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Original Investigation |

Usefulness of a Red Chromagen in the Diagnosis of Melanocytic Lesions of the Conjunctiva

Kailun Jiang, MD1,2,3; Seymour Brownstein, MD, FRCSC1,2,3; Kay Lam, MD1,2,3; Bruce Burns, MD, FRCPC2; James Farmer, MD, FRCSC, FRCPC1,2
[+] Author Affiliations
1Department of Ophthalmology, University of Ottawa, The Ottawa Hospital, Ottawa, Ontario, Canada
2Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa Hospital, The Ottawa Hospital, Ottawa, Ontario, Canada
3Ottawa Hospital Research Institute, Ottawa, Ontario, Canada
JAMA Ophthalmol. 2014;132(5):622-629. doi:10.1001/jamaophthalmol.2013.8216.
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Importance  Immunohistochemical analyses may assist in the diagnosis of precancerous and cancerous conjunctival lesions.

Objective  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.

Results  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.

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Figure 1.
Panmelanocytic Marker

A, A case of primary acquired melanosis (PAM) with moderate atypia containing extensive melanin pigmentation (hematoxylin-eosin, original magnification ×400). B, Components of double panmelanoma cocktail (dPANMEL) can be developed only with 3,3′-diaminobenzidine (DAB) (dark brown chromagen), which can be easily confounded with melanin pigmentation (original magnification ×400). C, Staining of melanoma antigen recognized by T cells 1 (Melan-A) developed with Vector Red is readily distinguishable from the melanin pigmentation in the same cells (original magnification ×400). D, In a noninferiority comparison, antigenicity for Melan-A is similar to that of dPANMEL (antibodies against tyrosinase, S100, and MART-1) in all benign and malignant melanocytic cells. The extent of staining is indicated as 1+ representing less than 25%, 2+ indicating less than 50%, 3+ showing less than 75%, and 4+ representing 100% of the cells. The limit lines indicate SE; limit lines are not shown where SE values were too small.

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Figure 2.
Markers of Atypia

A, In row 1, a representative case of primary acquired melanosis (PAM) with mild atypia showing extensive absence of immunogenicity for S100 Vector Red (VR) (a), human melanoma black 45 (HMB45) VR (b), and Ki67 VR (c) compared with the panmelanocytic marker, melanoma antigen recognized by T cells 1 (Melan-A) VR (d) (for all, original magnification ×400). Row 2 shows another specimen of PAM with moderate and thus worsening atypia than in row 1, with a larger proportion of the melanocytic cells stained positive for the 4 markers (S100 VR in a; HMB45 VR in b; Ki67 VR in c; and Melan-A VR in d) (for all images, original magnification ×400). In row 3, a case of PAM with severe atypia, antigenicity for S100 VR (a), HMB45 VR (b), Ki67 VR (c), and Melan-A VR (d) is more extensive in greater than 75% of melanocytic cells, with Ki67 being less informative, although it is expressed in approximately 75% of the thickness of the lesion (for all images, original magnification ×400). B, Antigenicity against S100 VR, HMB45 VR, and Ki67 VR increase in PAM with increasing atypia. However, S100 is extensively positive in benign melanocytes as exemplified in specimens of normal conjunctiva and choroid as well as in nevi. The usefulness of Ki67 is confounded by nonspecific staining in active basal epithelium, infiltrating lymphocytes, and nevus cells in the junctional region. The extent of staining is indicated as 1+ representing less than 25%, 2+ indicating less than 50%, 3+ showing less than 75%, and 4+ representing 100% of the cells. The limit lines indicate SE.aP < .001 for S100 and HMB45, PAM without to mild atypia vs PAM with moderate to severe atypia, and comparing PAM without to mild atypia vs conjunctival melanoma.bP = .02 for Ki67 (in PAM without to mild atypia vs in PAM with moderate to severe atypia).cP = .001 for Ki67 (in PAM without to mild atypia vs in conjunctival melanoma).

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Figure 3.
Vector Red (VR) Substrate and Bleaching

A case of primary acquired melanosis with moderate to severe atypia showing extensive melanin pigmentation. Hematoxylin-eosin (A) requiring bleaching with either permanganate (B) or hydrogen peroxide, 4% (H2O2) (C). Bleaching with 4% H2O2 minimally affects antigenicity with human melanoma black 45 (HMB45) 3,3′-diaminobenzidine (DAB) (D) and HMB45 DAB with 4% H2O2 (E). Bleaching is not required when developed with VR (HMB45 VR) (F). For all images, original magnification ×400.

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