1 Department of Epidemiology, University of Washington, Seattle, WA.
2 Department of Obstetrics and Gynecology, Duke University, Durham, NC.
3 Department of Pathology, University of Washington, Seattle, WA.
4 Department of Obstetrics and Gynecology, University of Washington, Seattle, WA.
Received for publication March 31, 2003; accepted for publication November 25, 2003.
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ABSTRACT |
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case-control studies; cervical intraepithelial neoplasia; cervix uteri; female; Ki-67 antigen; papillomavirus, human; smoking; tobacco smoke pollution
Abbreviations: Abbreviations: aOR, adjusted odds ratio; ASC-US, atypical squamous cells of uncertain significance; CI, confidence interval; CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; OR, odds ratio.
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INTRODUCTION |
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Understanding what factors other than HPV play a role in cervical cancer development is important. If exposures other than HPV infection, especially modifiable exposures, are significant in precancerous lesion development, women can be educated as to the risk associated with these exposures. This information is also important in the future application of HPV DNA testing to cervical cancer screening. If HPV DNA testing is implemented, it will be important to identify the subset of HPV-positive women that is at greatest risk of having a high-grade precancerous lesion, since HPV testing has significantly lower specificity than does cytology (2). Such women might be triaged immediately to colposcopy and biopsy versus follow-up by repeated cytology or HPV testing. Additionally, in resource-poor settings where HPV testing may be the only cervical cancer screening method available, the identification of those at greatest risk is important in allocating limited resources.
Few studies have considered risk factors for cervical disease among HPV DNA-positive women (39). Some (35, 79) but not all (6) previous studies of women with HPV infection found smoking to be positively associated with cervical disease. None of the previous studies, however, used the same methods to verify the disease status of all women, and only two studies (4, 8) restricted all women to those with oncogenic HPV infection. The aim of this study was to examine the relation between smoking and CIN among oncogenic HPV DNA-positive women. To limit potential bias, we defined all outcome groups in the same manner, included histologically negative women who at screening were oncogenic HPV DNA positive, and drew our cases and controls from women presenting for routine cytologic screening. Additionally, we performed Ki-67 (a marker for proliferation/metaplasia) immunohistochemistry testing on transformation zone biopsy samples from a subset of women.
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MATERIALS AND METHODS |
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Women with 1) atypical squamous cells of uncertain significance (ASC-US) and low-grade or high-grade squamous intraepithelial lesions on cytology and 2) negative cytology and a positive oncogenic HPV DNA test, as well as 3) a random sample of women with negative cytology and negative HPV DNA test results, were contacted and offered colposcopy and biopsy with the study.
After written, informed consent was obtained at the colposcopy-biopsy study visit, personnel administered a detailed questionnaire regarding demographic, reproductive/gynecologic, sexual history, smoking, and general medical information to participants. A pelvic examination was performed, and cervical specimens were collected as described for the screening visit. A colposcopic examination of the cervix was performed, and punch biopsies were taken from the most abnormal appearing areas or at the 12 oclock position if no abnormal areas were seen. Women with lesions that extended into the endocervical canal or women with squamous intraepithelial lesions on screening cytology and no colposcopically visible lesions also underwent endocervical curettage. Biopsy and endocervical curettage specimens were placed in formalin.
Women with negative results or low-grade disease (atypia or CIN1) were referred back to their regular provider for follow-up and treatment if necessary. Women with high-grade disease (CIN23) were advised of the indication for treatment and offered a loop electrosurgical excision procedure by a study clinician. Women with cancer were referred to a gynecologic oncologist for staging and appropriate treatment of their cancer. At the clinicians discretion (e.g., high-grade squamous intraepithelial lesion cytology and negative histology), 33 women were referred for repeated colposcopies, biopsies, and/or endocervical curettages. HPV testing was not performed at these repeated visits.
