ORIGINAL PAPER

Tobacco and alcohol use increases the risk of both HPV-associatedand HPV-independent head and neck cancers

Elaine M. Smith • Linda M. Rubenstein •

Thomas H. Haugen • Eva Hamsikova •

Lubomir P. Turek

Received: 4 December 2009 / Accepted: 2 April 2010

� Springer Science+Business Media B.V. 2010

Abstract Tobacco, alcohol, and human papillomavirus

(HPV) are major risk factors for head and neck cancer

(HNC), but it is unclear whether there are two distinct HNC

risk groups, one associated with HPV and the other with

tobacco/alcohol. Because HPV-positive HNC are clinically

distinct from HPV-negative cases in treatment response

and with more favorable prognoses, determining whether

these differences result from infection alone or in associ-

ation with other HNC risk factors is important for devel-

oping future therapeutic strategies. Incident cases of HNC

(n = 201) and age-gender frequency-matched controls

(n = 324) were recruited to assess anti-HPV VLP (virus

like particles) antibodies 16, 18, 31, and 33. Multivariate

logistic regression and stratified analyses were used to

calculate adjusted odds ratios (OR). HPV-seronegative and

seropositive/heavy tobacco users had similar increased

adjusted risks of HNC (HPV-seronegative OR = 2.6,

1.4–5.0; HPV-seropositive OR = 2.3, 1.1–4.8), as did

HPV-seronegative (OR = 4.3, 2.1–9.1) versus HPV-sero-

positive/heavy alcohol users (OR = 3.9, 1.6–9.4). Similar

HPV/tobacco/alcohol risk profiles also were seen in oro-

pharyngeal and oral cavity tumor sites. Our finding that

tobacco/alcohol use increased the risk of HNC in both

HPV-seropositive and HPV-seronegative individuals is

consistent with the observation that HPV infection is not a

sufficient cause of HNC but requires the accumulation of

additional cellular changes.

Keywords Head and neck neoplasms � Tobacco �Alcohol � Human papillomavirus � HPV VLP

Introduction

Tobacco and alcohol are considered the two major risk

factors for head and neck cancer (HNC) [1, 2]. However,

about 25% of HNC are associated with human papillo-

mavirus (HPV) infection [3, 4]. A few studies have sug-

gested that there are two distinct HNC etiologic risk

groups, those who develop cancer in association with

tobacco and alcohol, and the others who develop HNC as a

result of HPV infection [5–7]. Yet other studies [8–10]

have shown either an increased risk in HPV-positive

tobacco/alcohol users or no differences in risk of tumor

development by HPV status and tobacco/alcohol use.

Based on these conflicting findings, the potential role of

tobacco and/or alcohol in the development of HPV-asso-

ciated HNC has not been established. HPV infection with

concomitant expression for the high-risk HPV E6 and E7

oncogenes alone causes extension of the cellular life span

(immortalization) and more rapid growth [11]. However,

it has been shown in cervical cancer that high-risk HPV

(HR-HPV) infection alone is not a sufficient cause of

cancer but requires the accumulation of additional genetic

and epigenetic changes in the cell. Although the nature of

these changes remains only partially characterized, it is

apparent that they occur over a period of time and may be

E. M. Smith (&) � L. M. Rubenstein

Department of Epidemiology, College of Public Health,

University of Iowa, Iowa City, IA 52242, USA

e-mail: elaine-smith@uiowa.edu

T. H. Haugen � L. P. Turek

Veterans Affairs Medical Center, and Department of Pathology,

Carver College of Medicine, University of Iowa,

Iowa City, IA 52242, USA

E. Hamsikova

Department of Experimental Virology, Institute of Hematology

and Blood Transfusion, Prague, Czech Republic

123

Cancer Causes Control

DOI 10.1007/s10552-010-9564-z

accelerated by the mutagenic and carcinogenic effects of

tobacco use, and in the case of HNC in association with

alcohol effects as well.

We hypothesize that the discordant conclusions

regarding the association between HPV and alcohol/

tobacco in head and neck cancers are likely due to differ-

ences in study population characteristics such as age dis-

tribution, since HPV is more frequently detected in

younger age HNC patients. Furthermore, findings may

show distinctions based on the HNC sites evaluated since

HPV prevalence varies widely by tumor location, or based

on differences in exposure and cumulative consumption of

alcohol and tobacco. Diverse study results also may be due

to analyses limited to individual effects of tobacco and

alcohol rather than to stratified effects when interaction

exists but is not adequately evaluated. We performed a

case–control study to determine whether there were two

distinct risk factor profiles for HNC, one associated with

HPV and the other associated with tobacco and alcohol,

and whether these profiles varied by tumor site. We also

examined whether there were differences in HNC risk

profiles based on use of anti-HPV VLP antibodies or HPV

DNA in tumor tissue. Because HPV-positive HNC are

clinically distinct from HPV-negative cases in treatment

response and more favorable prognostic outcomes, deter-

mining whether these differences are due to infection alone

or in association with other HNC risk factors is of para-

mount importance in developing future screening and

therapeutic strategies.

Methods

Patient characteristics

Patients ages 18 and older with a primary HNC diagnosed

between 2001 and 2004 at the University of Iowa Hospi-

tals, Department of Otolaryngology or the Iowa City Vet-

erans Administration Hospital were eligible for enrollment.

