Figure 5

Genes consistently differentially expressed in FNA obtained 3 h after the first and fourth dose of IL-2. The expression profile of aRNA samples amplified from FNAs of melanoma lesions from six patients was analyzed. Four FNA (patients C.F.1, K.F.1, L.F.1, M.F.1) were obtained following one dose and five (G.F.b4, H.F.4, K.F.4, L.F.4, M.F.4) following four doses of IL-2. The FNA material was co-hybridized in pairs with reference aRNA amplified from FNA (red bar) obtained pre IL-2 therapy from each individual lesion. (a) Clusterogram depicts 150 genes derived from a group of 2,393 genes expressed with at least a 3-fold difference between reference and test samples in at least 90% of the experiments. In addition, genes were ranked according to their median ratio of expression among all tumor samples (75 with the highest median value of gene expression, 75 with the lowest, across nine experiments). This was done to identify those genes that, independent of sample origin or number of IL-2 doses, were more consistently affected by IL-2 treatment. An arbitrary cut-off of the 75 most representative genes in both ranges of expression was selected, which excluded genes with a median ratio of differential expression of less than 2. Red exemplifies upregulation in test samples compared with pre-IL-2 reference sample and green the opposite. Individual genes are shown on the right. To control for an influence on gene expression of the trauma of FNA, a separate control experiment included two FNAs obtained from a lesion at a 24-h interval in the absence of IL-2 administration. No direct effect of FNA alone on gene expression was observed after IL-2 administration, except for the genes reported in parentheses. Eisen's algorithm [21] was applied to this set of genes to identify proximity of different samples by applying the clustering to individual array experiments as shown by the dendrogam. Samples from the same lesion clustered together independently of number of doses received. (b) Clusterogram depicting the same 150 genes selected for (a) and analyzed in leukocyte subsets (blue bar) (as in Figure 4), and in PBMC in vitro (yellow bar) and in vivo (green bar) (see Figure 3).