Cofactors With Human Papillomavirus in a Population-Based Study of Vulvar Cancer

JNCI Journal of the National Cancer Institute, Oct 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.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

https://jnci.oxfordjournals.org/content/89/20/1516.full.pdf

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. Data Collection 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 retrieved. 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 subsequent report. 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). Data Analysis 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. Results 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 invasive tumors. 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 Case subjects Control subjects In situ cancer Invasive 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)* Invasive tumors (n 4 55) HPV DNA *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 1.4–3.3). 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 group. Among current smokers, increasing amount and duration of smoking substantially increased the risk of disease (Table 5). Invasive cancer (n 4 110) Control subjects (n 4 1403) In situ cancer (n 4 400) Case subjects Control subjects (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 2.5–19.6 7.2–26.5 14.7–42.6 4.2–19.0 14.4–44.6 7.8–31.9 2.3–8.6 4.1–25.7 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 by PCR. 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 histologic type. 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 disease. 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 vulvar carcinogenesis. 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. References 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 15, 1997. (1) Andersen WA , Franquemont DW , Williams H , Taylor PT , Crum CP . Vulvar squamous cell carcinoma and papillomaviruses: two separate entities . Am J Obstet Gynecol 1991 ; 165 : 335 - 6 . (2) Bloss JD , Liao SY , Wilczynski SP , Macri C , Walker J , Peake M , et al. Clinical and histologic features of vulvar carcinomas analyzed for human papillomavirus status: evidence that squamous cell carcinoma of the vulva has more than one etiology . Hum Pathol 1991 ; 22 : 711 - 8 . (3) Nuovo GJ , Delvenne P , MacConnell P , Chalas E , Neto C , Mann WJ . Correlation of histology and detection of human papillomavirus DNA in vulvar cancers . Gynecol Oncol 1991 ; 43 : 275 - 80 . (4) Park JS , Jones RW , McLean MR Currie JL, Woodruff JD , Shah KV , et al. Possible etiologic heterogeneity of vulvar intraepithelial neoplasia. A correlation of pathologic characteristics with human papillomavirus detection by in situ hybridization and polymerase chain reaction . Cancer 1991 ; 67 : 1599 - 607 . (5) Toki T , Kurman RJ , Park JS Kessis T, Daniel RW , Shah KV . Probable nonpapillomavirus etiology of squamous cell carcinoma of the vulva in older women: a clinicopathologic study using in situ hybridization and polymerase chain reaction . Int J Gynecol Pathol 1991 ; 10 : 107 - 25 . (6) Hording U , Junge J , Daugaard S , Lundvall F , Poulsen H , Bock JE. Vulvar squamous cell carcinoma and papillomaviruses: indications for two different etiologies . Gynecol Oncol 1994 ; 52 : 241 - 6 . (7) Trimble CL , Hildesheim A , Brinton LA , Shah KV , Kurman RJ . Heterogeneous etiology of squamous carcinoma of the vulva . Obstet Gynecol 1996 ; 87 : 59 - 64 . (8) Haefner HK , Tate JE , McLachlin CM , Crum CP . Vulvar intraepithelial neoplasia: age, morphological phenotype, papillomavirus DNA, and coexisting invasive carcinoma . Hum Pathol 1995 ; 26 : 147 - 54 . (9) Junge J , Poulsen H , Horn T , Hording U , Lundvall F. Human papillomavirus (HPV) in vulvar dysplasia and carcinoma in situ . APMIS 1995 ; 103 : 501 - 10 . (10) van Beurden M , ten Kate FJ , Smits HL , Berkhout RJ , de Craen AJ , van der Vange N , et al. Multifocal vulvar intraepithelial neoplasia grade III and multicentric lower genital tract neoplasia is associated with transcriptionally active human papillomavirus . Cancer 1995 ; 75 : 2879 - 84 . (11) Sturgeon SR , Brinton LA , Devesa SS , Kurman RJ . In situ and invasive vulvar cancer incidence trends (1973 to 1987) . Am J Obstet Gynecol 1992 ; 166 : 1482 - 5 . (12) Newcomb PA , Weiss NS , Daling JR . Incidence of vulvar carcinoma in relation to menstrual, reproductive, and medical factors . J Natl Cancer Inst 1984 ; 73 : 391 - 6 . (13) Mabuchi K , Bross DS , Kessler II. Epidemiology of cancer of the vulva . A case-control study . Cancer 1985 ; 55 : 1843 - 88 . (14) Brinton LA , Nasca PC , Mallin K , Baptiste MS , Wilbanks GD , Richart RM . Case-control study of cancer of the vulva . Obstet Gynecol 1990 ; 75 : 859 - 66 . (15) Sherman KJ , Daling JR , Chu J , Weiss NS , Ashley RL , Corey L. Genital warts, other sexually transmitted diseases, and vulvar cancer . Epidemiology 1991 ; 2 : 257 - 62 . (16) Daling JR , Sherman KJ , Hislop TG , Maden C , Mandelson MT , Beckmann AM , et al. Cigarette smoking and the risk of anogenital cancer . Am J Epidemiol 1992 ; 135 : 180 - 9 . (17) Ries LA , Kosay CL , Hankey BF , Harras A , Miller BA , Edwards BD, editors. SEER cancer statistics review, 1973 - 1993 : tables and graphs, National Cancer Institute. Bethesda (MD) , 1996 . (18) Bosch FX , Manos MM , Munoz N , Sherman M , Jansen AM , Peto J , et al. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective . International Biological Study on Cervical Cancer (IBSCC) study group. J Natl Cancer Inst 1995 ; 87 : 796 - 802 . (19) Kurman RJ , Trimble CL , Shah KV . Human papillomavirus and the pathogenesis of vulvar carcinoma . Curr Opin Obstet Gynecol 1992 ; 4 : 582 - 5 . (20) Crum CP . Carcinoma of the vulva: epidemiology and pathogenesis . Obstet Gynecol 1992 ; 79 : 448 - 54 . (21) zur Hausen H. Human genital cancer: synergism between two virus infections or synergism between a virus infection and initiating events? Lancet 1982 ; 2 : 1370 - 2 . (22) World Health Organization. International Classification of Diseases for Oncology . 1st ed. Geneva, 1976 . (23) Hartge P , Brinton LA , Rosenthal JF , Cahill JI , Hoover RA , Waksberg J. Random digit dialing in selecting a population-based control group . Am J Epidemiol 1984 ; 120 : 825 - 33 . (24) Carter JJ , Wipf GC , Hagensee ME , McKnight B , Habel L , Lee SK , et al. Use of human papillomavirus type 6 capsids to detect antibodies in people with genital warts . J Infect Dis 1995 ; 172 : 11 - 8 . (25) Ashley RL , Militoni J. Use of densitometric analysis for interpreting HSV serologies based on Western blot . J Virol Methods 1987 ; 18 : 159 - 68 . (26) Manos MM , Ting Y , Wright DK , Lewis AJ , Broker TR , Wolinsky SM . Use of polymerase chain reaction for the detection of genital human papillomaviruses . Cancer Cells 1989 ; 7 : 209 - 14 . (27) Lungu O , Wright TC Jr, Silverstein S. Typing of human papillomaviruses by polymerase chain reaction amplification with L1 consensus primers and RFLP analysis . Mol Cell Probes 1992 ; 6 : 145 - 52 . (28) Beckmann AM , Acker R , Christiansen AE , Sherman KJ . Human papillomavirus infection in women with multicentric squamous cell neoplasia . Am J Obstet Gynecol 1991 ; 165 ( 5 Pt 1 ): 1431 - 7 . (29) Saiki RK , Scharf S , Faloona F , Mullis KB , Horn GT , Erlich HA , et al. Enzymatic amplification of b-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia . Science 1985 ; 230 : 1350 - 4 . (30) Rothman K. Modern epidemiology . Boston: Little, Brown, 1986 : 321 - 2 . (31) Hosmer DW , Lemeshow S. Confidence interval estimation of interaction . Epidemiology 1992 ; 3 : 452 - 6 . (32) Koutsky LA , Holmes KK , Critchlow CW , Stevens CE , Paavonen J , Beckmann AM , et al. A cohort study of the risk of cervical intraepithelial neoplasia grade 2 or 3 in relation to papillomavirus infection . N Engl J Med 1992 ; 327 : 1272 - 8 . (33) Lowy DR , Kirnbauer R , Schiller JT . Genital human papillomavirus infection . Proc Natl Acad Sci U S A 1994 ; 91 : 2436 - 40 . (34) Sun Y , Hildesheim A , Brinton LA , Nasca PC , Trimble CL , Kurman RJ , et al. Human papillomavirus-specific serologic response in vulvar neoplasia . Gynecol Oncol 1996 ; 63 : 200 - 3 . (35) zur Hausen H, de Villiers EM . Human papillomaviruses . Annu Rev Microbiol 1994 ; 48 : 427 - 47 . (36) McDougall JK. Cofactors in the progression of HPV-associated tumors . Antibiot Chemother 1994 ; 46 : 150 - 64 . (37) Yang X , Jin G , Nakao Y , Rahimtula M , Pater MM , Pater A. Malignant transformation of HPV 16-immortalized human endocervical cells by cigarette smoke condensate and characterization of multistage carcinogenesis . Int J Cancer 1996 ; 65 : 338 - 44 . (38) Wright SC , Zhong J , Zheng H , Larrick JW. Nicotine inhibition of apoptosis suggests a role in tumor promotion . FASEB J 1993 ; 7 : 1045 - 51 . (39) Fritsch M , Melbye M. Risk of lung cancer in pre-and post-menopausal women with ano-genital malignancies . Int J Cancer 1995 ; 62 : 508 - 11 . (40) Sturgeon SR , Curtis RE , Johnson K , Ries L , Brinton LA . Second primary cancers after vulvar and vaginal cancers . Am J Obstet Gynecol 1996 ; 4 : 929 - 33 . (41) Galloway D , McDougall JK . The oncogenic potential of herpes simplex viruses: evidence for a 'hit-and-run' mechanism . Nature 1983 ; 302 : 21 - 4 . (42) DiPaolo JA , Woodworth CD , Popescu NC , Koval DL , Doniger J. HSV -2- induced tumorigenicity in HPV16-immortalized human genital keratinocytes . Virology 1990 ; 177 : 777 - 9 .


This is a preview of a remote PDF: https://jnci.oxfordjournals.org/content/89/20/1516.full.pdf

Margaret M. Madeleine, Janet R. Daling, Stephen M. Schwartz, Joseph J. Carter, Gregory C. Wipf, Anna Marie Beckmann, Barbara McKnight, Robert J. Kurman, Michael E. Hagensee, Denise A. Galloway. Cofactors With Human Papillomavirus in a Population-Based Study of Vulvar Cancer, JNCI Journal of the National Cancer Institute, 1997, 1516-1523, DOI: 10.1093/jnci/89.20.1516