Included were all oral cavity (ICD-O: 0–9; n = 139) and

oropharyngeal (ICD-O: 10, 12–14; n = 60) sites as defined

by the AJCC [12]. There were 201 eligible cases enrolled

after exclusions for: illness (4%), refusal (\10%), or mis-

sed interview (6%). Gender and five-year age group fre-

quency-matched controls (n = 324) were recruited from

clinics in Family Medicine and in Internal Medicine at the

same hospital as the cases where they were seeking routine

medical care, routine screening, or prescriptions. Exclusion

criteria in the healthy controls included those under age 18,

prior history of HNC, or care or evaluation for an acute or

chronic serious disease. This information was verified from

hospital medical records and prior cancer from the Iowa

NCI-SEER Cancer Registry. Less than 1% were excluded

because of prior HNC, 2% did not complete the specimen

collection requirements, and 4% declined participation.

Data collection

Patients were administered an institutionally approved

informed consent form prior to interview. Participants

completed a self-administered risk factor questionnaire that

included information about sociodemographics, medical

history, tobacco habits, alcohol use, sexual practices and

history of HPV-related diseases, and oral lesions. Infor-

mation was collected from patient medical records and

pathology reports regarding previous cancer history, cur-

rent head and neck tumor site, treatments and staging.

Tobacco units were defined as one pack-year equal to

one pack of cigarettes/day/year, with 20 cigarettes in a

pack. One cigarette was equivalent to one cigar, one

pipeful of tobacco, one oz of snuff or one oz of chewing

tobacco. Alcohol exposure was characterized according to

the average number of drinks per week. One drink was

equivalent to a 12-oz can or bottle of beer, 4 oz glass of

wine, or 1� oz shot of hard liquor. Never users of tobacco

or alcohol were defined as not having consumed either of

these substances on a regular basis during their lifetime for

1 year or more. Former use was defined as not having used

tobacco or alcohol at least 1 year prior to cancer diagnosis

or interview. Current use included use of tobacco or

alcohol at or less than 1 year prior to the time of the cancer

diagnosis. Moderate tobacco users included those who used

tobacco B30 pack-years. Moderate alcohol consumers

were defined as consuming B21 drinks per week. Heavy

users of tobacco or alcohol were those who smoked [30

pack-years or consumed [21 average drinks per week.

Laboratory methods

A blood sample was collected from all participants at the

time of interview and prior to cancer treatment. The

specimen was spun down and the serum was separated

from the plasma within 2 h of collection and transferred to

a snap cap tube for freezing at -80�C until processed for

HPV VLPs. Detection of HPV-specific antibodies has been

described previously [13].

The presence of HPV-specific antibodies to HPV-16, 18,

31, and 33 was tested using the enzyme-linked immuno-

sorbent assay (ELISA) described elsewhere [14]. Known

positive and negative control sera were tested on each

plate. The mean and standard deviation (SD) were calcu-

lated for each antigen. The cutoff point was represented as

the mean absorbance plus 3 SD from the standard control

sera. All samples within 10% of the cutoff value were

tested twice more; those positive in all three runs were

counted positive.

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123

Among the HNC cases, 193 had paraffin-embedded

tumor tissue prepared as described previously [15]. Sample

preparation, PCR analyses, DNA hybridization, and HPV-

typing procedures for assessment of tumor tissue were

based on a standard protocol [3, 15]. Each PCR included

primers to amplify the b-globin gene [16] and verified

sufficient DNA and adequacy of the PCR amplifications of

the cells. Extracted DNA was PCR-amplified with MY09

and MY11 primers [17] to detect HPV and a primer

(HMB01) was designed to amplify HPV-51 to improve

detection [18]. An aliquot of the PCR product of all those

specimens that were HPV-negative after staining was

hybridized by the dot-blot method with 32P-labeled probes

for a more sensitive detection of HPV DNA. After mem-

brane hybridization, positive samples underwent hemi-

nested PCR-amplification with MY09 and GP5?primers

[19]. DNA sequencing was used to determine the HPV

types in each specimen, and sequences were compared to

GenBank sequences using the BLAST program [20]. High-

risk, oncogenic types (HR-HPV) detected in HNC tumor

tissue included HPV 16 and 33.

Statistics

Variables analyzed categorically were age, education,

number of sexual partners (0–1, 2–10, C11) tobacco pack-

years (never, moderate = B30, heavy = [30) and average

number of alcoholic drinks (never, moderate = B21,

heavy = [21). Ranges for moderate and heavy users were

based on the overall distributions or median values among

HPV-negative cases and controls. Odds ratios (OR) and

95% confidence intervals (CI) were calculated using mul-

tivariate logistic regression analyses. Demographics, risk

factors, and pathologic characteristics were adjusted for

age (continuous) and gender. All other analyses controlled

for age (continuous), gender, tobacco pack-years (contin-

uous), and average drinks per week (continuous).

Individual and joint effects of tobacco and alcohol

stratified by HPV status were examined in combined HNC

sites and by site-specific tumor location. Stratifying by

HPV status, never users of tobacco or alcohol (reference

groups) were compared to moderate or heavy tobacco or

alcohol users among cases and controls. Combinations of

joint effects between HPV status and tobacco/alcohol

consumption were constructed. Six combinations were

created among those who were HPV-negative and the same

six groupings among those who were HR-HPV positive

with the never tobacco/never alcohol as the reference

compared to each of the other joint tobacco/alcohol cate-

gories. Additive joints effects and 95% CIs were assessed

using the synergy index [20–22], while multiplicative

interactions were examined by including the appropriate

interaction term in the multivariate logistic regression

models. All p-values were two sided and values B0.05

were considered statistically significant. Statistical analyses

were performed using SAS version 9.1 (Carey, NC).

