ARTICLE

Human Papillomavirus in Oral Exfoliated Cells and Risk of Head and Neck Cancer

Elaine M. Smith, Justine M. Ritchie, Kurt F. Summersgill, Henry T. Hoffman, Dong Hong Wang, Thomas H. Haugen, Lubomir P. Turek

Affiliations of authors: Departments of Epidemiology (EMS, DHW) and Biostatistics (JMR), College of Public Health, and Departments of Otolaryngology (HTH) and Pathology (THH, LPT), College of Medicine, University of Iowa, Iowa City; Veterans Affairs Medical Center, Iowa City (DHW, THH, LPT); Department of Oral Medicine and Pathology, School of Dental Medicine, University of Pittsburgh, Pittsburgh, PA (KFS)

Correspondence to: Elaine M. Smith, PhD, MPH, Department of Epidemiology, C-21 C GH, College of Public Health, University of Iowa, Iowa City, IA 52242 (e-mail: elaine-smith{at}uiowa.edu)


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Background: Human papillomavirus (HPV) has been associated with the development of head and neck cancers. In this study, we investigated whether the risk factors for head and neck cancer in relation to HPV infection are different from those in the absence of HPV infection and whether HPV detected in oral exfoliated cells is an independent predictor of head and neck cancer risk. Methods: We conducted a case–control study in 201 head and neck cancer case patients and 333 control subjects, frequency matched for age and sex. Oral exfoliated cells and tumor tissue were evaluated for HPV using polymerase chain reaction and DNA sequencing to type HPV. Logistic regression was used to calculate odds ratios (ORs) for head and neck cancer with HPV infection and 95% confidence intervals (CIs), adjusted for age, tobacco use, and alcohol consumption. Results: Oncogenic, or high-risk (HR), HPV types were detected in oral cells from 22.9% of case patients and 10.8% of control subjects. HPV16 was the most frequently detected type (19% versus 10% of case patients and control subjects, respectively). After adjusting for age, tobacco use, and alcohol consumption, the risk of head and neck cancer was statistically significantly greater in individuals with HPV-HR types (adjusted OR = 2.6, 95% CI = 1.5 to 4.2) but not in individuals with nononcogenic HPV types (adjusted OR = 0.8, 95% CI = 0.4 to 1.7) compared with HPV-negative individuals. Compared with individuals who were HPV-negative and did not use alcohol or tobacco, there was a statistically significant synergistic effect between detection of HPV-HR and heavy alcohol consumption (OR = 18.8, 95% CI = 5.1 to 69.5) but an additive effect between detection of HPV-HR and tobacco use (OR = 5.5, 95% CI = 2.1 to 14.1). HPV-HR types detected in oral exfoliated cells were predictive of HPV-HR types in tumor tissue.Conclusion: Infection of oral exfoliated cells with HPV-HR types is a risk factor for head and neck cancer, independent of alcohol and tobacco use, and acts synergistically with alcohol consumption. HPV testing of an oral rinse may be predictive of an HPV-related head and neck cancer.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
An association between the presence of human papillomavirus (HPV) and the development of head and neck cancer has been established recently (14). The association is strengthened by the fact that the same oncogenic HPV types detected in cervical carcinomas have been identified in head and neck cancers (5). Furthermore, there is molecular evidence that the same viral oncoproteins, E6 and E7 of HPV high-risk (HR) types, which inactivate tumor suppressor gene pathways and promote genomic instability in cervical cancer, do the same in head and neck cancer (2,6). Infection with oncogenic HPV types and the other major risk factors for head and neck cancer, tobacco and alcohol, may represent alternative pathways in the development of these cancers, and this relationship needs clarification.

The association of head and neck cancers with clinically significant morbidity and disfiguration makes early detection of the diseases and biomarkers to identify individuals at high risk of great importance. Furthermore, one of the conclusions from a recent National Cancer Institute workshop convened to assess viruses associated with human cancers (7) was that future HPV research needs to focus on developing a sensitive, validated laboratory test to detect HPV in oral exfoliated cells that would reflect HPV-HR types in head and neck tumors. Unfortunately, few studies (6,8) have collected oral exfoliated cells prior to treatment for comparison with biopsy specimens. Most investigations have resorted to HPV assessment of either biopsy specimens or serum and have limited evaluation to a few HPV types. Interpretation of serologic findings is further complicated because it is not clear whether a positive test indicates a current or historical infection in the oral mucosa or elsewhere in the body.

