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Background Substantial molecular evidence suggests a role for human papillomavirus (HPV) in the pathogenesis of oropharyngeal squamous-cell carcinoma, but epidemiologic data have been inconsistent.
Methods We performed a hospital-based, case–control study of 100 patients with newly diagnosed oropharyngeal cancer and 200 control patients without cancer to evaluate associations between HPV infection and oropharyngeal cancer. Multivariate logistic-regression models were used for case–control comparisons.
Results A high lifetime number of vaginal-sex partners (26 or more) was associated with oropharyngeal cancer (odds ratio, 3.1; 95% confidence interval [CI], 1.5 to 6.5), as was a high lifetime number of oral-sex partners (6 or more) (odds ratio, 3.4; 95% CI, 1.3 to 8.8). The degree of association increased with the number of vaginal-sex and oral-sex partners (P values for trend, 0.002 and 0.009, respectively). Oropharyngeal cancer was significantly associated with oral HPV type 16 (HPV-16) infection (odds ratio, 14.6; 95% CI, 6.3 to 36.6), oral infection with any of 37 types of HPV (odds ratio, 12.3; 95% CI, 5.4 to 26.4), and seropositivity for the HPV-16 L1 capsid protein (odds ratio, 32.2; 95% CI, 14.6 to 71.3). HPV-16 DNA was detected in 72% (95% CI, 62 to 81) of 100 paraffin-embedded tumor specimens, and 64% of patients with cancer were seropositive for the HPV-16 oncoprotein E6, E7, or both. HPV-16 L1 seropositivity was highly associated with oropharyngeal cancer among subjects with a history of heavy tobacco and alcohol use (odds ratio, 19.4; 95% CI, 3.3 to 113.9) and among those without such a history (odds ratio, 33.6; 95% CI, 13.3 to 84.8). The association was similarly increased among subjects with oral HPV-16 infection, regardless of their tobacco and alcohol use. By contrast, tobacco and alcohol use increased the association with oropharyngeal cancer primarily among subjects without exposure to HPV-16.
Conclusions Oral HPV infection is strongly associated with oropharyngeal cancer among subjects with or without the established risk factors of tobacco and alcohol use.
The epidemiologic evidence of a causal role for HPV in a subgroup of squamous-cell carcinomas of the head and neck is less rigorous than the molecular evidence. The example of the relationship between HPV and cervical cancer5 indicates that high-risk sexual behavior and exposure to and infection with HPV will increase the risk of other cancers caused by HPV.6 Although each of these three factors has been found to increase the risk of squamous-cell carcinomas of the head and neck,7,8,9,10,11,12,13,14 no single study has shown an association of all three with the development of oropharyngeal cancer.
In this study, we focused exclusively on oropharyngeal cancer, for which the molecular evidence of a causal role for HPV is compelling. Strong epidemiologic data would provide additional support for a causal association between HPV and oropharyngeal cancers and might guide future cancer-prevention programs involving vaccination to prevent oral HPV infection or screening to detect it.
Methods
Patients
Our case–control study was nested within a longitudinal cohort study of patients with newly diagnosed squamous-cell carcinomas of the head and neck in the outpatient otolaryngology clinic of the Johns Hopkins Hospital in Baltimore from 2000 through 2005. Eligible case patients included those with a confirmed diagnosis of oropharyngeal squamous-cell carcinoma.
The control group consisted of patients without a history of cancer who were seen for benign conditions between 2000 and 2005 in the same clinic from which the case patients were enrolled (Table 1). Subsequent to enrollment of a case, eligible control patients within the same sex and 5-year age categories were approached until two control patients were individually matched to each case patient. The study protocol was approved by the institutional review board of the Johns Hopkins Hospital. Written, informed consent was obtained from all patients.
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Specimens were collected from case patients before therapy and from control patients at enrollment. Oral-mucosal specimens were collected with the use of a saline oral rinse and 5 to 10 strokes of a cytology brush (Oral CDx, CDx Laboratories) on the posterior oropharyngeal wall. Serum samples were collected and stored at –80°C. For case patients, formalin-fixed, paraffin-embedded tumor specimens and, if possible, snap-frozen fresh tumor specimens were obtained for the detection of HPV.
All patients completed an audio, computer-assisted self-administered interview that obtained information about demographic characteristics, oral hygiene, medical history, family history of cancer, lifetime sexual behaviors, and lifetime history of marijuana, tobacco, and alcohol use (see the Supplementary Appendix, available with the full text of this article at www.nejm.org).
