Wnt ligand expression in malignant melanoma: pilot study indicating correlation with histopathological features

Abstract

Aims: Secreted Wnt ligands are key proteins regulating cell-cell interactions and cell growth and differentiation. These proteins, along with other components of the Wnt signalling pathway, are involved in the malignant transformation of various human cancers, including malignant melanoma. This study defines the expression of several members of the Wnt ligand family and correlates their expression with histological characteristics.

Methods: The expression of Wnt2, Wnt5a, Wnt5b, Wnt7b, and Wnt10b was defined by in situ, antisense RNA hybridisation of paraffin wax embedded sections of benign naevi and malignant melanoma. Immunoperoxidase based antibody staining was used to define the expression of frizzled (Fz) receptors.

Results: All naevi tested strongly expressed Wnt2, Wnt5a, Wnt7b, and Wnt10b. Melanomas characterised by small, uniform cells expressed each of the Wnts in a pattern similar to that seen for benign naevi. In contrast, melanomas characterised by large, pleomorphic cells expressed Wnt10b but did not express Wnt2 and had low levels of expression of Wnt5a. Expression of Wnt7b was variable in these melanomas. Fz receptor expression was present at a low level in normal epithelium and all naevi and melanomas.

Conclusions: The expression pattern of Wnt ligands in malignant melanoma correlates with histopathological features and may provide a basis for the molecular classification of this disease.

Figure 1

In situ hybridisation demonstrating expression of Wnt ligands in benign naevi. (A) Haematoxylin and eosin staining; original magnification, ×400. (B–F) In situ hybridisation with antisense RNA probes for (B) Wnt2, (C) Wnt5a, (D) Wnt5b, (E) Wnt7b, and (F) Wnt10b on representative samples; original magnification, ×400. Dark purple staining indicates positive hybridisation by probe with specific mRNA. (D) Minimal staining with the Wnt5b probe is seen in this naevus. Brown colouration represents melanin and can be seen clearly in (A) and (D). The strong signal from the antisense RNA probe hybridisation in (B), (C), (E), and (F) obscures the melanin pigment.

Figure 2

In situ hybridisation demonstrating expression of Wnt2, Wnt5a, and Wnt10b in melanomas of varying histologies; original magnification, ×400. Haematoxylin and eosin staining of (A) small cell melanoma and (B) large pleomorphic cell melanoma. (A) The white arrows identify melanoma cells. (B) Melanoma cells make up most of the photomicrograph and are readily apparent. Wnt2 in situ hybridisation of (C) small cell melanoma and (D) large pleomorphic cell melanoma. The dark purple staining indicates positive hybridisation. (C) The white arrows identify positive staining in small cell melanoma. No staining is apparent in the large cell melanoma. Wnt5a in situ hybridisation of (E) small cell melanoma and (F) large pleomorphic cell melanoma. (E) The white arrows identify positive staining in small cell melanoma. (F) The insert depicts strong Wnt5a expression in a hair follicle on the same slide from which the large cell melanoma image is derived. The strong staining in the hair follicle contrasts with the very weak staining in the large melanoma cells. Wnt10b in situ hybridisation of (G) small cell melanoma and (H) large pleomorphic cell melanoma. Both histological subtypes display substantial staining with the antisense probe (identified by white arrows in each panel).

Figure 3

Frizzled receptor expression by immunoperoxidase staining with anti-Fz1/2 antibody in (A) benign naevi, (B) small cell melanoma, and (C) large pleomorphic cell melanoma. (D) Strong expression of the Fz receptor in a hair follicle. Positive expression is indicated by the pink coloured staining and is shown by white arrows in each panel. Black arrows indicate melanin pigment. Original magnification, ×400.