ABSTRACT: Introduction: Human Papilloma Virus (HPV) is associated with a subset of head and neck squamous cell carcinoma (HNSCC), between 15% and 35% of HNSCC harboring HPV, almost exclusively of subtype 16. Demographic and exposure differences between HPV-positive (+) and negative (-) HNSCCs suggest that HPV(+) tumors may constitute a subclass with different biology, while clinical differences have also been observed. In this study, gene expression profiles of HPV(+) and (-) tumors were compared to further explore the biological effect of HPV in HNSCC. Methods: Thirty-six HNSCC tumors were analyzed for gene expression using Affymetrix Human 133U Plus 2.0 GeneChip and for HPV using consensus primers for HPV L1, E6 and E7 by PCR and RT-PCR. Results: Eight (22%) of 36 tumors were positive for HPV, all of the HPV 16 subtype, and the HPV positive samples also expressed viral HPV E6 mRNA determined by RT-PCR. Patients with HPV(+) HNSCCs were on average younger than those with HPV(-) tumors (mean age 50.2 vs. 58.7). Statistical analysis using Significance Analysis of Microarrays (SAM) based on HPV status as a supervising parameter resulted in a list of 91 genes that were differentially expressed with statistical significance. Results for a sub-set of these genes were verified by RT-PCR. Genes highly expressed in HPV(+) samples included cell cycle regulators (p16INK4A, p18 and CDK2) and transcription factors (TAF7L, RFC4, RPA2 and TFDP2). The microarray data were also investigated using DIGMap to map genes by chromosomal location. A large number of genes on chromosome 3q24-qter was found to be overrepresented in HPV(+) tumors. Conclusion: The gene expression profile associated with HPV reflects alterations in cell cycle and proliferation signals. Further investigation of differentially expressed genes may reveal the unique pathways in HPV(+) tumors that may explain the different natural history and biological properties of these tumors. These properties may be exploited as a target of novel therapeutic agents in HNSCC treatment. Experiment Overall Design: Patient selection and specimen collection. Thirty-six freshly frozen tumor samples were prospectively collected from patients undergoing surgery or biopsy for HNSCC at the University of North Carolina (UNC) at Chapel Hill (21 patients) and Vanderbilt University (15 patients). All tissues were snap-frozen in liquid nitrogen within 30 minutes of surgical resection or biopsy, and kept at -80oC until further processing. All patients consented to participation in this study under protocols approved by IRB at the two institutions. Experiment Overall Design: HPV detection and DNA sequencing. Tumor DNAs were tested for the presence of HPV DNA using a previously established PCR-based method [11]. This method employs degenerate PCR primers (MY09 and MY11, WD72/76 and WD66/67/154) that are designed to represent highly conserved HPV L1 and E6 sequences present in all major types of HPV. In addition, all HPV-positive samples were also tested with a HPV16-specific PCR for E7 (primer A: 5'-GGA CCG GTC GAT GTA TGT CT-3' and primer B: 3'-TAA AAC CAT CCA TTA CAT CCC G-5'). As a positive control for amplification, primers for beta-globin are included with each sample [11]. Optimal conditions for this combined PCR were determined using DNA from the cervical carcinoma cell line SiHa, which harbors on average 2 copies of HPV16 DNA per cell [11]. Other positive control cell lines were CaSki (HPV16) and HeLa (HPV18). For each case, 200 nanograms of tumor DNA were tested for the presence of HPV DNA. PCR samples which showed amplification products indicating the presence of HPV were purified using PCR purification columns (Qiagen, Valencia, CA) and subjected to bi-directional sequence analysis. In all of such cases, a positive identification of the HPV type could be made. Experiment Overall Design: RNA isolation and DNA microarray analysis. Each tumor was examined by H&E staining to ensure presence of tumor and enriched by macrodissection to achieve a minimum of 70% tumor cells in each preparation. Total RNA was purified from frozen tumors using Qiagen RNeasy Mini Kit according to the manufacturer's recommendations (Qiagen, Valencia, CA) using approximately 10-20 milligram of wet tissue from each sample. Fifty nanograms of the total RNA was amplified using NuGen RNA Amplification kit (NuGen, San Carlos, CA) and labeled ENZO BioArray High Yield RNA Transcript Labeling Kit (Affymetrix, Santa Clara, CA) according to the manufacturer's recommendations. Fifteen micrograms of biotin-labeled aRNA was fragmented and the quality of the RNA was reconfirmed using the Agilent RNA 6000 Nano LabChip Kit and Agilent 2100 bioanalyzer. The fragmented, biotin-labeled aRNA was combined with the hybridization mix and loaded on to the Affymetrix Human Genome U133 plus 2.0 GeneChip. After hybridization, the GeneChip was washed, stained with Strepavidin/phycoerythrin conjugate and biotinylated antibody, and scanned according to the manufacturer's recommendations. The raw microarray data was normalized using Perfect Match software for further statistical analyses.
