HPV-Associated Squamous Cell Carcinoma of the Eyelid: Diagnostic Utility of p16 Immunohistochemistry and mRNA In Situ Hybridization

Abstract

Background: High-risk (HR) Human papillomavirus (HPV) has been implicated in pathogenesis of squamous cell carcinomas (SCC) at several sites with mucocutaneous junctions, including the head and neck. SCC is the second most common eyelid malignancy. However, its association with transcriptionally active HR-HPV has not been adequately studied.

Methods: Two index cases of eyelid HPV-associated SCC are described in detail. A retrospective cohort of eyelid SCC was examined for p16 immunoexpression. Cases demonstrating p16 positivity or equivocal staining were subjected to high-risk HPV mRNA in situ hybridization (ISH). Quantitative real-time PCR (qPCR) was performed in mRNA ISH-positive cases for HPV genotyping.

Results: The two index patients were older adult females, with upper eyelid tumours. On histology, both tumours were non-keratinizing SCC with trabecular and nested architecture reminiscent of oropharyngeal HPV-associated non-keratinizing SCC, prompting p16 immunohistochemistry, which was positive. HR-HPV mRNA ISH was positive, and qPCR detected HPV16 in both cases. Three of 20 (15%) archival cases showed p16 immunopositivity and two (10%) showed equivocal staining. However, mRNA ISH was negative. All cases showing p16 immunostaining and lacking HR-HPV were keratinizing SCCs. Thus, 9% of all eyelid SCC examined demonstrated HR-HPV.

Conclusion: The prevalence of HR-HPV in eyelid SCC is low in Indian patients. HPV-associated SCC may mimic commoner eyelid carcinomas as it lacks overt keratinization. In basaloid-appearing eyelid carcinomas, p16 immunopositivity should be followed by reflex HR-HPV mRNA ISH, as p16 immunohistochemistry alone has low specificity. The prognostic role, if any, of HPV association needs further evaluation.

Keywords: Eyelid carcinoma; Human papillomavirus; Immunohistochemistry; Non-keratinizing squamous cell carcinoma; mRNA in situ hybridization.

Fig. 1

Case 1 showing trabeculae and large nests (A; HE, 40x) of basaloid tumour cells (B; HE, 100x) with clefting artefact and surrounding lymphocytic infiltrate (C; HE, 100x). Tumour cells have scant cytoplasm and ovoid hyperchromatic nuclei with frequent mitoses; keratinization is absent (D; HE, 400x). p16 shows diffuse positivity (E, 100x). mRNA ISH shows punctate red signals, indicative of HR-HPV positivity (F; 100x)

Fig. 2

Case 2 shows a tumour with trabecular architecture (A; HE, 20x), pushing borders, and central comedonecrosis (B; HE, 20x); at places, small infiltrative islands are present at the advancing edge (C; HE, 20x). Clefting artefact is prominent, with streaming appearance of cells (D; HE, 100x). A keratin pearl is seen within a tumour island (E; HE, 100x). Tumour cells have scant to moderate pale eosinophilic cytoplasm, round to ovoid hyperchromatic nuclei with frequent mitoses (F; HE, 200x). p16 (G, 100x) and mRNA ISH are positive (H; 100x)