The purpose of this study was to describe risk factors for head and neck cancers associated with oncogenic HPV detection. We also examined whether HPV-HR DNA, detected from an oral rinse of exfoliated cells at the time of diagnosis but before treatment, would predict patients at high risk for these cancers.


    PATIENTS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Patients

All newly-diagnosed patients with primary head and neck cancer aged 18 years or older who were seen at the University of Iowa Hospitals and Health Care (UIHC) or at the Veterans Affairs Medical Center, Iowa City, between 1994 and 1997 were eligible for enrollment. Of the eligible head and neck cancer patients, fewer than 2% were excluded because of mental incapacity or a language barrier, 1% did not complete the questionnaire or specimen collection procedures prior to treatment, and 2% refused to participate. Another 1% subsequently had inadequate DNA for HPV assessment. Thus, 201 case patients participated in the study. All sites in the oral cavity and oropharynx were examined because one of the purposes of this study was to determine whether HPV could be detected in each of these areas. Based on the American Joint Committee on Cancer (AJCC) criteria (9), the combined oral cavity sites included tumors from lip vermilion and inner mucosa, tongue, gingiva, floor of mouth, hard palate, other oral mucosa, parotid gland, and submandibular gland. The oropharyngeal sites included tumors of the base of tongue, soft palate, uvula, palatine tonsil fossa, pillar and overlapping lesions, oropharynx, and other unspecified oropharynx lesions. Staging was based on the AJCC criteria (9). Because the risk associated with HPV detection in histologic types (including acinic-cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, neuroendocrine carcinoma, and verrucous carcinoma) other than squamous-cell carcinoma (SCC) was unclear, we examined these histologic types in separate analyses. Of the 201 patients, 186 had SCC and 15 had other histologic types. The control subjects were recruited from family medicine and internal medicine clinics in the hospitals where they were being seen for routine screening visits. Inclusion criteria for the control subjects included being age 18 years or older and having no prior history of head and neck cancer. This information was verified from hospital medical records and the Iowa National Cancer Institute–Surveillance, Epidemiology, and End Results (SEER) Program1 Cancer Registry. Fewer than 1% of the control subjects were excluded because of prior head and neck cancer, 2% did not complete the specimen collection requirements, and 5% refused to participate. Control subjects were frequency-matched to case patients by 5-year age groups and by sex. The study enrolled 333 control subjects from whom an oral rinse of exfoliated cells was collected to test for HPV DNA.

Data Collection

After the university-approved institutional review board form was administered and signed, all patients completed a self-administered risk factor questionnaire with information about sociodemographics, medical history, smoking habits, alcohol consumption, sexual practices, and history of HPV-related diseases and oral lesions. From patient medical records and pathology reports, the research staff collected information regarding previous cancer history, cancer site, treatments, and staging. Before treatment, exfoliated cells were collected in expectorate from patients who had swished vigorously with an oral rinse of 10 mL of normal saline for 30 seconds. All specimens were reviewed using a hemocytometer to estimate the number of nucleated squamous cells and then were frozen at -80 °C. The cell count was used in preparing samples for polymerase chain reaction (PCR) analysis to ensure that a minimum number of nucleated cells would be included in each reaction.

Laboratory Methods

Procedures for extraction and PCR analysis of DNA from exfoliated cells have been described elsewhere (10). Each PCR mixture included DNA from 30 000 exfoliated cells. Methods used to determine HPV DNA types in tumor tissue have been described previously for laser capture microdissection (11) and tissue blocks (12). Each PCR mixture included primers that amplified mucosal HPV types (MY09/11) and primers that amplified a portion of the {beta}-globin gene (13), which was used to verify the presence of intact DNA and the adequacy of PCR amplifications. Any sample whose {beta}-globin PCR was negative was excluded from the study (three oral exfoliated specimens and two tumor specimens). The PCR product was examined for the presence of a 400-nucleotide amplified HPV band on ethidium bromide–stained 1% agarose gels. Products also were spotted on a nylon membrane and hybridized with 32P-labeled probes for more sensitive detection of HPV by dot blot hybridization (10). The dot blot detected fewer than 10 SiHa cells (obtained from American Type Culture Collection, Manassas, VA) in a spiked sample, or less than one copy of HPV per 1000 squamous cells in sample specimens used in the PCR analysis. Those samples found to be positive after the membrane hybridization underwent hemi-nested PCR amplification with universal HPV-specific MY09 and GP5+ primers (14). HPV PCR products were sequenced directly and compared with sequences in a DNA database to determine their HPV type (15,16). HPV types were considered high-risk (HPV-HR) if they have been associated with cervical cancer or were genetically related to high-risk types. All other HPV types were considered low-risk (HPV-LR). Samples were considered HPV-positive if a visible band was seen after the first round of PCR or after hemi-nested PCR.