Results

Demographic, risk factor, and pathologic characteristics

The average age was similar in cases and controls, 60 years

(Table 1). Males were more likely to be diagnosed with

HNC as were those with B12 years education. Compared

to those who never used tobacco or alcohol, the risk of

HNC was significantly elevated among cases who were

heavy smokers ([30 pack-years) or heavy drinkers ([21

drinks/week), and the adjusted risk was greater for heavy

alcohol users than for heavy tobacco users (OR = 4.8/2.9).

No differences were noted between cases and controls

for lifetime number of sex partners, oral-genital contact, or

oral-anal practices. Although cases were more likely to

report heavy alcohol use and/or heavy tobacco use com-

pared to controls (OR = 2.3, CI = 1.6–3.3), risky sexual

behaviors and heavy alcohol and/or heavy tobacco use

were strongly associated with each other (data not shown).

Compared to moderate and/or never users who reported no

risky sexual behaviors, heavy users were more likely to

have experienced oral anal or oral genital contact and have

more than 10 sexual partners (OR = 6.3, CI = 3.0–13.3).

HNC cases included 69% in the oral cavity and 31% in

the oropharynx. The prevalence of HPV VLP-seropositiv-

ity was higher in the oropharyngeal (57%) compared to

oral cavity (41%) cancer cases, and the age-and gender-

adjusted risk was significantly higher in the oropharynx

(OR = 2.7, 1.4–5.2). Among the pathological characteris-

tics, HPV-16 seropositivity was associated with later stage

(adj. OR = 2.4. 1.2–5.0) and positive nodal involvement

(adj. OR = 1.9, 1.02–3.5).

HPV prevalence and types

The prevalence of HPV VLP serologic types 16, 18, 31 or

33, was 46% in cases and 40% in controls (Table 1). The

most common HPV VLP types detected among cases and

controls were HPV-16 (33/22%) and HPV-31 (28/23%).

After adjusting for age and gender, a significant increased

risk of HNC was found among those who tested seropos-

itive for HPV-16.

Tobacco, alcohol, HPV, and HNC risk

Adjusting for age, gender, and alcohol, heavy ([30 pack-

years) tobacco users who were HPV VLP seronegative for

HPV-16. 18. 31, and/or 33 had a greater risk of HNC

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Table 1 Characteristics and

HPV VLP status of head and

neck cancer cases and controls

Percentages based on available

dataa Odds ratio adjusted for age

(continuous), and genderb Yearsc Standard deviationd Pack-yearse Average drinks/weekf HPV VLP negative: VLP

negative status for all VLP

types; VLP Positive: VLP-

positive status for HPV-16, 18,

31 and/or 33

Characteristic Cases n = 201 (%) Controls n = 324 (%) ORa (95% CI)

Ageb

Range 21–93 18-93

Mean (SDc) 59.6 (14.7) 59.6 (13.5)

B55 86 (42.8) 121 (37.3) 1.0

[55 115 (57.1) 203 (62.7) 0.8 (0.6–1.1)

Gender

Female 77 (38.3) 112 (34.6) 1.0

Male 124 (61.7) 212 (65.4) 0.9 (0.6–1.2)

Race

White 192 (95.5) 308 (95.4) 1.03 (0.4–2.4)

Other 9 (4.5) 15 (4.6) 1.0

Educationb

\12 45 (22.5) 36 (11.1) 3.8 (2.1–6.9)

12–15 117 (58.5) 175 (54.2) 2.0 (1.3–3.1)

C16 38 (19.0) 112 (34.7) 1.0

Tobaccod

Never 54 (27.0) 124 (38.8) 1.0

B30 51 (25.5) 107 (33.4) 1.2 (0.8–2.0)

[30 95 (47.5) 89 (27.8) 2.9 (1.8–4.5)

Alcohole

Never 60 (29.9) 134 (41.6) 1.0

B21 73 (36.3) 147 (45.7) 1.3 (0.8–2.0)

[21 68 (33.8) 41 (12.7) 4.8 (2.8–8.3)

# of Partners

0–1 65 (34.4) 106 (34.2) 1.0

2–10 84 (44.4) 145 (46.8) 0.97 (0.6–1.5)

C11 40 (21.2) 59 (19.0) 1.2 (0.7–2.1)

Oral-genital contact

Yes 48 (53.3) 142 (51.8) 1.1 (0.6–1.8)

No 42 (46.7) 132 (48.2) 1.0

Oral-anal contact

Yes 7 (7.9) 23 (8.4) 0.9 (0.4–2.3)

No 82 (92.1) 251 (91.6) 1.0

HPV VLPf

Negative 109 (54.2) 195 (60.2) 1.0

Positive 92 (45.8) 129 (39.8) 1.3 (0.9–1.8)

HPV-16 VLP

Negative 134 (66.7) 253 (78.1) 1.0

Positive 67 (33.3) 71 (21.9) 1.8 (1.2–2.6)

HPV-18 VLP

Negative 167 (83.1) 278 (85.8) 1.0

Positive 34 (16.9) 46 (14.2) 1.2 (0.7–2.0)

HPV-31 VLP

Negative 145 (72.1) 251 (77.5) 1.0

Positive 56 (27.9) 73 (22.5) 1.3 (0.9–2.0)

HPV-33 VLP

Negative 156 (77.6) 263 (81.2) 1.0

Positive 45 (22.4) 61 (18.8) 1.2 (0.8–1.9)