HPV DNA detection
HPV DNA processing and testing were performed at the HPV DNA Laboratory at Harborview Medical Center (Seattle, Washington). First, the cervical swab sample was digested with protease, and the DNA was precipitated with ethanol. Then the HPV L1 consensus primer (MY09/MY11 and HMB01) polymerase chain reaction amplification assay and a Roche reverse line blot hybridization (Roche Molecular Systems, Inc., Alameda, California) were used for amplification and typing of HPV DNA. This system allowed for the detection and typing of HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 55, 56, 58, 59, 68, 73, 82, and 83 (oncogenic) and HPV types 6, 11, 40, 42, 53, 54, 57, 66, and 84 (nononcogenic). Over 40 HPV types have been found in the anogenital mucosa, including the following uncharacterized types: 61, 62, 64, 67, 69, 70, 71, 72, 81, CP6108, and IS39, which were not included this system (10). The primers PC04 and GH20 were used for beta-globin detection (11). The process used one fiftieth of the original 1-ml sample for amplification by polymerase chain reaction.
Cytology and histology
All cytology and histology samples were processed and reviewed at Harborview Medical Center in Seattle, Washington. Cytology slides were stained with the Papanicolaou stain, screened by a cytotechnologist, and reviewed by pathologists without knowledge of colposcopy or HPV DNA results. Cytologic findings were recorded in terms of the Bethesda System classification (12). When more than 60 percent of the slide target area was without epithelial cells, the sample was considered to be unsatisfactory. A random 10 percent sample of all slides read out as negative was rescreened manually as mandated by federal law (13). All biopsies and endocervical curettages were classified as negative, atypia, CIN1, CIN2, CIN3, carcinoma in situ, adenocarcinoma in situ, microinvasive cancer, or invasive cancer.
Ki-67 antigen detection
Immunohistochemistry testing for Ki-67 antigen (a proliferation antigen associated with epithelial metaplasia and dysplasia) was performed on a consecutive series of the CIN1 and CIN23 cases and on a random subset of negative cases (n = 139) (14). Sections of formalin-fixed, paraffin-embedded tissues 4 µm in size were deparaffinized and rehydrated through graded alcohols. Immunohistochemistry testing for Ki-67 (MIB-1 clone at 1:50 dilution; DAKO Corporation, Carpenteria, California) was then performed using a modified avidin-biotin complex immunoperoxidase technique according to standard protocols with heat-induced epitope retrieval and 3,3'-diaminobenzidine as a chromogen with nickel chloride enhancement (15). The degree of Ki-67 staining was recorded as low (positive signal in the lower one third of the epithelium, generally confined to the basal layer of the epithelium) or intermediate to high (positive signal detected in the middle one third and/or upper one third of the epithelium) (16).
Statistical analysis
The analysis was restricted to women who were positive for an oncogenic HPV type at screening and who received a follow-up colposcopy and biopsy that were satisfactory for evaluation at the time of the analysis (n = 629). Prior to analysis, one control group and two case groups were defined on the basis of the most severe histologic diagnosis from any of the biopsy visits. Only five women received a more severe diagnosis at a second visit (median time between visits: 3 months; range: 16 months). The control group was composed of women with a negative histology diagnosis and negative or ASC-US Papanicolaou smears (one woman had a Papanicolaou smear diagnosis of atypical endocervical cells, favor reactive) at both the screening and colposcopy-biopsy visits (n = 181). The first case group was composed of women with normal or abnormal (ASC-US) Papanicolaou smears and a histologic diagnosis of CIN1 (n = 137). The second case group was composed of women with normal or abnormal (
ASC-US) Papanicolaou smears and a histologic diagnosis of
CIN23 (n = 143). In a subanalysis, 53 women with CIN2 and 90 women with
CIN3 were included as separate groups. There were no cases of invasive carcinoma diagnosed at biopsy or endocervical curettage. Two women with a biopsy diagnosis of carcinoma in situ were found to have invasive carcinoma in the loop electrosurgical excision procedure specimen. Overall, 36 percent of the 501 women with negative histology and
ASC-US cytology, 67 percent of the 204 women with CIN1, and 86 percent of the 167 women with
CIN23 were positive for oncogenic HPV DNA and were included in the analysis. Because there were two case groups, multinomial (polytomous) logistic regression was used. Stata 7 (STATA Corporation, College Station, Texas) software was used for all analyses (17).