Statistical Analysis

Tobacco exposure included use of cigarettes, cigars, pipes, chewing tobacco, and snuff. Total cumulative tobacco use was expressed in pack-years. One pack-year was equivalent to smoking one pack of cigarettes (20 cigarettes per pack) per day for 1 year, and tobacco equivalents were defined as one cigar equals one pipeful equals 1 ounce of snuff equals 1 ounce of chewing tobacco equals one cigarette. Alcohol consumption included intake of beer, wine, and liquor. Total alcohol consumption was defined as the average number of drinks per week. Alcohol equivalents were defined as one 12-ounce can or bottle of beer equals one 4-ounce glass of wine equals one 11/2-ounce shot of liquor. Study participants also were categorized as current, former, or never users; former users included individuals who had quit drinking alcohol and/or smoking at least 1 year before diagnosis or study entry for case patients and control subjects, respectively.

The Wilcoxon rank sum test and the chi-square test were used to compare quantitative and categorical variables between the two groups of patients, respectively. Multivariate logistic regression was used to assess the association between disease status and oncogenic mucosa HPV controlling for age, tobacco use, and alcohol consumption. Nonlinearity for quantitative variables was evaluated as discussed by Hosmer and Lemeshow (17). Cut points for tobacco use (never, <=30 [low], or >30 pack-years [high]) and alcohol consumption (never, <=21 [low], or >21 drinks per week [high]) were based on median levels among either the control subjects or the HPV-negative case patients.

Effect modification for oral HPV status by tobacco and alcohol use was evaluated on a multiplicative scale using the likelihood ratio test by including the appropriate interaction term in the multivariate logistic regression models. Additive joint effects were examined using the synergy index [SI; (1719)]. Five indicator variables representing the combination of the HPV result and tobacco or alcohol use were created: 1 = HPV-negative/low tobacco/alcohol exposure; 2 = HPV-negative/high tobacco/alcohol exposure; 3 = HPV-HR/no tobacco/alcohol exposure; 4 = HPV-HR/low tobacco/alcohol exposure; and 5 = HPV-HR/high tobacco/alcohol exposure. The reference group included HPV-negative patients with no tobacco use or no alcohol consumption. The synergy index was calculated as the ratio of the odds ratio (OR) estimate for the combination of the exposures minus 1 (e.g., OR5 - 1) to the sum of the odds ratio estimates for the individual exposures minus 2 (e.g., OR2 + OR3 - 2). The 95% confidence interval (CI) for the synergy index is based on the normal approximation and the variance and covariance estimates of the fitted coefficients for the indicator variables from the logistic regression models, as discussed by Hosmer and Lemeshow (18). When the synergy index and the lower limit of the corresponding 95% CI are both greater than one, the joint effect of the two exposures is statistically significantly more than that expected based on the sum of the individual effects. Analyses were performed using the SAS statistical package (20). All reported P values were two-sided.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
HPV Prevalence and Types

Based on HPV detected in oral exfoliated cells, the overall prevalence rate of HPV was greater in case patients than in control subjects (Table 1), and case patients were more than twice as likely to be detected with HPV-HR types (22.9% versus 10.8%, respectively). Compared with HPV-negative patients, only individuals detected with HPV-HR types had an increased risk of head and neck cancer (OR = 2.6, 95% CI = 1.5 to 4.2) The risk of head and neck cancer in participants with HPV-LR types (OR = 0.8, 95% CI = 0.4 to 1.7) was similar to that of HPV-negative patients. When the analysis was restricted to case patients with SCCs (n = 186), individuals with HPV-HR had an increased risk of head and neck cancer relative to HPV-negative individuals (OR = 2.7, 95% CI = 1.6 to 4.5), but individuals with HPV-LR types did not (OR = 0.9, 95% CI = 0.4 to 1.9).


View this table:
[in this window]
[in a new window]
 
Table 1. Human papillomavirus (HPV) prevalence and risk of head and neck cancer*

 
HPV16 was the most frequent oncogenic HPV type detected in exfoliated cells in both case patients and control subjects (19% versus 10%, respectively). The next most frequently detected high-risk HPV types were HPV18 and HPV33 among the case patients (3% and 1%, respectively) and HPV18, HPV31, and HPV58 among the control subjects (<1% each). No HPV-LR types predominated in either patient group. We detected HPV12, HPV13, HPV17, HPV22, HPV62, HPV72, and nine as-yet unnumbered HPV types. HPV16 was detected in two of the 15 case patients with non-SCC (one patient had acinar-cell carcinoma and one had mucoepidermoid carcinoma). All of the other HPV-HR and -LR types were found in oral exfoliated cells of individuals with SCC.

