Human Cancer Biology Gene Expression Differences Associated with Human Papillomavirus Status in Head and Neck Squamous Cell Carcinoma RobbertJ.C. Slebos,1, 2 Yajun Yi, 7 Kim Ely,3 Jesse Carter,8 Amy Evjen,1Xueqiong Zhang,4 Yu Shyr,4 Barbara M. Murphy,8 Anthony J. Cmelak,5 Brian B. Burkey,2 James L. Netterville,2 Shawn Levy,6 Wendell G. Yarbrough,1, 2 and Christine H. Chung8 Abstract Human papillomavirus (HPV) is associated with a subset of head and neck squamous cell carcinoma (HNSCC). Between 15% and 35% of HNSCCs harbor HPV DNA. Demographic and exposure differences between HPV-positive (HPV+) and negative (HPVÀ) HNSCCs suggest that HPV + tumors may constitute a subclass with different biology, whereas clinical differences have also been observed. Gene expression profiles of HPV+ and HPVÀ tumors were compared with further exploration of the biological effect of HPV in HNSCC. Thirty-six HNSCC tumors were analyzed using Affymetrix Human 133U Plus 2.0 GeneChip and for HPV by PCR and real-time PCR. Eight of 36 (22%) tumors were positive for HPV subtype 16. Statistical analysis using Significance Analysis of Microarrays based on HPV status as a supervising variable resulted in a list of 91genes that were differentially expressed with statistical significance. Results for a subset of these genes were verified by real-time PCR. Genes highly expressed in HPV+ samples included cell cycle regulators (p16INK4A, p18, and CDC7) and transcription factors (TAF7L, RFC4, RPA2, andTFDP2).The microarray data were also investigated by mapping genes by chromosomal loca- tion (DIGMAP).A large number of genes on chromosome 3q24-qter had high levels of expression in HPV+ tumors. 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. Head and neck cancer remains one of the most devastating subtypes of HPV have been described in humans, with HPV cancers in the United States. Development of the vast majority type 8, 11, 16, and 18 being associated with the majority of of these tumors has been attributed to use of tobacco and human disease. In the cervix, a distinction is made between ethanol products, but a significant portion of these tumors are ‘‘low-risk’’ (types 8 and 11) and ‘‘high-risk’’ (types 16 and 18) associated with human papillomavirus (HPV; refs. 1, 2). HPV, depending on their association with premalignant and Infection with HPV is associated with malignant and prema- malignant lesions, respectively. Reports of the prevalence of lignant lesions of the uterine, cervix, vulva, penis, conjunctiva, HPV infection in head and neck squamous cell carcinoma and upper aerodigestive tract (for review, see ref. 3). Over 100 (HNSCC) indicate that 15% to 35% of HNSCC may harbor HPV sequences, depending on the detection method used (4). DNA amplification by PCR remains the most sensitive technique to detect HPV, with almost 35% of HNSCCs yielding Authors’ Affiliations: Departments of 1Cancer Biology, 2Otolaryngology, HPV-specific amplification products, although this result 3 4 5 6 Pathology, Biostatistics, Radiation Oncology, and Biomedical Informatics; may be biased because of contamination problems associated Divisions of 7Genetic Medicine and 8Hematology/Oncology, Department of Medicine, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of with PCR. HPV is most commonly found in tonsillar tumors Medicine, Nashville, Tennessee (45-100%; ref. 5) with HPV type 16 (HPV16) being found in Received 9/14/05; revised 10/24/05; accepted 11/10/05. the vast majority and HPV18 associated with most others (6). Grant support: Barry Baker Research Endowment,Vanderbilt Physician-Scientist There are some indications that HPV-positive (HPV+) Development Award (C.H. Chung), Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation (C.H. Chung and W.G. Yarbrough), and Damon Runyon Cancer HNSCCs may represent a subclass with a different biology Research Foundation (C.H. Chung). and with different clinical properties. Molecular evidence that The costs of publication of this article were defrayed in part by the payment of page HPV status determines a separate class of HNSCC comes from charges. This article must therefore be hereby marked advertisement in accordance studies showing HPV+ tumors are associated with low rates with 18 U.S.C. Section 1734 solely to indicate this fact. INK4A Note: Supplementary data for this article are available at Clinical Cancer Research of p53 or p16 mutations as opposed to HPV-negative À INK4A Online (http://clincancerres.aacrjournals.org/). (HPV ) HNSCCs, where p53 and p16 alterations are Requests for reprints: Christine H. Chung, Division of Hematology/Oncology, common (50% and 80%, respectively; refs. 7–9). Comparative Department of Medicine, Vanderbilt University School of Medicine, 2220 Pierce genomic hybridization analysis showed specific patterns of Avenue, 777 Preston Research Building, Nashville,TN 