Laboratory Studies
In Situ Hybridization for HPV-16 Detection
We looked for HPV-16 in formalin-fixed and paraffin-embedded tumors from all case subjects, using in situ hybridization–catalyzed signal amplification for biotinylated probes (Dako GenPoint).15 The HPV-16-positive status of a tumor was defined as specific staining of tumor-cell nuclei for HPV-16.
DNA Purification and Analysis
DNA from oral specimens16 and fresh-frozen tumors17 from a subgroup of case subjects was purified as previously described. The tumor specimens were microdissected to ensure that more than 70% of the sample was DNA from the tumor.
We analyzed purified DNA for 37 types of HPV by means of a multiplex polymerase-chain-reaction (PCR) assay targeted to the L1 region of the viral genome, using PGMY09/11 L1 primer pools and primers for 
-globin, followed by hybridization to a linear probe array (Roche Molecular Systems).18 The HPV-16 viral load in purified DNA from oral-mucosal specimens and fresh-frozen tumor specimens was determined with the use of a sensitive real-time PCR assay targeted to the E6 coding region.16,19 The viral load was reported for positive samples (those with 
1 copy of the virus) and was adjusted to the total number of human cells tested with the use of a real-time PCR assay targeted to a single copy of a human gene (for endogenous retrovirus 3, ERV3).16
Serologic Analysis
Serum antibodies to the HPV-16 L1 protein were detected with the use of an enzyme-linked immunosorbent assay (ELISA) based on virus-like particles.20 Antibodies against HPV-16 E6 and E7 oncoproteins were detected with the use of ELISA and bacterially expressed full-length E6 or E7 as the antigen.21
Statistical Analysis
Cumulative alcohol use was calculated as follows. We defined a drink-equivalent as one 12-oz beer, one 6-oz glass of wine, one 3-oz mixed drink, or one 1.5-oz shot of liquor. The number of drink-equivalents per week was determined for each patient within each 5-year age interval and combined into a measure of lifetime alcohol use, defined as the number of years during which 15 or more drink-equivalents (hereafter called "drinks") per week were consumed.
We calculated cumulative tobacco use in pack-years using information about the frequency of use (number of cigarettes, pipes, or cigars smoked per day) and duration of use (during 5-year age intervals) and accounting for gaps in use. Four cigars or five pipes per day were deemed equivalent to one pack of cigarettes in the calculation of pack-years.22
Unconditional and conditional multivariate logistic-regression models were used to estimate odds ratios and the associated 95% confidence intervals (CIs). Results from the unconditional and conditional models were similar, and the results from the unconditional models are presented. Final multivariate models were created through stepwise elimination of variables of interest from univariate analysis while biologically relevant variables were retained. Owing to the colinearity of sexual behaviors, the effect of each behavior on the risk of cancer was evaluated in separate multivariate models adjusted for alcohol use, tobacco use, presence or absence of a family history of head and neck cancer, oral hygiene, age, and sex. To evaluate trends in odds, ordinal variables were modeled as single, continuous, independent variables. Multiplicative interactions among exposure to HPV, tobacco use, and alcohol use were evaluated by including an interaction term in the regression model, and statistical significance was determined with the use of the likelihood-ratio test. For comparison of our results with those in previous reports,9,10 additive interactions were evaluated with the use of a synergy index, calculated as (odds ratio for tobacco or alcohol use and HPV–1)÷([odds ratio for tobacco or alcohol use+odds ratio for HPV]–2).23 The odds ratio for HPV was for either seropositivity or infection. Attributable risk was calculated as previously described.24 P values of less than 0.05 for associations were considered to indicate statistical significance. Stata 8.0 software (Stata) was used for all analyses.
Results
We enrolled 130 consecutive patients with newly diagnosed oropharyngeal cancer in the longitudinal cohort study from 2000 through 2005, and 100 patients (77%) agreed to participate in our nested case–control study. Case patients who declined enrollment were similar to those who were enrolled with regard to age, race or ethnic group, and anatomical site of the tumor but were more likely to be female (P=0.001). Approximately 70% of eligible control patients (200) agreed to participate.
In the univariate analysis, case and control patients were similar with regard to age, sex, race or ethnic group, and education, but case patients were more likely than control patients to live outside of Maryland (Table 1). A history of squamous-cell carcinoma of the head and neck in a first-degree relative, a history of cancer in a sibling, a history of oral papillomas, and poor long-term oral hygiene (some or complete tooth loss or infrequent toothbrushing) were all associated with oropharyngeal cancer (Table 1). A history of heavy tobacco use (20 pack-years or more), a history of heavy alcohol use (15 drinks or more per week for 15 years or more), and a history of regular marijuana use were also associated with oropharyngeal cancer (Table 1). Similar percentages of case and control patients had no history of tobacco or alcohol use (13% and 14%, respectively; odds ratio, 1.0; 95% CI, 0.5 to 1.9).