Demographic and Risk Factor Characteristics

Patient demographic and risk factors were examined and odds ratios were calculated, adjusting for age, tobacco use, alcohol consumption, and oral exfoliated cell HPV-HR status (Table 2). We considered all histologic types together; however, virtually identical results were obtained when analyses were restricted to SCC. Risk of head and neck cancer increased with age (<=55 years versus >55 years, adjusted OR = 1.5, 95% CI = 0.96 to 2.2). Male case patients infected with HPV were younger on average than female case patients detected with HPV (57 years versus 64 years, P = .05), and risk of head and neck cancer was higher in patients with less education.


View this table:
[in this window]
[in a new window]
 
Table 2. Demographic characteristics and risk factors for head and neck cancer*

 
Statistically significantly increased risks of head and neck cancer were limited to current tobacco users (OR = 3.1, 95% CI = 1.8 to 5.5) and current alcohol users (OR = 2.4, 95% CI = 1.4 to 3.9) compared with never-users of tobacco and alcohol, respectively. When evaluated by dose–duration effects, only heavy use of tobacco (>30 pack-years) was associated with statistically significantly increased odds of head and neck cancer, whereas both light (<=21 drinks per week) and heavy drinking (>21 drinks per week) was associated with higher risk, independent of HPV status (Table 2). When the joint effects of tobacco and alcohol were examined (Table 2), only heavy tobacco use and either level of alcohol consumption increased the risk of head and neck cancer. However, no statistically significant interaction between tobacco and alcohol was seen.

Analyses of potential biologic interaction effects between HPV-HR types and exposure to tobacco and alcohol are shown in Table 3. Compared with individuals who never used tobacco and who were HPV-negative, individuals who were heavy tobacco users and detected with HPV-HR had a greater risk of head and neck cancer than individuals who used tobacco or were detected with HPV-HR. However, there was no statistically significant effect modification from the joint exposures (SI = 4.5, 95% CI = 0.7 to 27.4), suggesting that the risk was limited to an additive effect of the two exposures. Among heavy alcohol users detected with the virus, the risk of head and neck cancer was statistically significantly increased relative to that of HPV-negative never drinkers, and there was a statistically significant synergistic effect between alcohol and HPV (SI = 7.4, 95% CI = 1.7 to 33.4). When we considered the joint effects of heavy tobacco and heavy alcohol use with HPV-HR, the results were similar to those obtained for individuals who were heavy alcohol users detected with HPV-HR (SI = 6.0, 95% CI = 1.1 to 32.1), suggesting that tobacco use, even heavy use, did not play a major role in the synergistic effect between HPV-HR and alcohol. Interactions between HPV-HR and tobacco and alcohol use in the logistic regression models were not statistically significant (all P>=.20), suggesting that there was no effect modification on a multiplicative scale.


View this table:
[in this window]
[in a new window]
 
Table 3. Risk of head and neck cancer associated with human papillomavirus (HPV) DNA in oral exfoliated cells by tobacco and alcohol use

 
Oral Cavity and Oropharynx Risk

Because some studies (4,6) have shown differences in risk factors and HPV detection rates for cancers of the oral cavity and oropharynx, we examined these sites separately. Compared with those of control subjects, demographic characteristics were similar for case patients with either type of cancer and for those with SCC only. HPV DNA was detected in 15% of oral cavity cancers and in 38% of oropharyngeal cancers. Patients with oropharyngeal cancer (n = 71) had a greater risk of harboring HPV-HR in oral exfoliated cells than patients with cancers of the oral cavity (n = 130) (adjusted OR = 3.6, 95% CI = 1.8 to 7.1). Rates of HPV-HR types in oral exfoliated cells were similar between the oropharyngeal and oral cavity cancers, with HPV16 the most prevalent type (85% versus 84%) followed by HPV18 (11% versus 11%) and HPV33 (4% versus 5%). HPV DNA was not detected in cancers of the lip but was detected in all other oral cavity and oropharyngeal sites, including in one patient with a cancer in the parotid salivary gland (HPV16).

