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Series GSE3292 Query DataSets for GSE3292
Status Public on Nov 28, 2005
Title Gene expression signature of HPV in head and neck squamous cell carcinoma
Organism Homo sapiens
Experiment type Expression profiling by array
Summary 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.
Keywords: HPV, HNSCC, head and neck cancer, human, human papilloma virus
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.
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’). 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.
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.
Contributor(s) Slebos R, Yi Y, Ely K, Carter J, Evjen A, Zhang X, Shyr Y, Murphy BM, Cmelak AJ, Burkey BB, Netterville JL, Levy S, Yarbrough WG, Chung CH
Citation(s) 16467079, 16943533
Submission date Sep 12, 2005
Last update date Mar 25, 2019
Contact name Christine H. Chung
Phone 615-322-4967
Fax 615343-7602
Organization name Vanderbilt University
Department Internal Medicine
Lab Chung
Street address 777 PRB
City Nashville
State/province TN
ZIP/Postal code 37232-6307
Country USA
Platforms (1)
GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array
Samples (36)
GSM73653 VU HNSCCV300142
GSM73654 VU HNSCCV300171
GSM73655 VU HNSCCV300251
BioProject PRJNA92797

Significant Genes List header descriptions
Gene Name
Gene ID
Fold Change

Data table
Rank Gene Name Gene ID Fold Change
Increased in HPV positive
1 233320_at Homo sapiens LOC374817 (LOC374817), mRNA --- 5.5257
2 1557570_a_at Homo sapiens, clone IMAGE:5244459, mRNA --- 3.2382
3 220325_at TAF7-like RNA polymerase II, TATA box binding protein (TBP)-associated factor, 50kDa TAF7L 3.3099
4 205691_at synaptogyrin 3 SYNGR3 2.5369
5 211156_at cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) CDKN2A 5.2947
6 226832_at Homo sapiens, clone IMAGE:5286726, mRNA --- 3.6660
7 231164_at Homo sapiens, clone IMAGE:5271371, mRNA --- 3.7015
8 201756_at replication protein A2, 32kDa RPA2 2.8151
9 241730_at myoneurin MYNN 2.3719
10 1558217_at hypothetical protein FLJ31952 FLJ31952 2.8412
11 206526_at DKFZP566F0546 protein DKFZP566F0546 2.6313
12 230011_at hypothetical protein MGC40042 MGC40042 2.2832
13 230301_at hypothetical protein LOC340277 LOC340277 2.4832
14 238977_at MCM6 minichromosome maintenance deficient 6 (MIS5 homolog, S. pombe) (S. cerevisiae) MCM6 2.7490
15 236236_at Homo sapiens cDNA FLJ42662 fis, clone BRAMY2019546 --- 3.1076
16 204023_at replication factor C (activator 1) 4, 37kDa RFC4 3.6455
17 225768_at nuclear receptor subfamily 1, group D, member 2 NR1D2 2.5845
18 226456_at hypothetical protein MGC24665 MGC24665 3.6016
19 205222_at enoyl-Coenzyme A, hydratase/3-hydroxyacyl Coenzyme A dehydrogenase EHHADH 2.9072

Total number of rows: 94

Table truncated, full table size 6 Kbytes.

Patient Data header descriptions
Institute VU: Vanderbilt University; UNC: University of North Carolina at Chapel Hill
Sex M: Male; F: Female
Race W: White; B: Black; O: Other
Site HP: Hypopharynx; L: Larynx; OC: Oral Cavity; OP: Oropharynx
Tobacco 0 = no; 1 = yes
Alcohol 0 = no; 1 = yes; . = missing data
cStage Clinical Stage
cStage Clinical Stage
cLN Clinical Cervical Lymph Nodes

Data table
Institute ID Age Sex Race Site Tobacco Alcohol cStage cStage cLN pStage pTNM Grade HPV
VU 300142 68 F W OC 1 0 3 T3N0M0 0 1 T1N0M0 Moderate Negative
VU 300148 50 M W L 0 0 3 T3N0M0 0 3 T3N0M0 Moderate Positive
VU 300171 42 M W OP 0 0 4 T4N2cM0 1 4 T4N2cM0 Moderate Positive
VU 300251 77 M O OP 1 1 4 T3N3M0 1 4 T3N3M0 Moderate Negative
VU 300373 77 F W OC 0 0 1 T1N0M0 0 1 T1N0M0 Well Negative
VU 300383 71 F W OC 1 0 4 T4N1M0 1 . . Moderate Negative
VU 300400 89 M B L 0 0 3 T3N0M0 0 3 T3N0M0 Moderate Negative
VU 300423 41 M W L 1 0 3 T3N0M0 0 3 T3N0M0 Moderate Negative
VU 300431 65 M W OP 1 0 3 T3N1M0 1 3 T3N1M0 Poor Positive
VU 300520 47 M W OP 1 1 3 T3N1M0 1 3 T3N1M0 Well Positive
VU 300533 73 F B OC 1 0 4 T2N2bM0 1 4 T4N2bM0 Moderate Negative
VU 300637 69 M W OC 1 1 3 T2N1M0 1 3 T1N1M0 Moderate Negative
VU 300663 47 F W OC 1 0 2 T2N0M0 0 2 T2N0M0 Moderate Negative
VU 300688 47 M W OC 1 1 4 T2N2bM0 1 4 T2N2bM0 Moderate Negative
VU 300710 58 M W OC 1 1 1 T1N0M0 0 3 T1N1M0 Moderate Negative
UNC 318 56 M B L 1 1 4 T4N0M0 0 4 T4N1M0 Moderate Negative
UNC 10291 72 M B L 1 1 4 T3N2aM0 1 4 T3N2aM0 Poor Negative
UNC 10393 47 M O OP 1 0 4 T3N2bM0 1 4 T3N2bM0 Moderate Negative
UNC 10394 48 M W OP 1 1 4 T1N3M0 1 4 T2N3M0 Moderate Positive
UNC 10446 59 M B L 1 1 3 T3N0M0 0 4 T3N2cM0 Poor Negative

Total number of rows: 36

Table truncated, full table size 2 Kbytes.

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE3292_RAW.tar 169.9 Mb (http)(custom) TAR (of CEL)

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