No evidence for ErbB4 gene amplification in malignant melanoma.

Malignant melanoma (MM) represents the most severe skin cancer (1). Somatic ErbB4 gene mutations have been identified in 19% of individuals with MM (2). These mutations result in hyperactivation of the ErbB4 receptor (2, 3). This finding indicates a pivotal role of the ErbB4 receptor tyrosine kinase (RTK) in the tumourigenesis of MM. However, oncogenic receptor activation of ErbB RTKs in human cancer is not limited to activating point mutations. Amplifications of ErbB receptor genes (ErbB1–4), resulting in receptor overexpression and ligand-independent activation have been observed in a variety of human malignancies (4). Based on this rationale we analysed potential ErbB4 gene amplification in 28 melanoma samples using fluorescence-in-situ-hybridization (FISH). PATIEnTS And METHodS In total, 28 MM samples were analysed (17 superficial spreading melanomas, 9 nodular MMs, 2 secondary nodular MMs on the basis of a superficial spreading melanoma). The melanomas derived from 11 male and 17 female patients (mean age 60 years, age range 27–87 years). The study was performed according to the guidelines of the local ethics committee and the declaration of Helsinki. Sections 4 µm thick were prepared from formalin-fixed paraffin embedded melanoma samples, as described in detail else where (5, 6). In brief, for each tumour a representative tumour section was selected from a haematoxylin and eosin-stained section of the donor block. Core cylinders with a diameter of 1.5 mm each were punched from this area and deposited into a recipient paraffin block. Tissue microarray (TMA) sections were mounted on charged slides (SuperFrost™Plus; Menzel GmbH, Braunschweig, Germany). Haematoxylin and eosin stained TMA sections were used for reference histology. FISH was performed with the use of directly labelled ZytoLight SPEC HER4/2q11 dual-colour probe (ZytoVision Ltd, Bremerhaven, Germany) (Fig. 1). After probe hybridization nuclei were counterstained with anti-fading 4',6-diamidino-2-phenylindole (dAPI) Vectashield (Vector Laboratories, Burlingame, CA, USA) and analysed by epifluo-rescence microscopy using the AxioImager-Z1 (Zeiss, Göttingen, Germany). Hybridization signals of 25 nuclei were manually counted on single cell basis by two independent observers. RESULTS And dISCUSSIon dual-colour FISH, revealed no evidence for ErbB4 gene amplification (Table I). The highest ErbB4 gene/ centromer-2 ratio found was 1.17. Three cases showed a low degree of chromosome-2 polysomy (nos. 5, 6 and 20). only one specimen appeared suspicious, showing a low degree of chromosome-2 loss (no. 19). overall we did not observe any significant alterations in ErbB4 gene copy number. Although the quantity of tissues investigated in this study is limited …