A dose–duration effect was seen for tobacco and alcohol among oral cavity cancer patients who were heavy tobacco/alcohol users (OR = 3.2, 95% CI = 1.7 to 5.9) and among patients with cancer of the oropharynx who were either heavy tobacco/light alcohol users (OR = 10.3, 95% CI = 3.3 to 31.5) or heavy tobacco/heavy alcohol users (OR = 12.5, 95% CI = 4.3 to 36.6). Tobacco and alcohol had greater effects on the risk of oropharyngeal cancers than on the risk of cancer of the oral cavity. However, interactions between HPV-HR and alcohol/tobacco levels were similar for patients with cancers of the oropharynx and oral cavity.

Comparison of HPV Detection From Cancer Tissue and Oral Exfoliated Cells

We next determined whether the HPV type detected in oral exfoliated cells was representative of that detected in the cancer tissue at the time of diagnosis (Table 4). Tumor tissue was detected with HPV16, HPV18, or HPV33—the same three HPV-HR types found in the oral exfoliated cells of case patients. Patients whose tumor tissue contained HPV-HR types had a statistically significantly higher risk than control subjects of HPV-HR in oral exfoliated cells (adjusted OR = 11.5, 95% CI = 5.2 to 25.7), whereas case patients whose tumor tissue was HPV-negative did not (adjusted OR = 1.7, 95% CI = 0.9 to 3.1). These results suggest that an assessment of HPV detected from oral exfoliated cells collected at the time of diagnosis but before treatment may be predictive of an HPV-related head and neck cancer.


View this table:
[in this window]
[in a new window]
 
Table 4. Head and neck cancer associated with human papillomavirus (HPV) DNA status in exfoliated oral cells and tumor tissue

 

    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
We found that individuals whose oral exfoliated cells were detected with oncogenic HPV types had an independent increased risk of head and neck cancer. By contrast, there was no such association with head and neck cancer among patients detected with non-oncogenic HPV types, after adjusting for other potential risks factors. In addition, we demonstrated that case patients with an HPV-HR type in their tumor tissue were at higher risk of being detected with HPV-HR in their oral exfoliated cells than were case patients with HPV-negative cancer tissue.

Some investigators have used swabs or scrapes to collect oral mucosal cells from a limited number of oral mucosal sites (6,21), whereas we used an oral rinse, which is more likely to have sampled the tumor site and the localized field of HPV infection. This collection procedure was simple to perform, even from patients with bulky oral tumors. We and others (22) have established that an oral rinse is likely to yield a higher number of and a more representative sample of DNA-containing nucleated cells than other methods. Furthermore, we counted the oral exfoliated cells with a hemocytometer to ascertain that every sample contained an adequate number of cells for each PCR to detect HPV DNA. Adding cell counts reduces the potential for false-negative results from inadequate cell numbers or from the predominance of superficial, anucleate squamous cells in the oral mucosa. Finally, rather than screening for a limited number of HPV types by DNA hybridization with type-specific probes, we characterized all HPVs amplified by the universal HPV MY09/11 and GP5+ primers and DNA sequencing.

Nonetheless, not all patients who had HPV-HR types in oral exfoliated cells were detected with HPV DNA in the primary tumor. It is possible that the oral mucosa became infected with HPV after the tumor developed or that not all individuals with HPV-HR types detected in the oral mucosa develop cancer, as witnessed by detection of the virus in a small number of control subjects. Conversely, not all case patients with HPV-HR tumor tissue were detected with an HPV-HR type in oral exfoliated cells. This may result from the absence or low viral copy number in the oral mucosal cells, possibly reflecting the limited surface exposure of some tumors. Alternatively, the original HPV-HR infection may have cleared from the mucosa before the development of an HPV-HR cancer. Although our HPV detection method was highly sensitive, it would not detect fewer than 30 cells containing one HPV genome per cell.

A major purpose of this study was to assess whether HPV-HR types detected in oral exfoliated cells could be used as a biomarker of head and neck cancer risk associated with HPV-HR infection. Our results establish a statistically significant association between the detection of HPV-HR types in oral exfoliated cells and the presence of HPV-HR types in tumor tissue. We did not seek to establish specific concordance of the HPV-HR types but sought to determine whether the detection of HPV-HR types in oral exfoliated cells might be predictive for individuals who potentially could develop an HPV-associated head and neck cancer. HPV-HR DNA detected in the oral exfoliated cell samples may originate from HPV-positive tumor cells, from any associated HPV-HR infection that led to the development of head and neck cancer, or from an independent HPV-HR infection. Yet in any of these situations, detection of HPV-HR DNA may help identify individuals, including those with 1) any genetic predisposition to acquire HPV-HR infection and/or 2) a limited immunologic ability to eliminate the virus, who are at risk for the development of head and neck cancer because they are susceptible to HPV-HR infection in the head and neck area.

