Cofactors With Human Papillomavirus in a Population-Based Study of Vulvar Cancer
Journal of the National Cancer Institute
Cofactors With Human Papillomavirus in a Population-Based Study of Vulvar Cancer
Margaret M. Madeleine 0 2
Janet R. Daling 0 2
Joseph J. Carter 0 2
Gregory C. Wipf 0 2
Stephen M. Schwartz 0 2
Barbara McKnight 0 2
Robert J. Kurman 0 2
Anna Marie Beckmann 0 2
Michael E. Hagensee 0 2
Denise A. Galloway 0 1 2
0 Oxford University Press
1 Affiliations of authors: M. M. Madeleine , J. R. Daling, S. M. Schwartz , Division of Public Health Sciences, Fred Hutchinson Cancer Research Center , Seattle, WA , and Department of Epidemiology, University of Washington , Se- attle; J. J. Carter, G. C. Wipf, A. M. Beckman , Division of Public Health Sciences, Fred Hutchinson Cancer Research Center; B. McKnight, Department of Biostatistics, University of Washington; R. J. Kurman, Department of Pathol- ogy, The Johns Hopkins University , Baltimore, MD; M. E. Hagensee , Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, and De- partment of Medicine, University of Washington; D. A. Galloway, Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, and Depart- ment of Microbiology, University of Washington. Sciences, Fred Hutchinson Cancer Research Center, Weiss/Daling Studies (MP381) , 1124 Columbia St., Seattle, WA 98104-2092. See ''Notes'' following ''References.''
2 Journal of the National Cancer Institute , Vol. 89, No. 20, October 15, 1997
Background: Human papillomavirus (HPV) has been previously associated with vulvar cancer. In a population-based study, we examined whether exposure to HPV, cigarette smoking, or herpes simplex virus 2 (HSV2) increases the risk of this cancer. Methods: Incident cases of in situ (n = 400) and invasive (n = 110) squamous cell vulvar cancer diagnosed among women living in the Seattle area from 1980 through 1994 were identified. Serum samples were analyzed for antibodies against specific HPV types and HSV2. HPV DNA in tumor tissue was detected by means of the polymerase chain reaction. In most analyses, case subjects were compared with population-based control subjects (n = 1403). Relative risks of developing vulvar cancer were estimated by use of adjusted odds ratios (ORs) and 95% confidence intervals (CIs). Results: Increased risks of in situ or invasive vulvar cancer were associated with HPV16 seropositivity (ORs = 3.6 [95% CI = 2.6-4.8] and 2.8 [95% CI = 1.7-4.7], respectively), current cigarette smoking (ORs = 6.4 [95% CI = 4.4-9.3] and 3.0 [95% CI = 1.7-5.3], respectively), and HSV2 seropositivity (ORs = 1.9 [95% CI = 1.4-2.6] and 1.5 [95% CI = 0.9-2.6], respectively). When the analysis was restricted to HPV16 DNA-positive tumors (in situ or invasive), the OR associated with HPV16 seropositivity was 4.5 (95% CI = 3.0-6.8). The OR for vulvar cancer was 18.8 (95% CI = 11.9-29.8) among current smokers who were HPV16 seropositive in comparison with never smokers who were HPV16 seronegative. Conclusions: Current smoking, infection with HPV16, and infection with HSV2 are risk factors for vulvar cancer. Risk appears particularly strong among women who are both current smokers and HPV16 seropositive. [J Natl Cancer Inst 1997;89:1516-23]
In 1991, several reports (1–5) characterized pathologically
distinct forms of squamous cell vulvar cancer. The majority of
invasive squamous cell vulvar carcinomas are classified as
keratinizing squamous cell carcinomas. Only 4%–21% of
keratinizing squamous cell tumors are reported to be human
papillomavirus (HPV) DNA positive (1,5–7). Other invasive squamous
cell tumors are classified chiefly as basaloid, warty, or mixed
basaloid and warty carcinomas, of which 82%–86% contain
HPV DNA (1,5–7). In contrast to invasive vulvar cancer, in situ
vulvar cancer occurs more often among younger women, and
80%–96% of in situ lesions are reported to contain HPV DNA
(7–10). The incidence of in situ vulvar cancer has been
increasing, especially among younger women (11).