Certain kinds of sexual behavior were significantly associated with oropharyngeal cancer after adjustment for confounding variables (Table 2). The association with oropharyngeal cancer increased significantly with the number of vaginal-sex partners or oral-sex partners (P for trend=0.002 and 0.009, respectively) and was markedly elevated among patients with a high lifetime number of such partners (Table 2).
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The association between HPV-16 exposure and oropharyngeal cancer was investigated among patients with varied use of tobacco and alcohol. The association was greatly increased among patients without a history of smoking or drinking who were seropositive for HPV-16 L1 (odds ratio, 44.8; 95% CI, 5.9 to 338.5) or had an oral HPV-16 infection (odds ratio, 43.7; 95% CI, 4.2 to 452.7). HPV-16 L1 seropositivity and oral HPV-16 infection were also highly associated with oropharyngeal cancer among patients with a history of heavy tobacco and alcohol use and those without such a history (Table 4). Thus, measures of both lifetime and prevalent oral HPV-16 infection were associated with an increased risk of oropharyngeal cancer, whether or not there was a history of use of tobacco, alcohol, or both.
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In the multivariate analysis, oropharyngeal cancer was independently associated with HPV-16 L1 seropositivity (odds ratio, 32.2; 95% CI, 14.6 to 71.3), poor dentition (odds ratio, 4.1; 95% CI, 1.6 to 10.6), infrequent toothbrushing (odds ratio, 6.9; 95% CI, 1.6 to 30.3), history of squamous-cell carcinomas of the head and neck in a first-degree family member (odds ratio, 5.4; 95% CI, 1.0 to 30.8), and heavy tobacco use (odds ratio, 2.5; 95% CI, 1.1 to 6.0) after adjustment for age, sex, and alcohol use. These factors were collectively estimated to be responsible for 90% of cases of oropharyngeal cancers (the attributable risk; 95% CI, 72 to 96), with 55% of cases (95% CI, 45 to 63) attributable to HPV-16 exposure alone.
The percentage of oropharyngeal cancers in which HPV-16 genomic DNA was detected by in situ hybridization was 72% (95% CI, 62 to 81) (Table 3 and Figure 1). Of the 60 specimens of available fresh-frozen tumor, 35 (58%; 95% CI, 45 to 71) were positive for HPV-16, with a median of 1.2 viral copies per cell (interquartile range, 0.02 to 11) analyzed. Five fresh-frozen specimens were positive for a high-risk type of HPV other than HPV-16 (two for HPV-33, one for HPV-35, and two for both HPV-33 and HPV-16).
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Discussion
This epidemiologic study provides support for the association between HPV and a subgroup of oropharyngeal cancers. The strength of the evidence is underscored by the associations of high-risk sexual behaviors, oral HPV infection, and HPV-16 exposure (as determined from the results of serologic tests) with oropharyngeal cancer. Furthermore, we found that HPV-16 DNA was specifically localized to tumor-cell nuclei in 72% of 100 paraffin-embedded specimens of oropharyngeal cancers, a finding corroborated by the high prevalence of antibodies for HPV-16 oncoprotein E6, E7, or both (64%) in the patients with oropharyngeal cancer. Although a cause-and-effect relationship cannot be inferred from a single study, our findings confirm and extend those of other case–control studies.7,8,9,10,11,12,13,14 Our results are also consistent with a previous report of an increase in the subsequent risk of oropharyngeal cancer by a factor of 14 among HPV-16 L1 seropositive subjects,26 which provides strong evidence that exposure to HPV can precede the appearance of oropharyngeal cancer by 10 years or more.
The degree to which oral HPV infection may interact with tobacco use, alcohol use, or both to increase the risk of squamous-cell carcinomas of the head and neck has been unclear. A greater-than-additive risk has been reported, albeit inconsistently,8,9,10 for patients exposed to both HPV and tobacco9 and those exposed to both HPV and alcohol.10 We found that exposure to HPV increased the association with oropharyngeal cancer regardless of tobacco and alcohol use, but we uncovered no evidence of synergy between exposure to HPV and tobacco or alcohol use. For these reasons, our data suggest two distinct pathways for the development of oropharyngeal cancer: one driven predominantly by the carcinogenic effects of tobacco or alcohol (or both) and another by HPV-induced genomic instability.