Whether oral exfoliated HPV-HR status is predictive of cancer before invasion or progression in patients with head and neck cancer is unknown, but the answer could be clarified easily and inexpensively by repeated assessment in HPV-HR cases over time. The use of oral exfoliated cells could be extended to evaluate other molecular markers of early carcinogenesis in head and neck tumors in addition to HPV, including alterations in tumor suppressor gene pathways, changes in gene expression profiles, and microsatellite markers to increase their power to predict early-stage cancer. Serologic assessment of HPV in head and neck cancer may be less sensitive than assessment of oral exfoliated cells, but few studies have examined this issue to date. However, a blood draw is less acceptable to patients, more difficult to administer and preserve, and currently does not allow the evaluation of a wide variety of HPV types. Finally, HPV seropositivity is potentially indicative not only of current oral infection but also of any past infection not limited to the oral cavity or oropharynx, including infections in the anogenital area that are thought to be the most likely source of HPV in the majority of individuals with an HPV-positive serologic test.

Differences across studies in HPV detection rates of head and neck cancers may be related to the frequency of specific head and neck cancer sites evaluated, patient characteristics, and different assay methods used. The HPV-HR types we have detected in head and neck cancer are the same as those identified in invasive cervical cancer (23). Although several investigations (4,6,24,25) have reported finding HPV-LR types in paraffin-embedded or frozen tumor tissue from head and neck cancers, we did not obtain similar findings using laser capture microdissection. Our finding suggests that the HPV-LR types reported by others were likely associated with infection of adjacent, noncancerous tissue rather than infection of the malignant tumor cells and indicates that only HPV-HR types are associated with head and neck cancer development.

There were no differences in risk factors or HPV types between patients with head and neck cancer that included all histologic types and patients with SCC only. We did detect HPV-HR DNA in the tumor tissue of a mucoepidermoid carcinoma and an oral verrucous carcinoma. Other studies (24,26) also have detected HPV-HR in verrucous carcinoma of the head and neck. To date, however, only a few studies have examined other histologic types of head and neck cancer for HPV, possibly because most head and neck cancers are SCCs or because of the belief that only SCC is associated with HPV.

Some (11,21,23,27,28), but not all (4,2931), investigations have suggested that individuals with HPV-positive head and neck cancer have a substantially lower exposure to tobacco or alcohol, the major etiologic agents for head and neck cancer. Unlike other studies, however, we restricted the unexposed group to those who had never used tobacco or alcohol, excluding both past and low-dosage users, to reduce potential misclassification associated with carcinogenic effects from these exposures. We found no evidence to support the hypothesis that HPV-HR case patients were less likely than uninfected case patients to drink alcohol or smoke. Furthermore, dose-durations among those exposed to alcohol or tobacco were similar between HPV-HR and uninfected case patients.

Whether the risk of HPV DNA status in head and neck cancer is altered further by interaction between alcohol and tobacco remains unclear. Our study demonstrated that HPV detection has both an independent and an interaction effect with alcohol. The synergistic statistic was significant among the infected patients who were heavy alcohol users, and the result was similar to that of HPV-infected patients who were heavy tobacco/heavy alcohol users, suggesting that any biologic interaction effect with HPV is associated primarily with alcohol consumption and not with tobacco use. By contrast, Schwartz et al. (6) reported a potential interaction effect among infected case patients who were current smokers, whereas Herrero et al. (21) did not. We evaluated oral mucosal cells for HPV using DNA analyses, whereas Schwartz et al. (6) assessed antibodies to HPV, which may be indicative of both current and past infection in HPV-related lesions elsewhere in the body and not in the head and neck. It is biologically plausible that an interaction effect between HPV and alcohol may occur. Alcohol can biologically modify mucosal tissue, potentially increasing its permeability to viral infection, or it could influence the immune response to HPV (31). Studies (32) also indicate a possible chronic inflammatory risk for epithelial cancers by alcohol, resulting in the generation of free radicals, which in turn may damage DNA and play a role in the initiation and progression of cancers.