Vulvar cancer is a rare tumor that is often described as having
epidemiologic parallels with squamous cell cancer of the cervix,
including a link to HPV infection (7,12–16). The incidence of
invasive vulvar cancer is only a fifth of that of invasive cervical
cancer in the United States (17). Parallels between vulvar and
cervical cancers include a history of a high number of sexual
partners, smoking, an abnormal Pap smear, genital warts, and
other sexually transmitted diseases (7,12–16). Although HPV
has been associated with more than 90% of squamous cell
cervical cancers (18), it has been suggested that there are etiologic
pathways leading to the development of vulvar cancer that do
not involve HPV, especially among older women (19,20). HPV
infections may play a major role in the pathogenesis of vulvar
cancer among younger women and women with in situ disease.
Two risk factors associated with vulvar cancer, smoking and
infection with herpes simplex virus type 2 (HSV2), have been
suggested as possible cofactors in the pathogenesis of
HPVrelated genital cancers (21). Earlier reports from this ongoing
case–control study (15,16) and from a case–control study by
Brinton et al. (14) found high risks of vulvar cancer associated
with a history of genital warts, particularly among cigarette
smokers (14,16). An increased risk of vulvar cancer was also
found to be associated with seropositivity to HSV2 in a previous
report from this study (15) and for a self-reported history of
genital herpes in the study by Brinton et al. (14). In the analysis
described here, we examined, in our ongoing population-based
study, whether exposure to HPV, cigarette smoking, or HSV2
independently or together increases the risk of vulvar cancer.
Subject Eligibility, Identification, and Recruitment
Case subjects were women aged 18–79 years who resided in the Seattle area
and were diagnosed with incident vulvar cancer from January 1980 through June
1994. They were identified through the Cancer Surveillance System, a
population-based registry located in western Washington. We restricted this
investigation to epithelial tumors coded by the registry with International Classification of
Disease subjects for Oncology (ICDO) topography codes 184.1 through 184.4
and ICDO morphology codes 8010 through 8081 (22). Case subjects were
determined to have either in situ or invasive disease on the basis of the fifth-digit
ICDO behavior code.
We recruited 67.7% of the 830 eligible case subjects to participate in an
inperson interview. The response rate was somewhat higher for women with in
situ (70.5%) compared with invasive disease (60.8%). Reasons for
nonparticipation included patient refusal (21.8%), doctor refusal (5.8%), and death (4.7%).
A higher proportion of eligible women with invasive cancer (12.5%) than with
in situ cancer (1.5%) died before interview, but the rate of patient and doctor
refusals did not differ by lesion type. We excluded 52 case subjects who were
identified by the registry as having nonsquamous cell vulvar carcinomas, leaving
data from 510 women with squamous cell vulvar cancer available for analysis:
400 with in situ disease and 110 with invasive disease.
Population-based control subjects were identified by use of random-digit
telephone dialing (23), and these control subjects were frequency matched to the age
distribution of the case subjects in 5-year age intervals. To be eligible as a
control subject, a woman had to be a resident at the reference date of the
three-county area that includes Seattle, have a working telephone at the reference
date, be able to communicate, and have no history of vulvar cancer. A household
census was successfully conducted for 94.1% of all residential phone numbers
called. A total of 1415 (74.6%) women were interviewed among 1898 eligible
control subjects who were contacted. The overall response rate was 70.2% (the
screening response rate multiplied by the interview response rate). Twelve
control subjects were excluded because of a history of prior cancer, an inability to
communicate, or not being a resident in the three-county area at the reference
date, leaving data from 1403 control women available for analysis.
A team of interviewers administered a detailed interview in a standardized
way to case and control subjects. Topics covered in the interview included
demographic characteristics and reproductive, birth control, sexual, and smoking
histories. Smokers were women who had smoked more than 100 cigarettes,
former smokers had quit smoking before the reference date, and current smokers
were women who were still smoking at the reference date. Case subjects were
asked to refer to the time before diagnosis when answering questions during the
interview, and control subjects were matched to the case subjects on the year of
diagnosis and then assigned a randomly chosen month. For example, current
smoking at the reference date is defined by the date of diagnosis for the subjects
with vulvar cancer and by a comparable date for control subjects.
