Objective

This study utilized Next Generation Sequencing (NGS) to identify differentially expressed transcripts in orbital adipose tissue from patients with active Thyroid Eye Disease (TED) versus healthy controls.

Method

This prospective, case-control study enrolled three patients with severe, active thyroid eye disease undergoing orbital decompression, and three healthy controls undergoing routine eyelid surgery with removal of orbital fat. RNA Sequencing (RNA-Seq) was performed on freshly obtained orbital adipose tissue from study patients to analyze the transcriptome. Bioinformatics analysis was performed to determine pathways and processes enriched for the differential expression profile. Quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) was performed to validate the differential expression of selected genes identified by RNA-Seq.

Results

RNA-Seq identified 328 differentially expressed genes associated with active thyroid eye disease, many of which were responsible for mediating inflammation, cytokine signaling, adipogenesis, IGF-1 signaling, and glycosaminoglycan binding. The IL-5 and chemokine signaling pathways were highly enriched, and very-low-density-lipoprotein receptor activity and statin medications were implicated as having a potential role in TED.

Conclusion

This study is the first to use RNA-Seq technology to elucidate differential gene expression associated with active, severe TED. This study suggests a transcriptional basis for the role of statins in modulating differentially expressed genes that mediate the pathogenesis of thyroid eye disease. Furthermore, the identification of genes with altered levels of expression in active, severe TED may inform the molecular pathways central to this clinical phenotype and guide the development of novel therapeutic agents.

Background

p16 expression is a well established biomarker of cervical dysplasia and carcinoma arising from high risk human papilloma virus infection. Increased p16 expression is also seen in squamous neoplasms arising at other sites, including head, neck, and oropharyngeal tract. Squamous lesions are also frequently encountered at ocular surface and peri-orbital skin sites, but the prevalence of increased p16 expression in these lesions has been poorly studied.

Methods

We retrospectively surveyed 13 ocular surface and 16 orbital squamous lesions biopsied at UC San Diego Healthcare System and VA San Diego Healthcare System for p16 expression by immunohistochemistry. These cases included ocular surface lesions with diagnoses of conjunctival intraepithelial neoplasm (CIN) and squamous cell carcinoma in situ. Peri-orbital eyelid biopsies included lesions with diagnoses of SCCis and invasive squamous cell carcinoma. We performed multivariate logistic regression, followed by student's T-test or Fisher's exact test to determine if there were statistically significant associations between p16 immunoreactivity and patient age, gender, diagnosis, and ethnicity. Statistical significance was defined as p < 0.05.

Results

We found an unexpectedly large prevalence of strong nuclear and cytoplasmic p16 immunoreactivity in our cases. Almost all of the ocular surface squamous lesions were diffusely positive for p16 expression (12/13). All of the periorbital lesions showed diffuse p16 immunoreactivity (16/16). Altogether, 28/29 lesions tested showed strong and diffuse p16 expression. We found no statistically significant correlation between p16 expression and patient age, gender, ethnicity, or diagnosis. In 6 of the peri-orbital biopsies, we had sufficient tissue to assess high-risk HPV expression by in situ hybridization. Interestingly, all of these cases were negative for HPV, despite strong p16 expression.

Conclusion

Strong p16 expression was observed in virtually all of the ocular surface and peri-orbital squamous neoplasms in our study. The relationship between p16 expression and HPV infection in ocular surface and peri-orbital sites requires further investigation.

Two fraternal twin sisters developed cysticercosis localizing to the right lateral orbit over the same period after a presumed common-source exposure in China. This case demonstrates that cysticercosis can be related to travel. Similar temporal and spatial occurrences of these infections suggest a genetic tropism of the infecting organism in these twins.