Relevance of human papilloma virus (HPV) infection to carcinogenesis of oral tongue cancer

Article Outline

• Abstract
• Materials and methods
  • Tissue samples and DNA extraction
  • HPV genotyping
  • Real-time PCR
  • Evaluation of clinicopathological parameters
  • Statistical analysis
• Results
  • HPV prevalence
  • Viral status in head and neck cancers
  • Pathological correlation
  • Clinical correlation
• Discussion
• Funding
• Competing interests
• Ethical approval
• References
• Copyright

Abstract 

Human papilloma virus (HPV) infection is controversial as a causative factor in oral tongue cancer. This study aimed to clarify whether HPV directly affects the carcinogenesis and biological behaviour of oral tongue cancer by analyzing HPV prevalence, the physical status of the virus and clinicopathological parameters. Archival tissue was obtained from 36 patients diagnosed with T1 and T2 oral tongue cancer and 25 normal controls. HPV genotyping chip and real-time polymerase chain reaction were used to determine the prevalence, phenotype and physical status of HPV to clarify whether HPV directly affects oncogenesis. The results were also compared with clinicopathological parameters. HPV was detected in 36% (13/36) of oral tongue cancer patients, compared with 4% (1/25) of the control. In the HPV-positive group of oral tongue cancers, HPV-16 was the most common type and its prevalence rate was 85% (11/13). Of the HPV-16 infected oral tongue cancers, the integration rate of HPV-16 was 55% (6/11). The HPV-16 positive group showed shallower stromal invasion than the HPV-16 negative group (p=0.045). HPV-16 may be one of the causative factors in early squamous cell oral tongue carcinoma and be associated with its depth of invasion.

Keywords: human papilloma virus, integration, squamous cell carcinoma, tongue cancer

Cancers involving the oral cavity account for 2–3% of all malignancies and the tongue is the subsite with the highest incidence of cancer in the oral cavity²³. Tobacco smoking and alcohol consumption are major causative factors for head and neck squamous cell carcinoma (HNSCC) including oral tongue carcinoma, but viral infection such as high-risk human papilloma virus (HPV) may be an oncogenic factor in HNSCC¹⁰, ¹⁸.

HPV is a double-stranded DNA virus and may play a role in the pathogenesis of HNSCC given the similarities in morphology and susceptibility to HPV exposure between the tissues involved in head and neck cancers and uterine cervical cancer⁹, ¹³. The tongue may be the first site of exposure to viral microorganisms in the aerodigestive tract and oral tongue cancer could be susceptible to HPV exposure, directly or indirectly. The prevalence of HPV in oral tongue cancer is extremely diverse, ranging from 0% to 100% in the literature, and the prevalence of HPV in HNSCC is not uncommon⁵, ¹⁰, ¹⁷, ²¹. The markedly different reports of prevalence of HPV in oral tongue cancer may be due to: mixed samples with the oropharynx; methodological differences for detecting HPV, including less accurate methods; various tumour stages and racial and geographical differences between the studies. Of these, the methodological difference is the most important because results from recent studies using advanced technology for the detection of HPV differ to a great extent compared with the results of previous less accurate studies¹¹. The role of HPV in HNSCC is mainly carcinogenesis, leading to the possibility that studies including both early and late stage cancers may be inaccurate.

The prevalence and physical status of HPV purely in oral tongue cancer, excluding cancer of the base of the tongue, especially in the early stages, has not been studied widely. This study aims to clarify whether HPV directly affects the carcinogenesis and biological behaviour of early oral tongue cancer by analyzing the prevalence of HPV, the physical status of the virus and the clinicopathological parameters, using the most up-to-date technology.

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Materials and methods 

Tissue samples and DNA extraction 

Paraffin-embedded tissues were obtained from 36 patients who were diagnosed with T1 or T2 early oral tongue cancer and received surgery as initial treatment between 1995 and 2005. For comparative analysis, 25 pathologically normal oral tongue tissue samples obtained from leukoplakia specimens were included as a control group. The Institutional Review Board of Yonsei University approved the protocol.