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compared to HPV-seronegative/never tobacco users

(Table 2). The risk was similar to those who were HPV-

seropositive/heavy tobacco users compared to seropositive/

never users. Controlling for age, gender, and tobacco,

heavy alcohol use also was associated with increased risks

of HNC and was similar in HPV VLP-seronegative cases

and seropositive cases. The risk of HNC among heavy

tobacco users was less than that among heavy alcohol users

regardless of HPV serology status. The effect of the joint

risk between tobacco–alcohol uses showed little difference

by HPV VLP status. There were no significant multipli-

cative interaction effects between HPV status and tobacco,

alcohol, or the combined tobacco/alcohol groups as shown

in Table 2. Thus, compared to never users of tobacco and

alcohol, the risk in heavy users of both were similar and

significantly elevated in HPV-seronegative and HPV-

seropositive individuals.

Site-specific HNC risk associated with tobacco,

alcohol, and HPV status

We evaluated whether the odds ratios associated with

tobacco, alcohol, and HPV status by tumor site, oral cavity

and oropharynx, adjusting for age, gender, HPV status,

tobacco, and alcohol (Table 3). Compared to controls, the

adjusted risk for tumors of the oral cavity was significantly

elevated among heavy users of tobacco (OR = 1.7, 1.03–

2.9), but the risk was lower than that associated with heavy

alcohol intake (OR = 3.8, 2.1–7.1). In contrast, the risk of

HNC in tumors of the oropharynx among heavy tobacco

users was higher than seen for heavy alcohol users

(OR = 7.9, 3.2–19.5/6.2, 2.5–15.4) compared to controls.

The risks associated with either heavy tobacco or alcohol

were greater than the risks seen for the oral cavity.

Whereas the joint effect of tobacco/alcohol on the risk of

cancer in the oral cavity was significantly elevated only

among heavy users of both, the risk in the oropharynx was

elevated for all combined tobacco/alcohol levels. The odds

were almost 4-fold higher for tumors in the oropharynx

than in the oral cavity among heavy users of both. There

also was greater risk of developing cancer in the orophar-

ynx among those who were seropositive for HPV-16 or -33

VLP, but no association was seen with the oral cavity.

Next we examined the relationship between the three

risk factors by tumor site, stratifying by HPV serology

status and controlling for the effect of tobacco or alcohol

(Table 4). Adjusting for age, gender, and alcohol, the risk

of cancer in the oral cavity was significantly elevated

Table 2 Risk of head and neck

cancer associated with HPV

VLP status by tobacco and

alcohol use

Percentages based on available

dataa HPV VLP negative: VLP

negative status for all VLP

types; VLP Positive: VLP-

positive status for HPV-16, 18,

31 and/or 33b Synergy indexc Adjusted for age (continuous),

gender, and average drinks/

week (continuous)d Adjusted for age (continuous)

and tobacco pack-years

(continuous)e Adjusted for age (continuous)

HPV VLPa Risk factor Cases n = 201 (%) Controls n = 324 (%) OR (95% CI)

Tobaccoc

Negative Never 29 (14.5) 71 (22.2) 1.0

B30 28 (14.0) 65 (20.3) 1.3 (0.7–2.4)

[30 51 (25.5) 56 (17.5) 2.6 (1.4–5.0)

Positive Never 25 (12.5) 53 (16.6) 1.0

B30 23 (11.5) 42 (13.1) 1.1 (0.5–2.2)

[30 44 (22.0) 33 (10.3) 2.3 (1.1–4.8)

Alcohold

Negative Never 34 (16.9) 77 (23.9) 1.0

B21 40 (19.9) 93 (28.9) 1.2 (0.7–2.2)

[21 35 (17.4) 24 (7.4) 4.3 (2.1–9.1)

Positive Never 26 (12.9) 57 (17.7) 1.0

B21 33 (16.4) 54 (16.8) 1.3 (0.7–2.6)

[21 33 (16.4) 17 (5.3) 3.9 (1.6–9.4)

Tobacco/alcohole

Negative Never/never 21 (10.5) 43 (13.5) 1.0

B30/B21 15 (7.5) 40 (12.6) 1.2 (0.5–2.8)

B30/[21 8 (4.0) 10 (3.1) 2.8 (0.9–8.7)

[30/B21 17 (8.5) 27 (8.5) 2.0 (0.8–4.6)

[30/[21 26 (13.0) 12 (3.8) 8.4 (3.2–22.1)

Positive Never/never 18 (9.0) 33 (10.4) 1.0

B30/B21 12 (6.0) 20 (6.3) 1.1 (0.4–2.9)

B30/[21 5 (2.5) 8 (2.5) 1.3 (0.3–4.7)

[30/B21 15 (7.5) 15 (4.7) 2.0 (0.8–5.1)

[30/[21 27 (13.5) 7 (2.2) 7.7 (2.6–22.4)

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among heavy tobacco users who were HPV seronegative

but not among those who were seropositive. In contrast, the

risk of oropharyngeal cancer increased at each level of

tobacco use and was significantly elevated in both HPV-

seropositive and seronegative cases. The risk was higher

among the HPV VLP seronegative cases than among

seropositive cases (OR = 11.1, 2.3–54.6 vs. 6.4, 2.0–20.5).