At the conclusion of the interview, all subjects were asked to provide a serum
sample. Blood samples were collected by routine venipuncture from 93.9% (479
of 510) case subjects and 86.6% (1215 of 1403) control subjects and stored at
−70 °C. Case subjects were asked to give permission for tumor blocks to be
Seropositivity to HPV6, HPV16, and HPV18 capsid proteins was determined
by use of an antigen capture, enzyme-linked immunosorbent assay (ELISA).
This assay detected antibodies reacting with a conformational epitope on the L1
protein of vaccinia virus-expressed, HPV type-specific capsids [developed as
described by Carter (24)]. Monoclonal antibodies H11B2, H16V5, and H18J4
(provided by Neil Christiansen, Milton S. Hershey Medical Center, PA) were
used to detect HPV L1 capsid-types 6, 16, and 18, respectively. Optical density
values from six microtiter wells were normalized and combined into a single
value for each subject by subtracting the background readings from the wells
without capsids from those with capsids. Seropositive specimens were those
yielding a value greater than two standard deviations above the mean of ELISA
values from negative control serum specimens derived from a cohort of
university women who reported no history of sexual activity and whose cervical–
vaginal samples were HPV DNA negative (24). All study samples were run
concurrently with the negative control samples and a pool of positive control sera
obtained from people attending a sexually transmitted disease clinic.
Antibody response to HSV2 was assessed by means of a western blot assay to
discriminate between the immune response to HSV1 and HSV2 (25). For the
HSV2 variable, HSV2-seronegative women included women who were
seropositive for HSV1.
Since this is an ongoing project, samples are batched together for testing in the
serology laboratories. Not all samples have had all assays performed. For the
analyses presented here, 474 of 479 case subjects and 1204 of 1215 control
subjects have been tested for HSV2 antibodies; 364 of 479 case subjects and
1088 of 1215 control subjects have been tested for HPV18 antibodies; 345 of 479
case subjects and 1031 of 1215 control subjects have been tested for HPV16
antibodies; and 247 of 479 case subjects and 604 of 1215 control subjects have
been tested for HPV6 antibodies. All serologic tests were conducted without
knowledge of case–control status or other characteristics of the subjects.
To classify the vulvar tumor tissue with respect to the presence or absence of
HPV, polymerase chain reaction (PCR) methods were used to amplify HPV
DNA extracted from paraffin-embedded tissue. Consensus primers derived from
the L1 gene open reading frame [primers MY09/MY11 (26)] were used, and the
amplification products were typed by means of Southern hybridization with
oligonucleotide probes specific for HPV-types 6/11, 16, 18/45, and 31 (239 of
308 case subjects) or by means of restriction fragment-length polymorphism
analysis (69 of 308 case subjects) (27). Additional primers were derived from the
E6 gene open reading frame (28), and the identity of the HPV16, HPV18, and
HPV6 E6 products was confirmed by means of Southern hybridization. As a
control for HPV DNA-negative results, a fragment of the b-globin gene was
amplified to ensure that amplifiable DNA was present in the samples (29).
Positive and negative control reactions included the use of recombinant plasmids
containing HPV DNA and heart muscle tissue DNA, respectively. A detailed
comparison of the HPV DNA and serology testing will be the subject of a
Specimens from 42 case subjects were not included in the analysis because
they yielded no b-globin gene amplification product or the documentation
accompanying the block indicated lower grade disease than that reported to the
CSS. The tumor blocks used for PCR testing were chosen on the basis of
pathology reports. There were eight late consensus region (L1 gene)-positive
tumors that were not positive for any of the type-specific probes, referred to here
as HPV DNA positive/type unknown. Multiple blocks per person were available
for about half (45.5%) of the case subjects. Results were summarized across the
multiple blocks tested; if HPV DNA was detected in any of the blocks tested, the
tumor from that woman was called HPV positive for that type. PCR results were
available for 308 case subjects. A histologic review of slides cut from the
paraffin-embedded blocks for 34 case subjects with invasive disease was
performed by a board-certified pathologist (R. J. Kurman).