Ten micrometer sections were cut from the paraffin blocks and collected in 1.5ml Eppendorf tubes for DNA extraction. To prevent cross-contamination, each block was cut after thorough cleaning of the microtome blade. Paraffin-embedded samples were placed in xylene for 5min and centrifuged at 14,000rpm. DNA extraction was carried out using the QIAamp DNA Mini kit (Qiagen, CA, USA). The quality (ratio of 260nm/280nm) and quantity (absorbance at 260nm) of the isolated DNA were determined by optical density measurement. CasKi and SiHa cells were grown for approximately 5 days in the appropriate medium (CasKi cells and SiHa cells, RPMI 1600 [Gibco-BRL, Grand Island, NY, USA]). DNA isolation was performed with the QIAamp DNA Mini Kit according to the protocol for cultured cells grown in a monolayer.

HPV genotyping 

An HPV genotyping DNA chip (Biocore, Korea, Seoul) arrayed with multiple oligonucleotide probes of L1 sequences from 26 types of HPV was used according to the manufacturer's protocol. Consensus polymerase chain reaction (PCR) products for L1 were hybridized to the arrayed probes on the HPV chip, and HPV genotypes were identified using a fluorescence scanner (GenPix 4000B, Axon Instruments Inc., CA, USA) with a 532-nm laser for excitation of Cy3. The fluorescence intensity data of the specific probes were then printed out as an Excel spreadsheet.

Real-time PCR 

The copy numbers of the HPV E2 and E6 open reading frames (ORF) were assessed using a TaqMan-based 5′-exonuclease quantitative real-time PCR assay based on the DNA amplification of a 76-bp sequence of the E2 ORF and an 81-bp sequence of the E6 ORF, in the presence of HPV-16 E2- and E6-specific hybridization probes, respectively. The primers and probes for the E2 assay were designed to recognize the E2 hinge region of the E2 ORF, which is most often deleted upon HPV-16 integration in cervical carcinomas. For each specimen, identical amounts of DNA were quantified for the E6 and E2 sequence of HPV-16. Each specimen was assayed three times. PCR amplification was performed in a 25μl volume containing 1× iQ SuperMix (BioRad, Hercules, CA, USA), 200nM E2 and E6-specific primers (Table 1), 100nM dual-labelled (5′Hex and 3′BHQ2) E2 and (5′FAM and 3′BHQ1) E6 fluorogenic hybridization probe, and 200ng of the genomic DNA template. All experiments were performed using the real-time iCycler™ PCR platform (BioRad, Hercules, CA, USA). In each experiment, two standard curves were included obtained by amplification of a dilution series of the HPV viral copy number using CaSki (American Type Culture Collection, Manassas, VA, USA) cell line genomic DNA, which is known to have 600copies/genome equivalents (6.6pg of DNA/genome)¹. There was a linear relationship between the threshold cycle values plotted against the log of the copy number over the entire range of dilutions. The amplification ramp included two hold programmes of 2min at 50°C and 10min at 95°C, followed by a two-step PCR cycle with a melting step for 15s at 95°C and an annealing step for 1min at 60°C for a total of 45 cycles. The ratio of E2 to E6 copy numbers was calculated to determine the physical status of the HPV-16 viral gene. HPV-16 in the pure episomal form is expected to have equivalent copy numbers to those of E2 and E6 genes (i.e. E2/E6 ratio=1), whereas preferential disruption of E2 upon viral integration should result in fewer E2 gene copies than E6 genes. This means that an E2/E6 ratio of less than 1 would indicate the presence of both the integrated and episomal forms while a ratio of 0 would indicate the presence of only an integrated form. The copy number of the integrated E6 gene was calculated by subtracting the copy number of E2 (episomal) from the total copy number of E6 (episomal and integrated). The ratio of E2 to integrated E6 genes represents the amount of the episomal form in relation to the integrated form. Values less than one indicate the predominance of the integrated form. DNA extracted from the cervical carcinoma cell line SiHa, known to harbour a pure, integrated form of the HPV-16 gene in which the E2 and E4 ORFs are disrupted, was used as the control for E2 (negative) and E6 (positive) amplification⁴. The relative viral load can be estimated by calculating the ratio of copies of E6 in the sample and SiHa cells.

Table 1. Primers used for identifying HPV-16 physical status.

Evaluation of clinicopathological parameters 

The follow-up period ranged from 13 to 120 months with a mean of 61 months. Surviving patients were followed up for at least 24 months. In order to identify the factors that may be associated with HPV infection, the correlation between clinicopathological factors, such as TNM stage, depth of invasion, recurrence, survival, and HPV prevalence, was analyzed.

