Protein Expression Analysis of Melanocyte Differentiation Antigen TRP-2

Abstract

Melanocyte differentiation antigens, such as gp100, tyrosinase, and Melan-A and their corresponding antibodies HMB45, T311, and A103, are major diagnostic tools in surgical pathology. Little is known about tyrosinase-related protein 2 (TRP-2, or dopachrome tautomerase/DCT) another melanocyte differentiation antigen, which is an enzymatic component of melanogenesis. We identified a commercial reagent to TRP-2, monoclonal antibody (mAb) C-9 and undertook a comprehensive analysis to assess its specificity and usefulness for surgical pathology. Subsequently, we analyzed panels of normal tissues and tumors. We show that TRP-2 is regularly expressed in melanocytes of the normal skin. In cutaneous nevi, TRP-2 is present in junctional as well as in dermal nevocytes. In malignant tumors, C-9 reactivity is restricted to melanocytic and related lesions and present in 84% and 58% of primary and metastatic melanomas, respectively. Ten primary melanomas of the anorectal mucosa were all positive. Like the other melanocyte differentiation antigens, TRP-2 was absent in 6 desmoplastic melanomas. Also, only 2 of 9 angiomyolipomas were TRP-2 positive. We conclude that mAb C-9 is a valuable reagent for the analysis of TRP-2 expression in archival surgical pathology material. The expression pattern of TRP-2 in melanocytic and related lesions appears to parallel other melanocyte differentiation antigens, although the overall incidence is lower than other antigens, such as Melan-A or gp100.

Figure 1

Immunohistchochemical staining with anti-TRP-2 monoclonal antibody C-9 (avidin-biotin-method, diaminobenzidine chromogen) in skin showing strong immunopositivity of melanocytes in A) acetone-fixed frozen and B) formalin-fixed paraffin-embedded skin sections, C) high power magnification of skin with immunopositive melanocytes and D) immunonegative melanocytes after blocking with TRP-2 protein.

Figure 2

ELISA analysis of anti-TRP-2 mAb C-9 and anti-Melan-A mAb A103 in descending antibody concentrations with full-length protein preparations of Melan-A, Tyrosinase, and TRP-2 (1 μg/ml). Strong reactivity of mAb C-9 and mAb A103 was observed with TRP-2 and Melan-A respectively, with no cross-reactivity to other proteins.

Figure 3

Immunohistochemical staining of formalin-fixed paraffin-embedded pellets of melanoma cell lines with anti-TRP-2 mAb C-9 displaying A) homogeneous expression in SK-Mel-14, B) heterogeneous expression in SK-Mel-26, and C) no expression in SK-MEL-128 (avidin-biotin-method, diaminobenzidine chromogen).

Figure 4

Immunohistochemical staining of anti-TRP-2 mAb C-9 in A) lung displaying granular reactivity of alveolar macrophages, B) compound dermal nevus with strong staining of all nevocytes, homogenous strong TRP-2 in primary C) and metastatic D) melanoma, E) negative desmoplastic melanoma, and focal TRP-2 positive cells in F) renal angiomyolipoma.