The relative risk (RR) of cancer was estimated with the odds ratio (OR)
approximation by exponentiating coefficients obtained from multiple logistic
regression analysis. Subjects with missing values for any variables in a model
were excluded from that model. The following potential confounders were not
controlled because their control did not substantially affect the ORs of interest:
reference year, income (>$30 000, $15 000–$30 000, or <$15 000), alcohol use
(never, former, or current), education (ù13 or <13 years), marital status (married
or not married), parity status (nulliparous or number of live births plus number
of stillbirths), and race (white or nonwhite). The following confounders were
controlled in one or more of the ORs presented: age at reference date (<40,
40–59, or ù60 years), years of education (<12, 12, or ù13 years), body mass
index as weight in kilograms/height in meters squared (<25, 25–29, or ù30),
lifetime number of sex partners (1, 2–4, or ù5), smoking (never, former, or
current), and HPV16 seropositivity (negative or positive). Although most
analyses used binary logistic regression to compare in situ or invasive case groups
separately with the population control subjects, polytomous logistic regression
was sometimes used for simultaneous comparison of the two case groups and the
control subjects. To determine whether the combined effects of HPV
seropositivity and either cigarette smoking or HSV2 seropositivity were greater than that
predicted from the individual risk factors, we included interaction terms and
evaluated their contribution to the fit of the multiplicative and additive models
by means of likelihood ratio tests. We also calculated the synergy index and the
attributable proportion caused by interaction and their respective confidence
intervals (CIs) (30,31) to provide quantitative assessments of interaction under
an additive model.
Since we did not collect tissue from population-based control subjects for the
detection of HPV DNA, HPV16 antibody status was used as a marker of HPV
exposure for all subjects. To examine the RRs of HPV-negative and
HPVpositive vulvar cancer associated with HPV seropositivity, smoking, and HSV2
seropositivity in a more etiologically homogeneous case group, we performed a
subanalysis where the case groups were determined by their HPV DNA status.
Approximately one half (51.8%) of the women with invasive
disease were more than 60 years of age compared with 22.5% f
the women with in situ disease (Table 1). Among women with
either in situ disease or invasive vulvar cancer, the
agestandardized percentages of having 13 or more years of
education, an income more than $30 000 per year, being married, or
being parous were lower than among the population-based
control subjects. The case subjects had a higher age-standardized
proportion of women who had two or more sexual partners or
first intercourse at less than 17 years of age compared with the
control subjects. The distribution of case and control subjects
was similar by race. The age-standardized percentage of women
with invasive disease who reported being very overweight
(23.6%) was higher than that reported by either case subjects
with in situ disease (6.8%) or control subjects (8.2%).
Table 2 shows the PCR results obtained from tumor tissue of
308 of the 510 case subjects. Nearly 70% of the case subjects
(209 of 308) were positive for HPV DNA (181 [71.5%] of 253
in situ cancers and 28 [50.9%] of 55 invasive cancers). The most
commonly detected HPV type was HPV16, which was detected
in 61.7% of the in situ tumors and in 43.6% of the invasive
tumors. Oncogenic HPV types (16, 18, 31, 33, and 52) were
detected in 68.0% of the in situ cancers and in 47.3% of the
invasive cancers. Multiple HPV DNA types were found in 9.5%
of the in situ tumors (24 of 253) and in 1.8% (one of 55) of the
The RRs of vulvar cancer associated with seropositivity to
HPV6, 16, or 18 or seropositivity to HSV2 were similar when in
situ and invasive vulvar lesions were compared (Table 3). The
OR of vulvar cancer associated with HPV16 seropositivity was
3.6 (95% CI 4 2.6–4.8) for in situ disease and 2.8 (95% CI 4
1.7–4.7) for invasive disease. The prevalence of antibodies to
HPV6 and HPV18 in case subjects was similar to the prevalence
in control subjects. The most marked difference between the in
In situ cancer
n 4 1403
n 4 400
n 4 110
*Percentages in this table are adjusted to the age distribution of the control subjects.
†Body mass index 4 weight in kilograms divided by height in meters squared.
In situ tumors
(n 4 253)*
(n 4 55)
*n 4 number of case subjects.