Statistical analysis 

The relationship between HPV status and clinicopathological parameters was analyzed using cross-tabulations and Fisher's exact test with SAS software, version 9.1 (SAS Institute Inc., Cary, NC, USA). The survival rate of the patients was calculated using the Kaplan–Meier method and curves were compared using the log-rank test. A p-value less than 0.05 was considered statistically significant.

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Results 

HPV prevalence 

In the present study, HPV prevalence in early oral tongue cancer was 36% (13/36). In the HPV-positive tumours, 11 cases (84.8%) were infected with HPV-16 and the others were infected with non-16 high-risk type and low-risk type HPV each and multiple infections were not found in these cases. Corresponding control samples, neighbouring normal tissue of hyperplastic leukoplakia of the tongue, demonstrated rare incidence of HPV infection (1/25; 4%). There was a statistically significant difference in HPV prevalence between early oral tongue cancer and control samples (p<0.05) and HPV-16 was the single most common type.

Viral status in head and neck cancers 

Both validated assays of the real-time amplification systems for E2 and E6 ORFs using serially-diluted HPV-16 plasmid DNA showed similar amplification efficiencies as reflected by the almost identical slopes of the amplification curves (Fig. 1). The results for the physical states and viral copy numbers are summarized in Table 2. Integrated E6 was calculated by subtracting the numbers of E2 from E6. The ratio of E2 to integrated E6 represents the amount of the episomal form relative to the integrated form. A value of less than 1.0 indicates a predominance of the integrated form. In the 11 early oral tongue cancer samples with HPV-16 infection, six cases were integrated (55%) (Table 2).

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• Fig. 1. 

  Comparison of PCR amplification efficiencies for HPV-16 E2 and E6. A five-point 10-fold series of Caski cell line genomic DNA (2×10⁻² to 2×10²ng) with amplification efficiencies was found to be very similar for the two reactions.

Table 2. Physical status of HPV-16 infection in early oral tongue cancers.

TC: tongue cancer.

Pathological correlation 

Oral tongue cancers in this study were composed entirely of early-onset tumours; the incidence of pT1 (less than 2cm of superficial dimension) was 31% and of pT2 (less than 4cm) was 70%. Tumour invasion into the underlying musculature was presented in a tentacular or expansile pattern. Pathologically, there was a significant difference in the invasion characteristics, depending on the presence of HPV infection. 67% of HPV-positive cancer showed a pushing margin and less infiltrative pattern; 57% of HPV-negative cancer showed an infiltrative margin (Fig. 2).

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• Fig. 2. 

  Representative pathological pictures of HPV-16 positive tongue cancer (a) and HPV-16 negative tongue cancer (b). Pathologically, there was a difference in the invasion characteristics, depending on the presence of HPV-16 infection. 67% of HPV-16 positive cancer showed pushing margin and less infiltrative pattern. On the contrary, 57% of HPV-16 negative cancer showed infiltrative margins.

Invasion depths were compared according to HPV-16 infection in oral tongue cancer. The ratio of vertical invasion to horizontal width was significantly low in the HPV-16 positive group (p=0.045) (Fig. 3). This means HPV-16 integrated oral tongue cancer showed a significant association with shallower infiltration to the stroma. Pathological analysis was carried out by one head and neck dedicated pathologist.

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• Fig. 3. 

  Comparison of invasion depths according to HPV-16 infection in oral tongue cancer. The ratio of vertical invasion to the horizontal width was significantly low in the HPV-16 positive group (p=0.045).

Clinical correlation 

All cases in this study were composed of early T1 and T2 cancers: stage I (10 cases; 29%); stage II (16 cases, 44%); stage III (3 cases, 8%); and stage IV (7 cases, 19%). After a mean follow up of 61 months, recurrence was identified in six cases and the 5-year disease-free survival was 81%. There was no association with clinical factors including TNM stage and recurrence with HPV infection in early oral tongue cancer except for the invasion depth (Fig. 3). HPV infection was associated with favourable tumour-specific survival rate in early oral tongue cancer, although it was not statistically significant (p=0.24) (Fig. 4).

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• Fig. 4. 

  Comparison of tumour-specific survival according to HPV-16 infection. The HPV-16 positive group showed favourable disease-specific survival (p=0.24).