Future research needs to evaluate molecular mechanisms of head and neck carcinogenesis and to develop early biomarkers of cancer risk. With the advent of high-throughput technologies, we will be in a better position to identify signature clusters of genetic alterations and changes in gene expression associated with and independent of HPV-HR infection. Likewise, the detection of HPV-HR in oral exfoliated cells, its plasmid versus integrated state, as well as specific integration sites and gene expression patterns may serve as clonal markers to monitor the presence of residual tumor after surgery or radiation, cancer recurrence, and progression. The availability of an easily performed early screening test for a marker of cancer is essential for disease prevention and early diagnosis. Additional studies will be needed to determine whether the sensitivity and validity of a test can be increased to detect HPV DNA and head and neck cancer by using an oral rinse or whether there are inherent limitations in this procedure, precluding its use in screening.


    NOTES
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
1Editor’s note: SEER is a set of geographically defined, population-based, central cancer registries in the United States, operated by local nonprofit organizations under contract to the National Cancer Institute (NCI). Registry data are submitted electronically without personal identifiers to the NCI on a biannual basis, and the NCI makes the data available to the public for scientific research.

Supported by Public Health Service grants 5PODE10758 (to E. M. Smith and L. P. Turek) and RO1DE13110 (to E. M. Smith, J. M. Ritchie, D. H. Wang, T. H. Haugen, and L. P. Turek) from the National Institute of Dental Research and Craniofacial Research, National Institutes of Health, Department of Health and Human Services; a Veterans Affairs Dental Research Fellowship (to K. F. Summersgill); and Veterans Affairs Merit Review Funds (to T. H. Haugen and L. P. Turek).

We thank John T. Schiller, PhD, for his critical editorial review of this manuscript.


    REFERENCES
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

1 Franceschi S, Munoz N, Snijders PJ. How strong and how wide is the link between HPV and oropharyngeal cancer? Lancet 2000; 356: 871–2.[CrossRef][ISI][Medline]

2 Steenbergen RD, Hermsen MA, Walboomers JM, Joenje H, Arwert F, Meijer CJ, et al. Integrated human papillomavirus type 16 and loss of heterozygosity at 11q22 and 18q21 in an oral carcinoma and its derivative cell line. Cancer Res 1995; 55: 5465–71.[Abstract]

3 Smith EM, Hoffman HT, Summersgill KS, Kirchner HL, Turek LP, Haugen TH. Human papillomavirus and risk of oral cancer. Laryngoscope 1998; 108: 1098–103.[ISI][Medline]

4 Gillison ML, Koch WM, Capone RB, Spafford M, Westra WH, Wu L, et al. Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst 2000; 92: 709–20.[Abstract/Free Full Text]

5 Paz IB, Cook N, Odom-Maryon T, Xie Y, Wilczynski SP. Human papillomavirus (HPV) in head and neck cancer. An association of HPV 16 with squamous cell carcinoma of Waldeyer’s tonsillar ring. Cancer 1997; 79: 595–604.[CrossRef][ISI][Medline]

6 Schwartz SM, Daling JR, Doody DR, Wipf GC, Carter JJ, Madeleine MM, et al. Oral cancer risk in relation to sexual history and evidence of human papillomavirus infection. J Natl Cancer Inst 1998; 90: 1626–36.[Abstract/Free Full Text]

7 Wong M, Pagano JS, Schiller JT, Tevethia SS, Raab-Traub N, Gruber J. New associations of human papillomavirus, Simian virus 40, and Epstein-Barr virus with human cancer. J Natl Cancer Inst 2002; 94: 1832–6.[Free Full Text]

8 Giovannelli L, Campisi G, Lama A, Giambalvo O, Osborn J, Margiotta V, et al. Human papillomavirus DNA in oral mucosal lesions. J Infect Dis 2002; 185: 833–6.[CrossRef][ISI][Medline]

9 American Joint Committee on Cancer. AJCC cancer staging manual. 5th ed. Chicago (IL): Lippincott Williams and Wilkins; 1997.

10 Summersgill KF, Smith EM, Kirchner HL, Haugen TH, Turek LP. p53 polymorphism, human papillomavirus infection in the oral cavity, and oral cancer. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2000; 90: 334–9.[ISI][Medline]

11 Klussmann JP, Weissenborn SJ, Wieland U, Dries V, Kolligs J, Jungehuelsing M, et al. Prevalence, distribution, and viral load of human papillomavirus 16 DNA in tonsillar carcinomas. Cancer 2001; 92: 2875–84.[CrossRef][ISI][Medline]

12 Ritchie JM, Smith EM, Summersgill KF, Hoffman HT, Wang D, Klussmann JP, et al. Human papillomavirus infection as a prognostic factor in carcinomas of the oral cavity and oropharynx. Int J Cancer 2003; 104: 336–44.[CrossRef][ISI][Medline]