Controlling for age, gender, and tobacco, the risk of cancer

of the oral cavity was slightly increased among heavy

alcohol users regardless of HPV VLP status when com-

pared to never alcohol users. The elevated risk of oro-

pharyngeal cancer was similar in moderate alcohol users

regardless of HPV status but was much greater among

seronegative/heavy drinkers (OR = 9.5, 2.3–38.6) than

among seropositive/heavy alcohol users (OR = 5.0,

1.4–17.6) compared to controls. When the joint effects of

tobacco and alcohol were examined in the oral cavity, the

odds ratio was slightly higher in the seropositive/heavy

tobacco-alcohol cases than that in the seronegative/

heavy tobacco-alcohol cases. Because there were no

HPV-seronegative never tobacco/alcohol users among the

oropharyngeal cancer cases, the referent group included

seronegative cases who were either a never tobacco or

alcohol user. The risk associated with tobacco/alcohol use

Table 3 Site-specific head and neck cancer associated with tobacco, alcohol, and HPV VLP status

Characteristic Cases Controls

n = 324 (%)

Oral cavity

vs. controls ORa

(95% CI)

Oropharynx

vs. controls ORa

(95% CI)Oral cavity

n = 139 (%)

Oropharynx

n = 62 (%)

Tobacco

Never 47 (34.1) 7 (11.3) 124 (38.8) 1.0 1.0

B30 31 (22.4) 20 (32.3) 107 (33.4) 0.8 (0.5–1.4) 3.4 (1.4–8.6)

[30 60 (43.5) 35 (56.4) 89 (27.8) 1.7 (1.03–2.9) 7.9 (3.2–19.5)

Alcohol

Never 51 (36.7) 9 (14.5) 134 (41.6) 1.0 1.0

B21 43 (30.9) 30 (48.4) 147 (45.7) 0.9 (0.6–1.5) 2.9 (1.3–6.5)

[21 45 (32.4) 23 (37.1) 41 (12.7) 3.8 (2.1–7.1) 6.2 (2.5–15.4)

Tobacco/alcohol

Never/Never 36 (26.1) 3 (4.8) 76 (23.9) 1.0 1.0

B30/B21 15 (10.8) 12 (19.4) 60 (18.9) 0.7 (0.4–1.5) 5.3 (1.4–20.3)

B30/[21 9 (6.5) 4 (6.5) 18 (5.7) 1.7 (0.7–4.4) 5.5 (1.1–28.2)

[30/B21 17 (12.3) 15 (24.2) 42 (13.2) 1.1 (0.5–2.3) 11.4 (3.0–44.0)

[30/[21 35 (25.4) 18 (29.0) 19 (6.0) 6.6 (3.1–14.0) 24.8 (6.2–99.2)

HPV VLPb

Negative 82 (59.0) 27 (43.5) 195 (60.2) 1.0 1.0

Positive 57 (41.0) 35 (56.5) 129 (39.8) 1.0 (0.6–1.5) 2.1 (1.2–3.7)

HPV-16 VLP

Negative 103 (74.1) 31 (50.0) 253 (78.1) 1.0 1.0

Positive 36 (25.9) 31 (50.0) 71 (21.9) 1.2 (0.7–1.9) 3.6 (2.0–6.4)

HPV-18 VLP

Negative 117 (84.2) 50 (80.6) 278 (85.8) 1.0 1.0

Positive 22 (15.8) 12 (19.4) 46 (14.2) 1.0 (0.6–1.9) 1.4 (0.7–2.9)

HPV-31 VLP

Negative 102 (73.4) 43 (69.3) 251 (77.5) 1.0 1.0

Positive 37 (26.6) 19 (30.7) 73 (22.5) 1.2 (0.7–1.7). 1.5 (0.8–2.8)

HPV-33 VLP

Negative 115 (82.7) 41 (66.1) 263 (81.2) 1.0 1.0

Positive 24 (17.3) 21 (33.9) 61 (18.8) 0.9 (0.5–1.5) 2.2 (1.2–4.0)

Percentages based on available dataa Odds ratio adjusted by age (continuous), gender, HPV status, tobacco, and alcoholb HPV VLP negative status for all types, VLP-positive status for HPV-16, 18, 31, and/or 33

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increased with dose-duration regardless of HPV status.

Among the HPV-seropositive cases, risk was highest

among heavy tobacco/alcohol users. The risk was greater

among the HPV seronegative/heavy tobacco/alcohol group

than among the seropositive/heavy tobacco/alcohol group

(OR = 17.3, 4.8–62.7 vs. 10.5, 2.8–39.2). Tests for trend

in the oral cavity by HPV serology status suggested that an

increasing risk of oral cavity cancer was associated with

increasing tobacco/alcohol dose, regardless of serology

status.

Overall, the risk of cancer among HPV-seronegative/

tobacco users was much greater in the oropharynx than in

the oral cavity (Table 4), and there was a significant

interaction effect between HPV serology and tobacco but

only for cancer risk in the oropharynx (OR = 1.7, 1.3–2.3).

