BMC Cancer 2009, 9:11 http://www.biomedcentral.com/1471-2407/9/11 Table 2: Genes differentially expressed between oral tongue tumor and case-matched histologically normal mucosae (TN paired, n = 20) (Continued) AI885852 Z98946 AC002073 AF053356 AB028994 D42047 AB007972 AF007153 AI674208 AL080059 AL049381 AW021542 KIAA1071 KIAA0089 KIAA0503 EST EST EST EST EST EST EST EST EST Hierarchical clustering analysis Four separate clustering analyses were performed: (1) All tumor and normal samples (TN, n = 37), (2) the tumor and their matched normal samples (TN paired, n = 20); A B TFHNiigeurcarlruecsht1eicralsectlu,sNte=rin3g7of the gene expression data for the Hierarchical clustering of the gene expression data for the TN cluster set, N = 37. A; and the TN paired clus- ter set, N = 20. B. Approximately 12, 625 genes were clus- tered using the method described in the text. The genes shown represent the top 80 genes that were up-regulated (red) and down-regulated (green) in the sample sets. The normal tissue (N) and tumor tissue (T) are followed by their corresponding case numbers. 4.03 3.42 3.17 1.73 -6.08 -4.68 -3.96 -5.35 -3.57 -3.12 -2.93 -2.60 (3) the tumor samples by themselves (T, n = 31); and (4) the normal samples by themselves (N, n = 26). The data were clustered using the standard hierarchical method with ward linkage and using the Pearson correlation to determine the distance function. The distance between samples was dist = (1-p)/2 where p is the correlation coef- ficient. Before clustering, the data was filtered to remove genes that were scored Absent (A) by the MAS5.0 software in 75% or more of the samples as they are likely to be measuring noise in the system. To assess the robustness of the clustering results, a resampling method was used to create 1000 replica datasets by adding Guassian noise to each point. These 1000 data sets were individually clus- tered and then a consensus tree was built from them. The number at each node in the tree indicates how often that subtree appears in the 1000 replica trees. The higher the number, the more robust is the subtree. The samples from the TN cluster set clearly segregated the tumor from the normal samples (Figure 1A). Similarly, TN paired set sep- arated the tumor samples from their matched normal counterparts (Figure 1B). The two clusters exhibit nearly identical patterns of gene expression changes. As shown on Table 2, the genes that were overexpressed included those involved in tumor invasion, epithelial development and angiogenesis. In contrast, however, clustering analysis failed to show significant segregation of patients based on expression profiling in both the T and N cluster sets pos- Page 7 of 13 (page number not for citation purposes)