Moreover, we analyzed 28 melanoma tissues by TMA immunostaining for CTLA-4 and we evaluated CTLA-4 expression through the immunoreactive score (IRS; 0 = negative, 1-4 = low to intermediate and ≥6 = high), an index which takes into account both parameters of percentage of positively stained cells and staining intensity, according to their individual scores (34), as described in the Methods. This evidence concerns the gene CTLA4 and melanoma.