Among alcohol users, the risk was significantly elevated in

both HPV-seronegative and seropositive cases at both HNC

sites although again the risk was higher in the oropharyn-

geal cases. There was no interaction effect between HPV

VLP serology and alcohol and risk by tumor site. Risk

among all HPV-seropositive joint tobacco/alcohol groups

was much greater in tumors of the oropharynx than that in

the oral cavity and the risk increased with each level of

joint tobacco/alcohol use in the oropharyngeal cancers

whereas in the oral cavity it did not. The risk appeared to

be greater among HPV-seronegative cases in the orophar-

ynx whereas in the oral cavity this difference was not seen

by serology status. Tests for trend in the oropharynx by

serology status indicated that increasing risk of cancer in

the oropharynx was significantly associated with increasing

Table 4 Site-specific head and

neck cancer adjusted for HPV

VLP status, tobacco, and

alcohol use

Percentages based on available

dataa HPV VLP negative: VLP

negative for HPV-16, 18, 31,

and/or 33; VLP Positive: VLP-

positive status for HPV-16, 18,

31, and/or 33b Adjusted for age

(continuous), gender, and

average drinks/week

(continuous)c Adjusted for age (continuous),

gender, and tobacco pack-years

(continuous)d Adjusted for age (continuous)

and gendere The reference group for

oropharynx combined all never

alcohol or tobacco groups due to

zero cells; small samples

prevented estimation of odds

ratios and confidence intervals

HPV VLPa Risk factor Cases Controls

n = 324

(%)

Oral cavity

vs. controls

OR (95% CI)

Oropharynx vs.

controls OR

(95% CI)Oral cavity

n = 139 (%)

Oropharynx

n = 62 (%)

Tobaccob

Negative Never 27 (19.6) 2 (3.2) 71 (22.2) 1.0 1.0

B30 19 (13.8) 9 (14.5) 65 (20.3) 1.0 (0.5–2.0) 4.6 (0.9–22.6)

[30 35 (25.3) 16 (25.8) 56 (17.5) 2.0 (1.01–4.1) 11.1 (2.3–54.6)

Test for trend p-value 0.11 \0.001

Positive Never 20 (14.5) 5 (8.1) 53 (16.6) 1.0 1.0

B30 12 (8.7) 11 (17.7) 42 (13.1) 0.6 (0.3–1.5) 3.0 (0.9–9.8)

[30 25 (18.1) 19 (30.6) 33 (10.3) 1.5 (0.7–3.4) 6.4 (2.0–20.5)

Test for trend p-value 0.07 \0.002

Alcoholc

Negative Never 31 (22.3) 3 (4.8) 77 (23.9) 1.0 1.0

B21 28 (20.1) 12 (19.4) 93 (28.8) 1.0 (0.5–1.9) 3.3 (0.8–12.6)

[21 23 (16.5) 12 (19.4) 24 (7.5) 3.7 (1.6–8.3) 9.5 (2.3–38.6)

0.06 \0.001

Positive Never 20 (14.4) 6 (9.7) 57 (17.7) 1.0 1.0

B21 15 (10.8) 18 (29.0) 54 (16.8) 0.8 (0.4–1.8) 3.0 (1.1–8.3)

[21 22 (16.6) 11 (17.7) 17 (5.3) 3.8 (1.4–10.1) 5.0 (1.4–17.6)

Test for trend p-value 0.004 \0.001

Tobacco/alcohold

Negative Never/never 21 (15.2) 5 (8.1)e 43 (13.5) 1.0 1.0e

B30/B21 10 (7.2) 5 (8.1) 40 (12.6) 0.6 (0.2–1.3) 2.3 (0.6–8.8)

B30/[21 6 (4.3) 2 (3.2) 10 (3.1) 2.4 (0.7–8.2) 3.6 (0.6–21.6)

[30/B21 12 (8.7) 5 (8.1) 27 (8.5) 1.4 (0.6–3.6) 4.7 (1.2–8.2)

[30/[21 16 (11.6) 10 (16.1) 12 (3.8) 6.1 (2.2–17.2) 17.3 (4.8–62.7)

Test for trend p-value 0.10 \0.001

Positive Never/never 15 (10.9) 8 (12.9)e 33 (10.4) 1.0 1.0

B30/B21 5 (3.6) 7 (11.3) 20 (6.3) 0.5 (0.2–1.7) 3.4 (1.1–10.8)

B30/[21 3 (2.2) 2 (3.2) 8 (2.5)) 1.1 (0.2–4.9) 2.3 (0.4–13.4)

[30/B21 5 (3.6) 10 (16.1) 15 (4.7) 0.8 (0.2–2.7) 7.1 (2.3–22.1)

[30/[21 19 (13.8) 8 (12.9) 7 (2.2) 7.5 (2.4–23.6) 10.5 (2.8–39.2)

Test for trend p-value 0.02 \0.001

Cancer Causes Control

123

tobacco/alcohol dose, for patients who had either negative

or positive HPV serology status. Thus, there was no indi-

cation that HPV seropositivity was a risk factor only

among never smokers or never drinkers.

There were no significant differences in the adjusted risk

of HNC between HPV VLP-negative and VLP-positive

cases by tobacco, alcohol, or joint tobacco/alcohol use

(Table 5). Likewise, tobacco and alcohol were not signif-

icantly associated with HPV DNA types 16 (n = 48) or 33

(n = 3; Table 5) or HPV-16 tumor DNA alone (data not

shown), controlling for age, gender, tumor site, tobacco, or

alcohol. Examination of trends in dose of tobacco and

alcohol users and tumor HPV DNA status overall (Table 5)

or by site (data not shown) also were not statistically

significant.

We evaluated whether HPV anti-VLP antibody status

was a surrogate for HPV DNA in tumor tissue or whether

tobacco/alcohol status might affect that concordance.