†Values for the HPV-positive subcategories do not sum to the totals because
of multiple positivity for 25 samples. Twenty-one samples were positive for
HPV16 and one other type, two samples were positive for HPV16 and two other
types, and two samples were positive for HPV6/11 and one other type.
‡HPV types 16, 18/45, 31, 33, and 52 are combined in this category.
situ and invasive vulvar cancers was seen with cigarette
smoking. The OR associated with current smoking was 6.4 (95% CI
4 4.4–9.3) for in situ cancer and 3.0 (95% CI 4 1.7–5.3) for
invasive cancer. When women with invasive vulvar cancer
were used as the reference group, there was a significantly
increased risk of in situ cancer associated with current smoking
according to polytomous logistic regression (ratio 4 2.2; 95%
CI 4 1.2–4.2). When control subjects were used as the reference
group, former smoking did not increase the RR of invasive
cancer (OR 4 1.4; 95% CI 4 0.7–2.8), but it was associated with
an increased risk of in situ cancer (OR 4 2.1; 95% CI 4
To identify risk factors that may be specifically related to
HPV-associated disease, we divided the case groups according
to HPV DNA status, regardless of lesion grade (Table 4). The
RRs of vulvar cancer associated with HPV seropositivity,
smoking, and HSV2 seropositivity were similar when comparing
HPV DNA-negative and HPV16 DNA-positive case subjects
with polytomous logistic regression.
In Table 5, the combination of HPV16 seropositivity and
smoking history is examined, with women (case and control
subjects) who were HPV16 seronegative and who had never
smoked as the reference group. On the basis of a comparison
with this reference group, the OR for vulvar cancer associated
with HPV16 seropositivity and never smoking was found to be
2.9 (95% CI 4 1.7–5.0), the OR associated with current
smoking and HPV16 seronegativity was found to be 4.9 (95% CI 4
3.3–7.5), and the OR associated with current smoking and
HPV16 seropositivity was determined to be 18.8 (95% CI 4
11.9–29.8). Although there was no departure from the
multiplicative model, there was a significant interaction on the additive
scale (P<.001). The synergy index was calculated as the ratio of
the sum of the risk differences (18.8 − 1.0)/[(4.9 − 1) + (2.9 − 1)]
4 3.1 (95% CI 4 2.0–4.8), and the attributable proportion of
vulvar cancers explained by the combined exposure was (18.8 −
4.9 − 2.9 + 1)/18.8 4 0.63 (95% CI 4 0.49–0.78). When the
case groups were compared by use of polytomous logistic
regression, the OR for in situ vulvar cancer associated with current
smoking and HPV16 seropositivity was 2.3 (95% CI 4 1.0–5.4)
when women with invasive cancer were used as the reference
Among current smokers, increasing amount and duration of
smoking substantially increased the risk of disease (Table 5).
(n 4 110)
(n 4 1403)
In situ cancer
(n 4 400)
(n 4 1403)
HPV16 DNA-positive case subjects
(n 4 180)
HPV DNA-negative case subjects
(n 4 99)
Among former smokers, recent cessation of smoking was
associated with higher risks than cessation of smoking at 5 or more
years before the reference date. Starting to smoke at earlier ages
and pack-years (i.e., number of packs smoked per day multiplied
by the number of years of smoking) of cigarette smoking showed
a less consistent pattern of increased risk.
The combination of HPV16 and HSV2 seropositivity was
also examined for an indication of increased risk associated with
combined effects. With women who were HSV2 and HPV16
seronegative as the reference group, the age and
smokingadjusted OR for vulvar cancer associated with HSV2
seropositivity and HPV16 seronegativity was 1.9 (95% CI 4 1.3–2.7),
the OR for HPV16 seropositivity and HSV2 seronegativity was
3.2 (95% CI 4 2.2–4.6), and the OR for being seropositive for
both HSV2 and HPV16 was 5.7 (95% CI 4 3.8–8.4). However,
there was no indication that including an interaction term
improved the fit of the multiplicative or additive models.