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Discussion 

The key risk factors for head and neck cancer worldwide include tobacco smoking, alcohol abuse, and specifically a combination of the two, but viral infection is also considered a risk factor for head and neck cancer, and HPV is one of the most well known viruses. The molecular mechanism of viral oncogenesis of HPV has been established. After viral infection, disruption occurs most frequently in an E2 open reading frame of the HPV genome, which is called integration, and the breakage of E2 allows for the dysregulation of the E6/E7 oncoprotein. The viral protein, E6, promotes the degradation of p53 and E7 inactivates pRb, which leads to cell cycle dysregulation and eventually malignant transformation¹⁹, ²², ²⁵. Integration to the nucleus of the host cell after infection is a prerequisite for HPV-16 to induce cancer, though previous studies only investigated the presence of the infection using PCR, in situ hybridization or immunohistochemical staining. Episomal status in which integration to the host cell has not occurred after HPV-16 infection is regarded as a positive result in these studies. There is a need for research on the physical status that identifies the presence of integration to the nucleus of the host cell, and the present study does this.

Many studies have been carried out on the prevalence of HPV and its carcinogenic effect in oral tongue cancer, but debates remain about other subsites in the head and neck. In one of the largest multicentre case–control oral cancer studies, the incidence of HPV infection was 4% in 766 oral cavity cancers²⁴. Dahlgren et al. reported a very low prevalence of HPV (2%) in 85 mobile tongue cancers and HPV infection had no clinical significance in oral tongue cancer⁷. Liang et al. performed a PCR assay for HPV in 51 fresh-frozen oral tongue cancer tissues but the overall frequency of HPV was only 2% (1/51)¹⁶. Meta-analytic data on 5046 patients from 60 studies of head and neck cancers, showed an overall HPV prevalence of oral tongue cancer of 24%¹⁵. Kozomara et al. reported an HPV prevalence rate of 64% (32/50) in oral cavity cancer (mostly oral tongue). They also reported that most infections were high-risk HPVs and HPV infection was related to poor prognosis¹⁴. da Silva et al. reported a high prevalence rate for HPV (74%) in oral tongue cancer using a PCR assay with fresh tissue⁸.

One of the main causes of this discrepancy is the heterogenous primary site of the subjects. Many studies on the prevalence of HPV have looked at all oral cavity cancer including multiple primary sites, not oral tongue cancer alone. Even the studies for tongue cancer have included cancer of the base of the tongue, which is in the oropharynx not the oral cavity. In the present study, the authors investigated only oral tongue cancer patients, who were diagnosed with T1 or T2 early lesions and had not received any previous treatment. The diverse methods to detect HPV DNA also result in a wide range of data variation and inconsistency in determining HPV prevalence², ²⁰. In contrast to other meta-analyses or cumulative studies based on genotype-specific PCR or other classical methods, the present study was carried out using the HPV DNA chip, which is high throughput, state-of-the-art technology with the most sensitive and specific tool to detect 26 different HPV genotypes. In the present study, HPV was detected in 36% of early oral tongue cancer and this prevalence rate showed that HPV infection is not uncommon in early oral tongue cancer patients and there was a statistically significant difference between early oral tongue cancer and the normal control group.

Of the HPV-positive tumours in early oral tongue cancers, HPV-16 is the most common genotype. According to Kremier et al.¹⁵ HPV-16 accounts for most HPV-positive oral tongue cancer (68%). In the present study, the frequency of HPV-16 was 85% of all HPV-positive early oral tongue cancers. The overwhelming prevalence of HPV-16 high-risk type tumours alone in head and neck cancers is different from that in the uterine cervix; the latter demonstrated HPV-16 prevalence equivalent to 63% and multiple infections with more than two different genotypes was estimated to be 21%³.

The physical status of HPV has been analyzed using real-time PCR and state-of-the-art technology, with ease and great reproducibility. The data showed HPV-16 integration in 55% of HPV-16 positive tonsillar cancers, with 18% having complete integration and 36% having a mixture of integrated and episomal forms. In the authors’ previous study of tonsillar cancer, the HPV-16 integration rate was 94% in HPV-16 positive tonsil cancers with 41% having complete integration¹³. This site-specific difference in the physical status of HPV-16 is suggestive of either a direct or indirect role in carcinogenesis. Integration of HPV DNA in key points of the cell genome could cause carcinogenesis by an alternative mechanism unrelated to early gene transcription by blocking the transcription of tumour suppressor genes or activating proto-oncogene transcription². HPV infection may have a less direct role in the carcinogenesis of early oral tongue cancer than in tonsillar cancer.