Compared to HPV VLP status among controls, cases whose

tumor tissue contained HPV-16 or -33 were at significantly

greater risk of testing seropositive (adj. OR = 4.4,

2.3–8.2), whereas cases whose tumor tissue was HPV-

seronegative were not (OR = 0.9, 0.6–1.4). The risk was

even greater among the HPV-16 seropositive and HPV-16

DNA-positive tumors (OR = 6.0, 3.1–11.6) but not tumor

HPV-16 seronegative and HPV-16 tumor DNA-negative

cases (OR = 0.9, 0.6–1.5) compared to HPV VLP status

among controls. Because medical tests are often performed

with limited knowledge of patient risk factors or the

potential effects of risk factors which vary among exposed

individuals, we examined the unadjusted risks as well.

Among cases who were HPV seropositive/HPV DNA

tumor-positive, the OR was 2.9 (1.1–7.6); the OR for

HPV-16 seropositive/HPV-16 DNA-positive was 5.0 (2.3–

11.2). The risk was not increased among VLP-seronega-

tive/HPV DNA tumor-negative or among HPV-16 sero-

negative/HPV-16 tumor-negative cases (OR = 0.8, 0.5–1.3

both) compared to controls. These results suggest that

testing for HPV antibodies at the time of diagnosis is

predictive of an HR-HPV related head and neck tumor

regardless of tobacco or alcohol status.

Discussion

In contrast to studies suggesting that there are two distinct

risk groups for HNC, one associated with HR-HPV infec-

tion and another related to tobacco and alcohol use, our

study population showed no such distinctions in risk pro-

files. Compared to those who never smoked nor drank

alcohol, those who were heavy users of both had a similar,

increased risk of HNC in both HPV-seropositive (OR =

7.7) and seronegative (OR = 8.4) individuals. We previ-

ously reported comparable results based on oral exfoliated

cells in HNC cases and controls from this population [3].

Our findings support the well-established public health

premise that risky health behaviors go together in indi-

viduals: whereas 76% of HPV DNA-negative cases were

Table 5 Tobacco and alcohol status in head and neck cancer cases by HPV VLP and tumor DNA status

Risk Factor VLP? n = 87 (%) VLP- n = 104 (%) OR (95% CI) DNA? n = 51 (%) DNA- n = 140 (%) OR (95% CI)

Tobaccoa

Never 18 (24.0) 34 (29.6) 1.0 9 (17.7) 43 (30.9) 1.0

B30 19 (25.3) 30 (26.1) 0.8 (0.3–2.0) 20 (39.2) 29 (20.9) 1.7 (0.6–5.0)

[30 38 (50.7) 51 (44.4) 0.9 (0.4–2.2) 22 (43.1) 67 (48.2) 0.7 (0.2–2.0)

Alcoholb

Never 19 (25.3) 38 (32.8) 1.0 9 (17.7) 48 (34.3) 1.0

B21 27 (36.0) 43 (37.1) 1.0 (0.4–2.2) 25 (49.0) 45 (32.1) 1.7 (0.6–4.5)

[21 29 (38.7) 35 (30.2) 1.4 (0.6–3.5) 17 (33.3) 47 (33.6) 1.2 (0.4–3.9)

Tobacco/alcoholc

Never/never 14 (18.7) 24 (20.9) 1.0 5 (9.8) 33 (23.7) 1.0

B30/B21 11 (14.7) 16 (13.9) 0.9 (0.3–2.6) 13 (25.5) 14 (10.1) 2.8 (0.7–11.2)

B30/[21 4 (5.3) 8 (7.0) 0.7 (0.2–3.1) 4 (7.8) 8 (5.8) 1.5 (0.3–8.4)

[30/B21 13 (17.3) 17 (14.8) 1.1 (0.4–3.2) 9 (17.6) 21 (15.1) 1.0 (0.2–4.1)

[30/[21 24 (32.0) 26 (22.6) 1.4 (0.5–3.8) 12 (23.5) 38 (27.3) 0.9 (0.2–3.5)

HPV VLP: Percentages based on available data; VLP negative for HPV-16 and -33; VLP positive for HPV-16 and/or 33

Tumor DNA status: Percentages based on available data; DNA negative for all HPV DNA types; DNA positive for HPV-16 and/or 33a Adjusted for age (continuous), gender, tumor site (oral cavity, oropharynx), and average drinks/week (continuous)b Adjusted for age (continuous), gender, tumor site (oral cavity, oropharynx), and tobacco pack-years (continuous)c Adjusted for age (continuous), gender, and tumor site

Cancer Causes Control

123

tobacco or alcohol users, over 90% of HPV DNA-positive

cases were.

A large study based in Seattle using VLP serology and

dose-duration effects of tobacco and alcohol use supports

our current results [8]. A reanalysis of the Iowa data using

the Seattle study categories for serology, smoking, and

alcohol, and for the adjustment variables (sex, age con-

tinuous, pack-years, drinks/week) showed that the ORs for

the Iowa data were statistically significant, had similar

magnitudes for the ORs, and had overlapping CIs. In a

large IARC study of HNC, tobacco use was stratified as

never/ever adjusted for alcohol dose-duration [9]. Their

findings also did not support the hypothesis that never

smokers were more likely than smokers to have HPV-

related tumors. In fact this study showed that compared to

HPV-seronegative/never smokers, seropositive/smokers

had the greatest site-specific HNC risks, greater than in

seronegative/smokers or seropositive/never smokers.

In comparison, recent epidemiologic studies in Balti-

more and Boston [6, 7, 23] identified HPV and tobacco/

alcohol as two distinct risk factor groups. How can these

differences in findings about risk groups for HNC be rec-

onciled? Possibly differences in patient characteristics

including differences in frequency of exposures in patient

groups or associated with tumor site may have led to these

conclusions. The rate of never alcohol users in Iowa cases

and controls was higher (30/42%) than in the Baltimore

(17/22%; ref. 6) or Boston (19/28%; ref. 7) study groups.

