacrimal sac carcinomas, this interpretation of p16 positivity is not applicable to periocular SC.enHuman Papillomavirus (HPV)High riskConjunctivaLacrimal sacEyelidThesis