A small subsample of slides from 34 case subjects coded in
the registry as having invasive squamous cell tumors were
obtained for histopathologic review. There were 26 (76.5%) case
subjects classified as having keratinizing squamous cell
carcinoma and eight (23.5%) case subjects identified having as
baHPV16 antibody status
saloid or warty carcinoma. HPV16 DNA was found in 75.0%
(six of eight) of the basaloid or warty carcinomas and in 22.7%
(five of 22) of the keratinizing squamous cell carcinomas tested
One limitation of this population-based study is the low
participation level (~70%) for case and control subjects, which may
be partly explained by the sensitive nature of the questions being
asked. If women who did not participate were different in a
consistent way with respect to their exposure history from those
who did participate, the results of this study will be biased.
However, strong associations such as we found are unlikely to
be caused by this potential source of bias.
Another limitation is that only a subset of invasive cancers
(34 of 110) were reviewed histopathologically to distinguish
between different types of invasive squamous cell lesions;
therefore, the invasive case group is probably comprised of a mixture
of HPV-related and non-HPV-related case subjects. It has been
hypothesized that the majority of keratinizing squamous cell
carcinomas, which are usually found in older women, are likely
to be non-HPV related (19,20). The subsample that was
reviewed histopathologically supports this hypothesis, since 75%
of the tested basaloid or warty tumors, but only 23.5% of the
keratinizing squamous cell tumors, were HPV16 DNA positive.
Since we did not have information about specific squamous cell
histopathology for the majority of case subjects with invasive
disease, we were not able to address this hypothesis directly.
There were, however, significantly increased risks of invasive
vulvar cancer among women more than 60 years of age
associated with HPV16 seropositivity and having had more than two
sexual partners, and HPV16 DNA was found in 42.5% (34 of 80)
of case subjects more than 60 years of age. Since these results
would argue for a substantial HPV-related cause in at least some
older women, histopathologic review of all case subjects is
needed. It may be that the increased risks for these sexual factors
among older women is restricted to the basaloid or warty
The lack of HPV DNA testing on control subjects is a
limitation of this study. In both the in situ and the invasive case
groups, the majority of tumor tissues tested by PCR were HPV
DNA positive; therefore, the results presented here are most
generalizable to women with HPV-related vulvar tumors. It is
likely that the 308 of the 510 case subjects who were tested for
HPV DNA by PCR are representative of the entire case group,
since tissue was sought for all case subjects and the tissues were
tested in the order that they were received in the laboratory.
Also, the prevalence of HPV DNA may be underestimated, since
there may be other HPV types not represented in our testing
protocol that are important in vulvar cancer.
As expected from the high prevalence of HPV16 DNA,
HPV16 seroprevalence was strongly associated with vulvar
cancer. Also, the low prevalence of HPV6 and HPV18 DNA was
mirrored by the lack of association between HPV6 and HPV18
antibodies and vulvar cancer. Short of knowing the natural
history of each HPV infection and the corresponding immune
response, however, the serologic test may be only interpreted as
indicating a previous or current exposure to HPV. The
seropositive case subjects whose tumors are HPV DNA negative could
be revealing a response to an HPV infection that is no longer
required for tumor maintenance, an old or newly acquired
infection not associated with the tumor, a false-positive serology
result, or a false-negative PCR result. A seronegative case
subject who was HPV DNA positive could be demonstrating the
loss of the L1 gene after the integration of HPV, the greater
sensitivity of PCR in comparison with ELISA to detect the
presence of HPV, or a false-negative serology result. Since most
women probably have only a transient, subclinical infection with
high-risk HPV types at young ages (32,33), seropositivity
among the control subjects may indicate persistent infections or
cleared HPV infections. The seropositive control subjects with
persistent infections may also represent women who, at present,
lack the complement of cofactors necessary to progress to
Sun et al. (34) used a different HPV16 virus-like particle
ELISA, which was also directed to capsid proteins, than was
employed here. Both studies reported similar seroprevalences of
HPV16 in the control subjects: 18.2% of 44 control subjects in
the study by Sun et al. compared with 22.2% of 1031 control
subjects in this study. The seroprevalence among women with
vulvar intraepithelial neoplasia (59.1% of 22) in the study by
Sun et al. was similar to the seroprevalence among the in situ
case subjects in this study (53.3% in 272). In both studies, there
was a lower seroprevalence with invasive cancer. Sun et al. were
able to distinguish invasive keratinizing squamous cell
carcinomas by histopathology and found a lower seroprevalence of
HPV16 among women with these tumors (22.2% of 18) than
was reported in this study (46.2% of 13). Overall, the
seroprevalence of HPV16 was 43.8% among the subjects with invasive
vulvar cancer in this study. Histopathologic review of the tumors
from these women is now part of the design of this ongoing
project, which may help to resolve this issue.
