We used an integrative bioinformatic approach to identify genes in the consensus cancer coding sequence,17 located in areas of chromosomal loss or gain, which had differential expression in HNSCC (Figure 1). The process had 2 parts: target discovery and target validation. Three data sets were incorporated into target discovery: (1) genes found in the consensus cancer coding sequence survey of 13 023 possible cancer-causing genes and DNA mutations,17 (2) regional DNA loss or gain of the whole cancer genome (using a literature search of known HNSCC amplicons and deletions with aCGH data from Sparano et al30), and (3) RNA expression differences of 33 000 genes measured from expression microarray (Affymetrix, U133A and U133B plates) of 6 primary normal samples and 8 primary tumor specimens. Validation of targets was initially based on literature search, qRT-PCR for expression on primary tumor and normal samples, qPCR for validation of exact gene amplification or deletion, and/or primary tumor sequencing. The Table shows the 20 genes from the consensus cancer coding sequence,17 which are in areas of HNSCC chromosomal loss or gain. Of these 20 genes, 8 were part of areas of chromosomal deletion, and 12 were part of chromosomal amplification. Although all genes were validated somatic mutations found in breast or colon cancer, only 5 genes were CAN genes found in the final validation set by Sjöblom et al.17 These genes and their cancer mutation prevalence score, which reflects the probability that the number of mutations observed in a gene reflects a mutation frequency that is higher than that expected to be observed by chance (frequency >1.0 was considered significant), were CHL1 (1.26) (GenBank 10752), CMYA1 (1.36), ITGA9 (1.055), RUNX1T1 (2.42), and TGFBR2 (GenBank 7048) (2.85). These genes can be theoretically altered by mutation (missense, nonsense, etc), gene amplification or deletion, or via epigenetic activation or inactivation. All 20 genes may have functionally altering mutations in sporadic tumors, but gene amplification and deletion events tend to occur at a higher frequency in nonfamilial cancers, resulting in gene activation or inactivation. Five of these genes—BRCA2 (P = .003), CHL1 (P = .05), DLEC1 (P = .002), RTP1 (GenBank 132112) (P = .05), and TGFBR2 (P = .03)—showed statistically significant differences in Texp, compared with Nexp, that correlated with amplification (upregulated) or deletion (downregulated) status, based on Mann-Whitney U test. These genes are shown in Figure 2. One additional gene from the Table, EIF4A2 (GenBank 1974), is contained within a well-described 3q amplicon in HNSCC but showed statistically significant downregulation (median Texp/Nexp = 0.82; P = .02), which may reflect an area of chromosomal disruption. Also shown in Figure 2 are other genes with trends toward differential expression but which were not statistically significant: LIFR (median Texp/Nexp = 0.84; P = .61), KCNB2 (median Texp/Nexp = 1.23; P = .19), ITGA9 (median Texp/Nexp = 0.80; P = .15), and RFC4 (median Texp/Nexp = 1.54; P = .20).