Immunohistochemical analyses may assist in the diagnosis of precancerous and cancerous conjunctival lesions.
To use Vector Red (VR) to identify an immunologic marker that is sensitive for all melanocytes and another that is sensitive and specific for activated and/or atypical conjunctival melanocytic lesions (MLs).
Design, Setting, and Participants
Eight specimens each of control lesions (normal conjunctiva and normal uvea as well as choroidal melanoma) and 8 from the diagnostic categories (conjunctival nevus, primary acquired melanosis with mild or no atypia, primary acquired melanosis with moderate to severe atypia, and conjunctival melanoma) that provided sufficient quantity and quality of tissue were available for processing. The specimens were obtained from the Ophthalmic Pathology Laboratory, The Ottawa Hospital, from 2005 to 2013. The specimens were immunolabeled with human melanoma black 45 (HMB45), melanoma antigen recognized by T cells 1 (Melan-A), S100, and Ki67 using VR and a double panmelanoma cocktail (dPANMEL) using 3,3′-diaminobenzidine (DAB) and VR. The HMB45-immunolabeled specimens were additionally developed with DAB, with and without overnight bleaching with hydrogen peroxide, 4%. Data were collected by 2 pathologists who were masked to sample grouping.
Main Outcomes and Measures
Differentiation between benign and malignant MLs based on immunomarker profile.
Immunoreactivity was best visualized in specimens with VR. Melan-A labeled all melanocytes (100% sensitivity; panmelanocyte marker) without discriminating between benign and malignant lesions (0% specificity). Atypical melanocytes were most specifically labeled with HMB45 (96% specificity, 97% sensitivity; atypia marker). In primary acquired melanosis specimens, we found that the percentage of HMB45 (P < .001), S100 (P < .001), and Ki67 (P ≤ .02) positivity increased significantly with worsening atypia.
Conclusions and Relevance
We recommend VR, which rarely requires specimen bleaching, as the standard substrate for immunohistochemical analysis of conjunctival MLs. We found Melan-A and HMB45 to best characterize MLs. In conjunctival MLs, the use of VR with Melan-A and HMB45 provides substantial sensitivity for all melanocytes and for atypical melanocytes, respectively, and reduces specimen-processing time for laboratories performing immunohistochemistry on MLs.