37232-6307. Phone: 615- chromosomal alterations associated with HPV+ tonsillar 322-4967; Fax: 615-343-7602; E-mail: [email protected]. F 2006 American Association for Cancer Research. tumors, which were more likely to have gain on chromosome doi:10.1158/1078-0432.CCR-05-2017 3q, or have an absence of gains on chromosome 7q relative to www.aacrjournals.org 701 Clin Cancer Res 2006;12(3) February 1, 2006 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Human Cancer Biology HPVÀ tumors (5). HPV status is also associated with specific RNeasy Mini kit according to the manufacturer’s recommendations demographics: patients with HPV+ HNSCCs are usually (Qiagen) using f10 to 20 mg of wet tissue from each sample. Fifty younger and are less likely to have tobacco exposure than nanograms of the total RNA were amplified using NuGEN Ovation those with HPVÀ tumors. Several studies have suggested that Biotin RNA Amplification and Labeling kit (NuGen, San Carlos, CA) according to the manufacturer’s recommendations. The NuGEN HPV+ tumors are associated with favorable survival (4, 10). Ovation amplification methodology uses an isothermal linear Despite these indications that HPV status is associated with amplification using random hexamers. This technology provides molecular and clinical differences, all HNSCCs are clinically sensitive and rapid whole-genome amplification without introducing managed irrespective of their HPV status. Understanding of a significant bias toward the 3V end of the transcripts (14). Fifteen the differences between HPV+ and HPVÀ HNSCC tumors may micrograms of biotin-labeled aRNA were fragmented, and the quality allow us to develop biomarkers for early detection or recurrence of the RNA was reconfirmed using the Agilent RNA 6000 Nano surveillance, to identify therapeutic targets, and to begin LabChip kit and Agilent 2100 bioanalyzer. The fragmented, biotin- individualization of treatment based on the biology of these labeled aRNA was combined with the hybridization mix and loaded tumors. The aim of this study was to identify the differences in on to the Affymetrix Human Genome U133 plus 2.0 GeneChip. After the gene expression profiles of HPV+ and HPVÀ HNSCCs and to hybridization, the GeneChip was washed, stained with streptavidin/ phycoerythrin conjugate and biotinylated antibody, and scanned better understand the biological effect of HPV infection in HNSCC. We found that there is a distinct gene expression profile that is associated with HPV status analyzed by Ta b l e 1. Patient characteristics in HPV+ and HPVÀ Significance Analysis of Microarrays. In addition, the expression cases data was analyzed using differential gene locus mapping (DIGMAP; ref. 11) to investigate the correlation between HPV + HPVÀ Total previously published chromosomal abnormalities and gene (n =8) (n =28) (N =36) expression patterns. These analyses revealed that HPV+ tumors had increased levels of expression of genes on chromosome Age (median, range)* 49 (41-65) 58 (30-89) 55 (30-89) 3q24-qter compared with HPVÀ tumors. Sex Male 8 2 1 29 Female 0 7 7 Materials and Methods Racec White 8 16 24 Patient selection and specimen collection. Thirty-six freshly frozen Black 0 9 9 tumor samples were prospectively collected from patients undergoing Other 0 3 3 surgery or biopsy for HNSCC at the University of North Carolina at Chapel Hill (21 patients) and Vanderbilt University (15 patients; see To b a c c o u s e Table 1 and Supplementary Data). All tissues were snap-frozen in liquid Ever 6 26 32 nitrogen within 30 minutes of surgical resection or biopsy and kept at Never 2 2 4 À80jC until further processing. All patients consented to participation Alcohol use in this study under protocols approved by the Institutional Review Ye s 4 1 8 2 2 Boards at the two institutions. A previous gene expression study (12) No 4 10 14 included the 21 tumors from University of North Carolina reported Tu m o r s i t e b here, but to allow comparison with the specimens from Vanderbilt and Oral cavity 0 15 15 because the Agilent platform used in the prior study was discontinued, a Oropharynx 7 2 9 completely new expression analysis was done using the Affymetrix Larynx 1 8 9 platform (see below). HPV detection and DNA sequencing. Tumor DNAs were tested for Hypopharynx 0 3 3 the presence of HPV DNA using a previously established PCR-based Clinical stage method (13). This method employs degenerate PCR primers (MY09 I-II 0 3 3 and MY11, WD72/76 and WD66/67/154) that are designed to III 4 9 13 represent highly conserved HPV L1 and E6 sequences present in all IV 4 16 20 major types of HPV. In addition, all HPV-positive samples were also Clinical cervical lymph nodex tested with a HPV16-specific PCR for E7 (primer A, 5V-GGACCGGTC- Positive 7 18 25 V V GATGTATGTCT-3 and primer B, 3-TAAAACCATCCATTACATCCCG- Negative 1 10 11 5V).