BMC Cancer 2009, 9:11 http://www.biomedcentral.com/1471-2407/9/11 recurrence (P=0.038). HSAL2 was positively associated with depth of invasion (P=0.015) and advanced T stage (P=0.047). In survival studies, only GLUT3 showed a prognostic value with disease-free (P=0.049), relapse-free (P=0.002) and overall survival (P=0.003). PACE4 mRNA expression failed to show correlation with any of the relevant parameters. Conclusion: The characterization of genes identified to be significant predictors of prognosis by oligonucleotide microarray and further validation by real-time RT-PCR offers a powerful strategy for identification of novel targets for prognostication and treatment of oral tongue carcinoma. Background Cancer arising from the oral cavity accounts for approx- imately 1.6% of all cancers diagnosed in the United States with an incidence of 22,000 new cases per year [1]. Despite the advances in multimodality treatment, the overall prognosis for patients with oral squamous cell carcinoma (SCC) has remained unchanged in the past three decades. Furthermore, variability in the clin- ical course of patients with oral SCC remains unex- plained and conventional clinicopathological parameters fail to answer all questions. Identification of novel prognostic factors may allow a rational selec- tion of the most appropriate therapeutic options for individual patients. The cellular and molecular hetero- geneity of oral SCC and the large number of genes potentially involved in oral carcinogenesis and progres- sion emphasize the importance of studying multiple gene alterations on a global scale. Gene expression pro- filing by high-throughput technologies have proven to be valuable tools for prognostication of outcome and progression in human malignancies including head and neck cancer [2-10]. These technologies permit us to classify individual cancers and enhance our under- standing of molecular cancer pathogenesis. There are several distinct subsites within the oral cavity cancer including buccal mucosa, oral tongue, floor of mouth, gingiva, retromolar trigone and hard palate. Since they differ in their biological and clinical behav- iors, the present study focused on one subsite – the oral tongue. This study utilized high-density oligonucle- otide array to generate a molecular portrait of oral tongue SCC and to explore the correlations between gene expression patterns and clinically relevant param- eters. We performed hierarchical clustering analysis, analyzed gene expression profiles by comparing pri- mary tumor and their matched normal mucosa and compared different patient groups based on lymph node status and tumor stage to identify clinically signif- icant genes. Data from the microarray analysis were then validated by real-time RT-PCR. The present study is the first to demonstrate the ability of gene expression profiling to predict clinical outcome in one cancer sub- site within the oral cavity. Methods Tumor Selection Following guidelines established by the Institutional Review Board at Memorial Sloan-Kettering Cancer Center (MSKCC), fresh tissue samples were sequentially collected after obtaining written informed consent from 49 patients undergoing therapeutic surgical resection for SCC of the oral tongue at the Head and Neck Service, MSKCC from January 28, 1998 to January 2, 2002. Post-operative adju- vant treatment was given to selected patients following the institutional protocol. In each case, the portion of tumor was resected near the advancing edge of the tumor to avoid its necrotic center. After excision, the tissues were immediately snap-frozen and stored in liquid nitrogen until use. Histologically normal mucosae of the upper aerodigestive tract, resected 5 cm away from the tumor area, were obtained in all cases and used as controls. Tumors were staged according to the AJCC/UICC TNM classification 5th edition [11]. "Node-positive cases" in this study refers to the presence of positive cervical nodes based on a histological diagnosis after a neck dissection, while the patients who experienced no metastasis for at least 12 months post-operatively were scored as "node- negative cases." The clinical and pathological characteris- tics of all patients analyzed in the study are summarized in Table 1. Oligonucleotide microarray analysis Tumor and normal tissues from 37 of the 49 patients were used for the oligonucleotide microarray analysis. Twenty (TN paired) of the 37 patients had primary tumor samples and matched normal mucosa available for analysis. Total RNA from snap-frozen tissue samples from the 37 patients was extracted with TRIsol™ reagent (Gibco BRL) following the manufacturer's protocol and re-purified by the RNAeasy Mini-spin column (Qiagen). Five to 10 μg of total RNA was reverse transcribed in the presence of an oligo dT-T7 primer. The cDNA was used for in vitro transcription amplification reaction in the presence of biotinylated nucleotides. Fifteen μg of labeled cRNA was fragmented and then hybridized against the Affymetrix HG_U95Av2 oligonucleotide arrays (Affymetrix, Santa Clara, CA). The arrays were scanned using a Hewlett Packard confocal laser scanner and analyzed using MicroArray Suite 5.0 (Affymetrix). Page 2 of 13 (page number not for citation purposes)