The pathological T stage of tongue cancer depends entirely on the greatest dimension, irrespective of vertical dimension or muscle invasion. Invasion depth of the tongue muscle alone is not considered in pT even though the tongue musculature is well supplied with blood vessels that form numerous anastomoses and the lymphatics drain mainly into the submandibular and deep cervical lymph nodes. In surveys, early-onset tongue cancers are entirely composed of pT1 or pT2, and a higher relative ratio of the vertical dimension to the horizontal width is significantly associated with a poor outcome¹². In the present study, regarding HPV infection, early oral tongue cancer cases with HPV infection revealed fewer invasions into the intrinsic musculature (p=0.045). These findings are compatible with previous results, which suggested that patients with HPV-positive head and neck cancers have better survival rates than those with HPV-negative cancers⁶, ¹⁷.

In conclusion, although HPV prevalence in early oral tongue cancer was lower than that in oropharyngeal cancer, there was a significantly higher prevalence of HPV than in the normal control group, and HPV infection was related to a shallower vertical invasion depth of oral tongue muscle. Most HPV infections occurred due to HPV-16 and integrations were found in more than half of the cases. The authors conclude that HPV infection has a role in the oncogenesis of oral tongue squamous cell carcinoma.

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Funding 

This research was supported by academic research program funded by Korean Society of Otorhinolaryngology-Head and Neck Surgery.

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Competing interests 

None declared.

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Ethical approval 

Not required.