Our data suggest that oral HPV infection is sexually acquired. Oral–genital contact was strongly associated with oropharyngeal cancer, but we cannot rule out transmission through direct mouth-to-mouth contact or other means. Certain sexual behaviors13,14 and a history of oral HPV infection7,10 were associated with an increased risk of squamous-cell carcinomas of the head and neck in previous studies in which 25% or more of the tumors from patients were positive for HPV DNA but not those in which less than 25% of the tumors from patients were positive for HPV DNA.8,9 Discrepant findings may be explained by the heterogeneity of the case populations, with variable percentages of cancer cases attributable primarily to tobacco and alcohol use, as compared with HPV infection. In our study, the heterogeneity of case patients was minimized by restricting enrollment to patients with oropharyngeal cancer, 90% of whom had tumors on the tonsil or base of the tongue.
Although HPV-16 alone accounts for more than 90% of cases of HPV-positive squamous-cell carcinomas of the head and neck,8 a more accurate and probably higher proportion might be found by testing for other types of HPV (e.g., types 18, 31, 33, and 35), which are infrequently detected in oropharyngeal cancers.
In our study, oropharyngeal cancer was independently associated with a family history of squamous-cell carcinoma of the head and neck and poor oral hygiene, findings that are consistent with other reports.27 The risk of cervical cancer is also increased in women with a family history of that cancer.28,29 Until specific genetic markers for the risk of an HPV-associated cancer are identified, familial aggregation due to shared environmental exposures cannot be ruled out as an explanation for these findings. Poor dentition,30,31 infrequent toothbrushing,31,32 and infrequent dental visits30,33 have been associated with an increased risk of squamous-cell carcinomas of the head and neck. Because tooth loss is commonly caused by chronic bacterial infections (e.g., periodontitis), it may serve as a surrogate for chronic infection and inflammation, which may be important in the pathogenesis of cancer. Particular coinfections in the cervix (e.g., infection with Chlamydia trachomatis) increase the risk of cancer,34 and our results suggest that bacterial coinfections could play a similar role in the oral region. The absence of data on diet, which is associated with the risk of squamous-cell carcinomas of the head and neck,35 is a limitation of our study but is unlikely to explain the observed associations with HPV infection.
The public health implications of our findings are underscored by the annual increases in the incidence of tonsillar and base-of-tongue cancers in the United States since 1973.36,37 The widespread oral sexual practices among adolescents may be a contributing factor in this increase.38 Our results and those of other studies provide a rationale for HPV vaccination in both boys and girls — since oropharyngeal cancers occur in men and women. If vaccination is as effective in preventing oral HPV-16 infection as it is in preventing cervical infection,39 a substantial reduction in the incidence of oropharyngeal cancer in vaccinated populations would provide the ultimate evidence of causality.
Supported in part by grants from the Damon Runyon Cancer Research Foundation (Clinical Investigator Award, to Dr. Gillison), the State of Maryland Cigarette Restitution Fund (to Dr. Gillison), the National Institute of Dental and Craniofacial Research (DE016631-01, to Dr. Gillison), and the National Institutes of Health (Training Grant T32AI50056, to Dr. D'Souza).
No potential conflict of interest relevant to this article was reported.
We thank Janell Alden, Nicole Benoit, Barbara Clayman, Erlinda Embuscado, Eleni Goshu, Megan Hyle, Zubair Khan, Katherine Locke, Elizabeth O'Leary, Catherine Pevtsova, Carolyn Plescia, and Weihong Xiao for their roles in data collection and laboratory analysis; Dr. Shanaz Begum for technical expertise; Dr. Elizabeth Garrett-Mayer for statistical advice; and Dr. David E. Symer for comments on the manuscript.
Source Information
From the Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health (G.D.); the Departments of Pediatrics (R.V.), Otolaryngology–Head and Neck Surgery (C.F., W.M.K.), and Pathology (W.H.W.), Johns Hopkins Hospital; and the Division of Viral Oncology, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University (M.L.G.) — all in Baltimore; the Division of Cancer Prevention, National Cancer Institute, Bethesda, MD (A.R.K.); and the Infection and Cancer Control Program, German Cancer Research Center, Heidelberg, Germany (M.P.).
Address reprint requests to Dr. Gillison at Johns Hopkins University, Cancer Research Bldg. I, Rm. 3M 54A, 1650 Orleans St., Baltimore, MD 21231, or to gillima{at}jhmi.edu.
References
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Related Letters:
Human Papillomavirus and Oropharyngeal Cancer
Ukpo O. C., Moore E. J., Smith D. I., Williams H., Higgins C. D., Crawford D. H., Braakhuis B. J., Snijders P. J., Leemans C. R., Gillison M. L.
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N Engl J Med 2007;
357:1156-1158, Sep 13, 2007.
Correspondence
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