BMC Cancer 2009, 9:11 http://www.biomedcentral.com/1471-2407/9/11 Table 1: Clinicopathological characteristics of the patients in the study and validation groups (Continued) I II III IV Unknown The microarray data have been deposited in NCBIs Gene Expression Omnibus (GEO) http://www.ncbi.nlm .nih.gov/geo/ and can be accessed through GEO Series accession number GSE13601. RNA preparation and real-time RT-PCR RT-PCR of GLUT3, HSAL2, and PACE4 was performed on the larger cohort of 49 patients. Two μg of total RNA was reverse transcribed with MultiScribe™ Reverse Tran- scriptase (Applied Biosystems, Inc.). Gene specific primers were designed using the Primer3 Program. Sequences of PCR primer sets (in 5'-3' direction) were as follows: GLUT3 forward: TAGAAAGCCTGTTCCCCTCA, GLUT3 backward: GTGGCGGGATTACTTCAAAA; HSAL2 for- ward: CCCTCCTATTTCAGCCTCCT, HSAL2 backward: TCTTCAGTACCGGCACCTTC; PACE4 forward: CCTGT- GTGACCCTCTGTCCT, PACE4 backward: GGTTCATC- CACGCACTTTTT. The sequence of PCR primer sets for 18S rRNA were previously described [12]. Quantification of transcripts was performed by the ICycler Detection Sys- tem (Bio-Rad Laboratories) using SYBR green detection. The relative quantification of a target gene in comparison to a reference (18S rRNA) was performed as described [13]. Unless otherwise stated, each assay included dupli- cate reactions for each sample and was repeated twice. Statistical analysis All correlation and outcome analysis was performed using the JMP statistical software package version 4.0.0 (SAS Institute, Inc.). Disease-free survival is defined as the time from surgery to the day of the first recurrence or death. Relapse-free survival was defined as time from surgery to the day of the first recurrence. Overall survival was defined as time from surgery to the day of death or last follow-up. Results and discussion Gene expression patterns in oral tongue SCC We analyzed gene expression profiles in 20 patients with oral tongue SCC by comparison between primary tumor samples and their matched morphologically normal mucosa. Among 12,625 probe sets in the Affymetrix array, 77 probe sets had statistically significant difference 8 (21.6) 12 (32.4) 3 (8.1) 14 (37.9) 0 4 (20) 6 (30) 3 (15) 7 (35) 0 11 (22) 12 (25) 7 (14) 19 (39) 0 (P < 0.05) between all tumors and their matched normal tissues. There were 60 probe sets representing 45 genes and 11 ESTs that were increased and 17 probe sets repre- senting 9 genes and 8 ESTs that were decreased in tumors compared to normal controls. Table 2 lists the genes that were up-regulated or down-regulated along with the fold changes in gene expression in tumor compared to their normal counterparts. These include genes known to be relevant in oncogenesis such as cell proliferation, apop- tosis, development, angiogenesis, invasion and metasta- sis as well as genes that have not been implicated in oral tongue carcinogenesis. Among the 45 over-expressed genes, MMP-1 encoding interstitial collagenase showed the highest level of increase (average: 34.18 fold). MMP- 7 and MMP-12 were also found to be overexpressed. Matrix metalloproteinases (MMPs), a family of 23 human zinc-dependent extracellular endopeptidases involved in the degradation of extracellular matrix and basement membrane during tumor cell invasion, have been implicated in a number of different human tumors including head and neck SCC [14,15]. Not surprisingly, enhanced MMP-1 expression has been found to be asso- ciated with malignant progression as well as poor out- come in head and neck SCC [15-17]. Likewise, MMP-7 and MMP-12 have both been implicated in tumor aggressiveness in oral SCC [18]. Genes that have been shown to be involved in epithelial development and dif- ferentiation such as the cytokeratins KRT16 and KRT17 were found to be overexpessed. In their study investigat- ing RNA from head and neck SCC and normal tissues, Villaret et al. (2000) found KRT6 and KRT16 to be the genes most commonly expressed [19]. Similarly, KRT16 has also been found to be highly expressed in squamous cell carcinoma of the skin [20]. In addition, those that play a role in angiogenesis, such as hypoxia-inducible factor (HIF-1 ) and platelet-derived endothelial cell growth factor (ECGF1) were also overexpressed. Several transcripts were found to be significantly under- expressed or absent in tumor compared with matched normal tissues including those that encode for cell sur- face (CO-029), nuclear (ZAKI-4) and extracellular pro- teins (hSBP). Page 4 of 13 (page number not for citation purposes)