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References 

1. Alazawi W, Pett M, Arch B, Scott L, Freeman T, Stanley MA, et al. Changes in cervical keratinocyte gene expression associated with integration of human papillomavirus 16. Cancer Res. 2002;62:6959–6965
   • View In Article
   • MEDLINE
2. Almadori G, Cadoni G, Cattani P, Galli J, Bussu F, Ferrandina G, et al. Human papillomavirus infection and epidermal growth factor receptor expression in primary laryngeal squamous cell carcinoma. Clin Cancer Res. 2001;7:3988–3993
   • View In Article
   • MEDLINE
3. An HJ, Cho NH, Lee SY, Kim IH, Lee C, Kim SJ, et al. Correlation of cervical carcinoma and precancerous lesions with human papillomavirus (HPV) genotypes detected with the HPV DNA chip microarray method. Cancer. 2003;97:1672–1680
   • View In Article
   • MEDLINE
   • CrossRef
4. Baker CC, Phelps WC, Lindgren V, Braun MJ, Gonda MA, Howley PM. Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines. J Virol. 1987;61:962–971
   • View In Article
   • MEDLINE
5. Bouda M, Gorgoulis VG, Kastrinakis NG, Giannoudis A, Tsoli E, Danassi-Afentaki D, et al. “High risk” HPV types are frequently detected in potentially malignant and malignant oral lesion, but not in normal oral mucosa. Mod Pathol. 2000;13(6):644–653
   • View In Article
   • MEDLINE
   • CrossRef
6. Cruz IB, Snijders PJ, Steenbergern RD, Meijer CJ, Snow GB, Walboomers JM, et al. Age-dependence of human papillomavirus DNA presence in oral squamous cell carcinomas. Eur J Cancer B Oral Oncol. 1996;32B:55–62
   • View In Article
   • MEDLINE
7. Dahlgren L, Dahlstrand H, Lindquist D, Högmo A, Björnestål L, Lindholm J, et al. Human papillomavirus is more common in base of tongue than in mobile tongue cancer and is a favorable prognostic factor in base of tongue cancer patients. Int J Cancer. 2004;112:1015–1019
   • View In Article
   • MEDLINE
   • CrossRef
8. da Silva CE, da Silva ID, Cerri A, Weckx LL. Prevalence of human papillomavirus in squamous cell carcinoma of the tongue. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007;104(October (4)):497–500
   • View In Article
   • Abstract
   • Full Text
   • Full-Text PDF (133 KB)
   • CrossRef
9. Frisch M, Goodman MT. Human papillomavirus-associated carcinomas in Hawaii and the mainland. Cancer. 2000;88:1464–1469
   • View In Article
   • MEDLINE
   • CrossRef
10. Gillison ML, Shah KV. Human papillomavirus-associated head and neck squamous cell carcinoma: mounting evidence for an etiologic role for human papillomavirus in a subset of head and neck cancers. Curr Opin Oncol. 2001;13:183–188
    • View In Article
    • MEDLINE
    • CrossRef
11. Hobbs CG, Sterne JA, Bailey M, Heyderman RS, Birchall MA, Thomas SJ. Human papillomavirus and head and neck cancer: a systematis review and meta-analysis. Clin Otolaryngol. 2006;31:259–266
    • View In Article
    • MEDLINE
12. Johnson N, Franceschi S, Ferlay J. TNM classification of carcinomas of the oral cavity and oropharynx. In:  Barnes L,  Eveson JW,  Reichart P,  Sidransky D editor. Pathology and Genetics of Head and Neck Tumors. Lyon: IARC, Inc.; 2005;p. 164–175
    • View In Article
13. Kim SH, Koo BS, Kang S, Park K, Kim H, Lee KR, et al. HPV integration begins in the tonsillar crypt and leads to the alteration of p16, EGFR and c-myc during tumor formation. Int J Cancer. 2007;120:1418–1425
    • View In Article
    • MEDLINE
    • CrossRef
14. Kozomara R, Jović N, Magić Z, Branković-Magić M, Minić V. p53 mutation and human papillomavirus infection in oral squamous cell carcinomas: correlation with overall survival. J Craniomaxillofac Surg. 2005 Oct;33(5):342–348
    • View In Article
    • Abstract
    • Full Text
    • Full-Text PDF (185 KB)
    • CrossRef
15. Kremier AR, Clifford GM, Boyle P, Franceschi S. Human papillomavirus types in head and neck squamous cell carcinomas worldwide: a systemic review. Cancer Epidemiol Biomarkers Prev. 2005;14:467–475
    • View In Article
    • MEDLINE
    • CrossRef
16. Liang X, Lewis J, Foote R, Smith D, Kademani D. Prevalence and significance of human papillomavirus in oral tongue cancer: the Mayo clinic experience. J Oral Maxillofac Surg. 2008;66:1875–1880
    • View In Article
    • Abstract
    • Full Text
    • Full-Text PDF (165 KB)
    • CrossRef
17. Matzow T, Boysen M, Kalantari M, Johansson B, Hagmar B. Low detection rate of HPV in oral and laryngeal carcinomas. Acta Oncol. 1998;37:73–76
    • View In Article
    • MEDLINE
    • CrossRef
18. McKing RG, Baric RS, Olhsan AF. Human papillomavirus and head and neck cancer: epidemiology and molecular biology. Head Neck. 1998;20:250–265
    • View In Article
    • MEDLINE
    • CrossRef
19. Munger K, Werness BA, Dyson N, Phelps WC, Harlow E, Howley PM. Complex formation of human papillomavirus E7 protein with the retinoblastoma tumor suppressor gene product. EMBO J. 1989;8:4099–4105
    • View In Article
    • MEDLINE
20. Rees L, Birchall M, Bailey M, Thomas S. A systemic review of case–control studies of human papillomavirus infection in laryngeal squamous cell carcinoma. Clin Otolaryngol. 2004;29:301–306
    • View In Article
21. Ringström E, Peters E, Hasegawa M, Posner M, Liu M, Kelsey KT. Human papillomavirus type 16 and squamous cell carcinoma of the head and neck. Clin Cancer Res. 2002;8(10):3187–3192
    • View In Article
    • MEDLINE
22. Scheffner M, Werness BA, Huibregtse JM, Levine AJ, Howley PM. The E6 oncoprotein encoded by human papillomavirus type 16 and 18 promotes the degradation of p53. Cell. 1990;63:1129–1136
    • View In Article
    • MEDLINE
    • CrossRef
23. Silverman S. Demographics and occurrence of oral and pharyngeal cancers. The outcomes, the trends, the challenge. J Am Dent Assoc. 2001;132(Suppl.):7S
    • View In Article
24. Syrjanen S. Human papillomavirus (HPV) in head and neck cancer. J Clin Virol. 2005;32:59–66
    • View In Article
    • Abstract
    • Full Text
    • Full-Text PDF (112 KB)
    • CrossRef
25. Zur Hausen H. Papillomaviruses in human cancers. Proc Assoc Am Physicians. 1999;111:581–587
    • View In Article
    • MEDLINE
    • CrossRef