Detection of Human Papillomavirus in Squamous Lesions of the Conjunctiva Using RNA and DNA In-Situ Hybridization

In-situ hybridization provides a convenient and reliable method to detect human papillomavirus (HPV) infection in formalin-fixed paraffin-embedded tissue. Cases of conjunctival papillomas, conjunctival intraepithelial neoplasia (CIN), conjunctival carcinoma in situ (cCIS), and invasive squamous cell carcinoma (SCC), in which low-risk (LR) and/or high-risk (HR) HPV types were evaluated by RNA or DNA in-situ hybridization, were retrospectively identified. LR HPV types were frequently detected in conjunctival papillomas (25/30, 83%), including 17/18 (94%) with RNA probes, compared to 8/12 (75%) with DNA probes. None of the CIN/cCIS or SCC cases were positive for LR HPV by either method. HR HPV was detected by RNA in-situ hybridization in 1/16 (6%) of CIN/cCIS cases and 2/4 (50%) of SCC cases, while DNA in-situ hybridization failed to detect HPV infection in any of the CIN/cCIS lesions. Reactive atypia and dysplasia observed in papillomas was generally associated with the detection of LR HPV types. Collectively, our findings indicate RNA in-situ hybridization may provide a high-sensitivity approach for identifying HPV infection in squamous lesions of the conjunctiva and facilitate the distinction between reactive atypia and true dysplasia. There was no clear association between HPV infection and atopy in papillomas or dysplastic lesions

Transcriptomic and Immunohistochemical Analysis of Progressive Keratoconus Reveal Altered WNT10A in Epithelium and Bowman's Layer

PURPOSE. To identify global gene expression changes in the corneal epithelium of keratoconus (KC) patients compared to non-KC myopic controls. METHODS. RNA-sequencing was performed on corneal epithelium samples of five progressive KC and five myopic control patients. Selected results were validated using TaqMan quantitative PCR (qPCR) on 31 additional independent samples, and protein level validation was conducted using western blot analysis on a subset. Immunohistochemistry was performed on tissue microarrays containing cores from over 100 KC and control cases. WNT10A transcript levels in corneal epithelium were correlated with tomographic indicators of KC disease severity in 15 eyes. Additionally, W1V710A was overexpressed in vitro in immortalized corneal epithelial cells. RESULTS. WNT10A was found to be underexpressed in KC epithelium at the transcript (ratio KC/control = 0.59, P = 0.02 per RNA-sequencing study; ratio = 0.66, P = 0.03 per qPCR) and protein (ratio = 0.07, P = 0.06) levels. Immunohistochemical analysis also indicated WNT10A protein was decreased in Bowman's layer of KC patients. In contrast, WNT10A transcript level positively correlated with increased keratometry (Kmax rho = 0.57, P = 0.02). Finally, WNT10A positively regulated COL1A1 expression in corneal epithelial cells. CONCLUSIONS. A specific Wnt ligand, WNT10A, is reduced at the mRNA and protein level in KC epithelium and Bowman's layer. This ligand positively regulates collagen type I expression in corneal epithelial cells. The results suggest that WNT10A expression in the corneal epithelium may play a role in progressive KC