13 Chehab FF, Doherty M, Cai SP, Kan YW, Cooper S, Rubin EM. Detection of sickle cell anaemia and thalassaemias [published erratum appears in Nature 1987;329:678]. Nature 1987; 329: 293–4.[ISI][Medline]

14 de Roda Husman AM, Walboomers JM, van den Brule AJ, Meijer CJ, Snijders PJ. The use of general primers GP5 and GP6 elongated at their 3' ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR. J Gen Virol 1995; 76: 1057–62.[Abstract]

15 Summersgill KF, Smith EM, Levy BT, Allen JM, Haugen TH, Turek LP. Human papillomavirus in the oral cavities of children and adolescents. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001; 91: 62–9.[CrossRef][ISI][Medline]

16 Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997; 25: 3389–402.[Abstract/Free Full Text]

17 Hosmer DW, Lemeshow S. Applied logistic regression. New York (NY): John Wiley & Sons; 1989. p. 88–91.

18 Hosmer DW, Lemeshow S. Confidence interval estimation of interaction. Epidemiology 1992; 3: 452–6.[ISI][Medline]

19 Rothman KJ. The estimation of synergy or antagonism. Am J Epidemiol 1976; 103: 506–11.[ISI][Medline]

20 SAS System for Windows. Version 8.2. Cary (NC): SAS Institute; 2001.

21 Herrero R, Castellsagué X, Pawlita M, Lissowska J, Kee F, Balaram P, et al. Human papillomavirus and oral cancer: The International Agency for Research on Cancer Multicenter Study. J Natl Cancer Inst 2003; 95: 1772–83.[Abstract/Free Full Text]

22 Lawton G, Thomas S, Schonrock J, Monsour F, Frazer I. Human papillomaviruses in normal oral mucosa: a comparison of methods for sample collection. J Oral Pathol Med 1992; 21: 265–9.[ISI][Medline]

23 Bosch FX, Manos MM, Munoz N, Sherman M, Jansen AM, Peto J, et al. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group. J Natl Cancer Inst 1995; 87: 796–802.[Abstract]

24 Noble-Topham SE, Fliss DM, Hartwick WJ, McLachlin CM, Freeman JL, Noyek AM, et al. Detection and typing of human papillomavirus in verrucous carcinoma of the oral cavity using the polymerase chain reaction. Arch Otolaryngol Head Neck Surg 1993; 119: 1299–304.[ISI]

25 Cruz IB, Snijders PJ, Steenbergen 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.[Medline]

26 Miller CS, Johnstone BM. Human papillomavirus as a risk factor for oral squamous cell carcinoma: a meta-analysis, 1982–1997. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001; 91: 622–35.[ISI][Medline]

27 Schwartz SR, Yueh B, McDougall JK, Daling JR, Schwartz SM. Human papillomavirus infection and survival in oral squamous cell cancer: a population-based study. Otolaryngol Head Neck Surg 2001; 125: 1–9.[CrossRef][ISI][Medline]

28 Fouret P, Monceaux G, Temam S, Lacourreye L, St Guily JL. Human papillomavirus in head and neck squamous cell carcinomas in nonsmokers. Arch Otolaryngol Head Neck Surg 1997; 123: 513–6.[Abstract]

29 Ha PK, Pai SI, Westra WH, Gillison ML, Tong BC, Sidransky D, et al. Real-time quantitative PCR demonstrates low prevalence of human papillomavirus type 16 in premalignant and malignant lesions of the oral cavity. Clin Cancer Res 2002; 8: 1203–9.[Abstract/Free Full Text]

30 Koch WM, Lango M, Sewell D, Zahurak M, Sidransky D. Head and neck cancer in nonsmokers: a distinct clinical and molecular entity. Laryngoscope 1999; 109: 1544–51.[ISI][Medline]

31 Molina PE, McClain C, Valla D, Guidot D, Diehl AM, Lang CH, et al. Molecular pathology and clinical aspects of alcohol-induced tissue injury. Alcohol Clin Exp Res 2002; 26: 120–8.[ISI][Medline]

32 Brooks PJ. DNA damage, DNA repair, and alcohol toxicity--a review. Alcohol Clin Exp Res 1997; 21: 1073–82.[ISI][Medline]

Manuscript received March 28, 2003; revised January 5, 2004; accepted January 16, 2004.


This article has been cited by other articles in HighWire Press-hosted journals:


Correspondence about this Article

             
Copyright © 2004 Oxford University Press (unless otherwise stated)
Oxford University Press Privacy Policy and Legal Statement