Serial 4-μm-thick paraffin sections were stained with hematoxylin-eosin or used for immunohistochemical analysis. For the latter, after deparaffinization, endogenous peroxidase activity was blocked with 0.2% hydrogen peroxide (20 minutes) and the sections were incubated with normal goat serum (30 minutes). When necessary, pigment was bleached according to the method used by Orchard and Calonje.18 Sections were exposed for 1 hour to the following monoclonal antibodies: anti–HMB-45 (Ylem, Avezzano, Italy; 1:40), anti–melan-A (Neomarkers, Fremont, California; 1:50), anti–S-100 (Neomarkers; 1:200), antivimentin (Ylem; 1:40), anti–Ki-67 (Ylem; 1:50), anti-CD68 (Dako, Glostrup, Denmark; 1:1500), anti–GST-π (Novocastra, Newcastle, England; 1:100), and anti–nuclear factor–κB (NF-κB)–p65 subunit (Santa Cruz Biotechnology Inc, Santa Cruz, California; 1:50). Anti–GST-π immunostaining was repeated with rabbit polyclonal antibody (Santa Cruz Biotechnology Inc; 1:100), which gave similar results. Diaminobenzidine and red chromogen amino ethyl carbazole were used as final chromogens. As a control for immunohistochemical evaluation, a group (n = 10) of consecutive patients with superficial spreading melanomas in vertical growth phase (mean [SD] tumor thickness, 2 [0.65] mm) was enrolled. The mean (SD) age of the control patients (52  years) did not differ from that of the ATM group. Immunoreactivity was estimated at an original magnification of ×200 in at least 10 fields by 2 of the authors (S. Costantini and A.F.), with an intraobserver reproducibility greater than 95%. Immunostaining results were graded according to the percentage of positive cells as follows: 0, negative or less than 1% positive cells; 1, less than 25% positive cells; 2, between 25% and 50% positive cells; and 3, greater than 50% positive cells. For GST-π, the same score was also used to calculate nuclear staining.