HPV is thought to lead to transformation and expression of a
malignant phenotype that interferes with the ability of the host
genome to impede oncogenesis in proliferating cells (35). This
carcinogenesis is hypothesized to be a multistage process that
requires cofactors to achieve malignant transformation (35).
Smoking is hypothesized to be such a cofactor. Furthermore,
there is in vitro evidence that suggests that the byproducts of
smoke can transform HPV-immortalized cell lines (36,37).
Another mechanism by which smoking may promote
carcinogenesis is the inhibition of apoptosis, since it has been suggested
that nicotine inhibits apoptosis (38). The combination of
cigarette smoking and HPV could be particularly important in
abrogating control on two components of cell kinetics:
proliferation and programmed cell death.
An earlier report from this study (6) and reports from other
epidemiologic studies (12–14) of vulvar cancer have noted a
strongly increased risk associated with smoking, especially
current smoking. Women with anogenital cancers, particularly
premenopausal women, have been found to have an increased risk
of lung cancer (39). Sturgeon et al. (40) also found an excess risk
of smoking-related cancers after primary vulvar cancer. The
pattern of RRs associated with combinations of smoking and HPV
serology in this study suggest the existence of one or more
mechanisms by which smoking and HPV infection interact.
Furthermore, they suggest that 63% (95% CI 4 0.49–0.78) of the
vulvar neoplasias in the population of women who smoke and
are infected with HPV can be attributed to such mechanisms. We
also found that current smoking is a stronger risk factor for
vulvar cancer than former smoking, and former smokers who
quit more recently have a higher risk than women who quit 5 or
more years previously. Smoking was not associated with HPV16
seropositivity among the control subjects, which suggests that
smoking status did not affect acquisition of HPV infection.
Together, these data suggest that smoking may act at a late stage in
HSV2 is another potential cofactor with HPV in anogenital
carcinogenesis (21,41). In HPV16 immortalized keratinocytes,
HSV2 induced tumorigenicity, but it was not detectable in the
tumor cells (42). Our study found that HPV DNA-negative case
subjects and HPV16-positive case subjects had an increased risk
of vulvar cancer associated with seropositivity to HSV2.
However, we found no evidence for a joint effect of HSV2 and
HPV16 seropositvity in a comparison with women who were
seronegative for both HSV2 and HPV16. These data do not
support synergy between HSV2 and HPV, but suggest that some
vulvar cancers could be HSV2 related.
In this study, the increased risk of vulvar cancer associated
with smoking was focused among current smokers and was
strongest for women with in situ disease. We also found that
HPV16 seropositivity was a risk factor for in situ and invasive
vulvar cancer. In addition, there is a particularly strong risk
among women with both exposures when measured on the
additive scale. Since other investigators have also reported the
association between smoking and vulvar neoplasia, it may be of
public health importance to encourage women at risk of HPV
infection or early vulvar lesions to quit smoking.
Supported by Public Health Service grant 3P01-CA42792 and contract
N01CN05230 from the National Cancer Institute, National Institutes of Health,
Department of Health and Human Services, with additional support from the Fred
Hutchinson Cancer Research Center. Its contents are solely the responsibility of
the authors and do not necessarily represent the official views of the National
Cancer Institute. M. E. Madeleine is supported by a Physician’s Training grant
from Howard Hughes Medical Center.
We thank the women who participated in this study as case subjects and
control subjects and the interviewers. We also thank Drs. Karen J. Sherman and
Rhoda C. Ashley for their contributions and the staff of the Cancer Surveillance
System for their diligent efforts at patient ascertainment.
Manuscript received April 14, 1997; revised July 29, 1997; accepted August
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