The frequency of heavier alcohol use (C15 drinks per

week) in cases also was higher in Iowa than in Baltimore

(40 vs. 28%) but lower than in Boston (51%; ref. 25).

Smoking rates also differed. The rate of never smokers was

much lower in Iowa cases and controls (27/39%) than in

Baltimore (41/57%) but higher than in Boston (19/33%).

Iowa cases and controls also reported a higher rate

(55/40%) of heavier smoking (C20 pack years) compared

to Baltimore (47/25%), but a lower rate in cases and similar

rate in controls compared to Boston (62/39%). Whereas

HPV-16 VLP positivity among cases was similar in the

three studies (Iowa: 33%, Boston: 30%, Baltimore: 28%),

Iowa had C2 times the seropositivity rate in controls than

did Boston or Baltimore (22/11/8%). Among HPV-16

tumor negative patients, Iowa also had a seropositivity rate

similar to Boston but almost three times that of Baltimore

(23/21/8%). In the Baltimore study [6], HPV-16 status was

based on tumor tissue in HNC cases whereas it was based

on VLP serology in controls. This may have led to mis-

classification bias in controls but not cases because a

positive serology test may be due to HPV infection else-

where in the body.

A comparison of the site-specific stratified risk of cancer

by HPV serology shows major differences across studies as

well. The Boston investigation found no increased risk

associated with level of alcohol or tobacco use among

seropositive oropharyngeal cancer cases, whereas risk did

increase with level of exposure among the seronegative

group. This finding contrasts with the Iowa data in which

risk associated with tobacco or alcohol level increased in

both seropositive and seronegative subjects in oral cavity

and oropharyngeal cancers. The Baltimore study did not

evaluate HNC sites separately.

In our current study based on HPV VLP serology, there

was no interaction with alcohol in the oral cavity or oro-

pharynx, but there was a significant interaction between

HPV and tobacco among those with oropharyngeal cancer.

In contrast, significant interaction effects were detected in

the Boston study [17] with increasing risk associated with

greater alcohol and alcohol/tobacco dose among HPV-16

VLP seronegative pharyngeal cancer cases only. They also

showed an increased risk of pharyngeal cancer with

smoking among HPV-16 seronegative individuals but none

among seropositive/tobacco users at this site. Although the

study did not report tests for interactions stratified by HPV

serology, they indicated that multiplicative interaction

effects were not statistically significant; they did not report

on additive effects.

In a small study of never smokers and HNC, Koch et al.

[24] did not evaluate history of alcohol use and Fouret et al.

[25] had only few nonsmoker (4.8%) cases who also never/

rarely used alcohol. Both studies concluded that HR-HPV

status was associated with nonsmoking status. Strome cat-

egorized their lowest alcohol users as those who drank B1

drink/week [26] and found that tonsillar cases with HPV

DNA were significantly less likely to have a history of

smoking but again alcohol status was not reported. Dahl-

strom et al. [10] using similar definitions to our study for

never tobacco and alcohol use, adjusted for alcohol con-

sumption to assess risk of HNC in HPV-16 VLP cases who

were never and ever smokers, rather than combine never

alcohol users with the unexposed tobacco users. Like us,

they did not find an association between HPV and smoking

status, although they found an increased risk of HNC asso-

ciated with HPV-16 which was actually higher in their ever-

smoker cases versus ever smoking controls (OR = 9.9,

2.3–43.1) compared to their never-smoker case–control

groups (OR = 5.3, 2.0–14.4). Never smoking was not

associated with an increased risk of HPV-16 positivity

among case–case HNC-matched pairs (OR = 1.2). While

there may be methodological differences among the inves-

tigations that affect study conclusions, there also are distinct

differences in the seroprevalence of HPV-16, distribution of

alcohol and tobacco use, and age in the populations that may

better account for the disparate results in risk factor groups.

Taken together, studies indicate that there are not just

two distinct risk profiles of HNC etiology, but multiple

profiles, most likely due to differences in population age

Cancer Causes Control

123

characteristics, sexual behaviors, HPV prevalence, alcohol

consumption, and tobacco use. Most HR-HPV-associated

tumors are dependent on the expression of the viral onco-

genic proteins E6 and E7, which disrupt the p53, Rb and

possibly other, additional tumor suppressor gene pathways

in the cell. However, it is apparent that HR-HPV infection

alone is not sufficient as the cause of cancer in the uterine

cervix and most likely not in the head and neck either.

Progression of the HR-HPV-infected cell to cancer there-

fore involves additional, as yet undefined changes whose

frequency may be increased by mutagens and carcinogens

in tobacco smoke and/or alcohol, its metabolites or cong-

eners in alcoholic beverages. HPV E6 and E7, which are

considered to be more sensitive measures of active HPV

infection than VLPs, also should be examined in stratified

analyses with tobacco/alcohol exposures like that per-

formed in the current study, to confirm or clarify any dif-

ferences identified. Those investigations have not been

conducted. Larger or pooled studies will be necessary to

identify these distinct risk profiles of HNC risk factors.

Acknowledgments This study was supported by NIH DE13110

(EMS, LMR, THH, LPT), DE1311-S1 (EMS, LMR, THH, EH, LPT),

Veterans Affairs Merit Review Funds (THH, LPT).

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