Post on 30-Apr-2023
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.
Cancer Causes Control
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
Cancer Causes Control
123
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)
Cancer Causes Control
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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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123
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
Cancer Causes Control
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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).
References
1. Franceschi S, Talamini R, Baria S et al (1990) Smoking
and drinking in relation to cancers of the oral cavity, pharynx,
larynx and esophagus in Northern Italy. Cancer Res 50(20):6502–
6507
2. Blot WJ, McLaughlin JK, Winn DM et al (1988) Smoking and
drinking in relation to oral and pharyngeal cancer. Cancer Res
48(11):3282–3287
3. Smith EM, Ritchie JM, Summersgill KF et al (2004) Human
papillomavirus in oral exfoliated cells and risk of head and neck
cancer. J Natl Cancer Inst 96(6):449–455
4. Kreimer AR, Clifford GM, Boyle P et al (2005) Human papil-
lomavirus types in head and neck squamous cell carcinomas
worldwide: a systematic review. Cancer Epidemiol Biomark Prev
14(2):467–475
5. Gillison ML, Koch WM, Capone RB et al (2000) Evidence for a
causal association between human papillomavirus and a subset of
head and neck cancers. J Natl Cancer Inst 92(9):709–720
6. Gillison ML, D’Souza G, Westra W et al (2008) Distinct risk
factor profiles for human papillomavirus type 16-positive and
human papillomavirus type 16-negative head and neck cancers.
J Natl Cancer Inst 100(6):407–420
7. Applebaum KM, Furniss CS, Zeka A et al (2007) Lack of asso-
ciation of alcohol and tobacco with HPV16-associated head and
neck cancer. J Natl Cancer Inst 99(23):1801–1810
8. Schwartz SM, Daling JR, Doody DR et al (1998) Oral cancer risk
in relation to sexual history and evidence of human papilloma-
virus infection. J Natl Cancer Inst 90(21):1626–1636
9. Herrero R, Castellsaque X, Pawlita M et al (2003) Human papil-
lomavirus and oral cancer: the international agency for research on
cancer multicenter study. J Natl Cancer Instit 95(23):1772–1783
10. Dahlstrom KR, Little JA, Zafereo ME et al (2008) Squamous cell
carcinoma of the head and neck in never smoker-never drinkers: a
descriptive epidemiologic study. Head Neck 30(1):75–84
11. Psyrri A, DiMaio D (2008) Human papillomavirus in cervical and
head-and-neck-cancer. Nat Clin Prac Oncol 5(1):24–31
12. American Joint Committee on Cancer (1997) AJCC cancer
staging manual, 5th edn. Lippincott Williams and Wilkins,
Chicago
13. Smith EM, Rubenstein LM, Ritchie JM et al (2008) Does pre-
treatment seropositivity to human papillomavirus have prognostic
significance for head and neck cancers? Cancer Epidemiol Bio-
mark Prev 17(8):2087–2096
14. Hamsıkova E, Ludvıkova V, Smahel M et al (1998) Prevalence of
antibodies to human papillomaviruses in the general population
of the Czech Republic. Int J Cancer 77(5):689–694
15. Summersgill KF, Smith EM, Kirchner HL et al (2000) p53
polymorphism, human papillomavirus infection in the oral cavity,
and oral cancer. Oral Surg Oral Med Oral Pathol Oral Radiol
Endod 90(3):334–339
16. Chehab FF, Doherty M, Cai SP et al (1987) Detection of sickle cell
anaemia and thalassaemias [letter]. Nature 329(6141):293–294
17. Ting Y, Manos MM (1990) Detection and typing of genital
human papillomaviruses. In: Innis MA, Gelfand DH, Sninsky JJ,
White TJ (eds) PCR protocols, a guide to methods and applica-
tions. Academic Press, Berkeley, pp 356–367
18. Hildesheim A, Schiffman MH, Gravitt PE et al (1994) Persistence
of type-specific human papillomavirus infection among cytolog-
ically normal women. J Infec Dis 169(2):235–240
19. de Roda Husman A-M, Walboomers JMM, van den Brule AJC
et al (1995) 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 76(Pt 4):
1057–1062
20. Altschul SF, Madden TL, Schaeffer AA et al (1997) Gapped
BLAST and PSI-BLAST: a new generation of protein database
search programs. Nucleic Acid Res 25(17):3389–3402
21. Hosmer DW, Lemeshow S (1992) Confidence interval estimation
of interaction. Epidemiology 3(5):452–456
22. Kalilano L, Atashili J (2006) Measuring additive interaction
using odds ratios. Epi Perspectives 3:1–10
23. D’Souza G, Kreimer AR, Viscidi R et al (2007) Case-control
study of human papillomavirus in oropharyngeal cancer. N Engl J
Med 356(19):1944–1956
24. Koch WM, Lango M, Sewell D et al (1999) Head and neck
cancer in nonsmokers: a distinct clinical and molecular entity.
Laryngoscope 109(10):1544–1551
25. Fouret P, Monceaux G, Temam S et al (1997) Human papillo-
mavirus in head and neck squamous cell carcinomas in non-
smokers. Arch Otolaryngol Head Neck Surg 123(5):513–516
26. Strome SE, Savva A, Brissett AE et al (2002) Squamous cell
carcinoma of the tonsils: a molecular analysis of HPV associa-
tions. Clin Cancer Res 8(4):1093–1100
Cancer Causes Control
123