Smoking history, which was ascertained from the colposcopy-biopsy visit questionnaire, was analyzed using the following three definitions: smoking status (never (never smoked at the time of the colposcopy-biopsy visit), former (quit smoking prior to the colposcopy-biopsy visit), current (smoked at the time of the colposcopy-biopsy visit)); pack-years of exposure (0, 5, >5 pack-years); and number of cigarettes per day (never smokers or those who quit smoking more than 2 years prior to the colposcopy-biopsy visit (0 cigarettes/day), 110, >10). Additionally, to evaluate whether those who recently quit smoking were at an increased risk of CIN1 or
CIN23 compared with never smokers, we placed those who quit smoking 2 years or less before their colposcopy-biopsy visit into a separate group. Those who smoked 0.1 pack-year or less were classified as nonsmokers for all analyses.
The following covariates were evaluated as possible confounders: age at biopsy (continuous and 1819, 2024, 2529, 3050 years), screening clinic, education (<12th grade, 12th grade), race/ethnicity (White, Black, other), Papanicolaou screening history (number in past 5 years, history, and number of abnormal Papanicolaou smears), current use of hormonal birth control (yes/no), number of male partners (continuous), parity (0, 1,
2 births), self-reported history of sexually transmitted diseases (Chlamydia trachomatis, Neisseria gonorrhoeae, cervicitis), clinician assessment of ectopy at the colposcopy-biopsy visit (09, 1019, 2029,
30 percent, missing), and HPV DNA test results (number of oncogenic types and positivity for the same oncogenic HPV type at both visits). The presence of confounding was assessed using the following criteria as a guideline: change of greater than 10 percent in the odds ratio of the smoking history variable when the potential confounder was added to the univariate model and/or significant change in the log likelihood.
To avoid misclassification of case and control status, women were excluded from analysis if they had a final histologic diagnosis of negative and unsatisfactory cytology at either visit (n = 10), squamous intraepithelial lesions on cytology and a final histologic diagnosis of negative (n = 71), or a final histologic diagnosis of atypia (n = 77). To avoid misclassification of exposure status, women were excluded from analysis if they had incomplete smoking information (n = 1) or if their colposcopy-biopsy visit HPV DNA specimen was insufficient for testing (n = 9).
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RESULTS |
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There was a median of 54 days between the screening visit and the colposcopy-biopsy visit. Of the 461 subjects who were initially positive for oncogenic HPV DNA at screening at the colposcopy-biopsy visit, 316 (68 percent) were repeatedly positive for the same type of oncogenic HPV DNA, 31 (7 percent) were positive for a different type of oncogenic HPV DNA, and 114 (25 percent) were negative for oncogenic HPV DNA. At the colposcopy-biopsy visit, 33 (18 percent), 29 (21 percent), and 71 (50 percent) of the negative, CIN1, and CIN23 subjects, respectively, were positive for HPV type 16 DNA; 118 (80 percent) of the 147 women with HPV type 16 DNA detected at screening were positive for HPV type 16 DNA at the colposcopy-biopsy visit.
Women with CIN1 were more likely than women with negative histology findings to be current as opposed to never smokers (adjusted odds ratio (aOR) = 1.8, 95 percent confidence interval (CI): 1.1, 3.1) (table 2). Similarly, women with CIN23 were more likely than those with negative histology findings to be current smokers (aOR = 1.6, 95 percent CI: 1.0, 2.7). Compared with never smokers, women who had quit smoking less than 2 years earlier were at increased risk of
CIN23 (aOR = 2.2, 95 percent CI: 1.0, 5.2). The risk was not appreciably elevated among women who quit smoking more than 2 years ago (aOR = 1.3, 95 percent CI: 0.4, 4.9).
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When the analysis of each smoking variable was restricted to HPV type 16-positive women, the strength of the associations increased; however, because of small numbers, the width of the confidence intervals increased (data not shown). The analysis was also performed combining CIN1 and CIN23 and, finally, separately for CIN2 and CIN3. For the combined analysis, the risk estimates were between those of the two separate categories (data not shown). When the CIN2 and the CIN3 were separated, the risk estimates for CIN2 and CIN3 were similar (data not shown). A stratified analysis was performed to determine the extent to which the risk of CIN could be explained by an interaction between number of cigarettes per day and HPV type-specific repeated positivity (table 3). There was no clear evidence for interaction on a multiplicative scale.
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DISCUSSION |
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An association between smoking and CIN among women with HPV infection has been reported in some previous (35, 79), but not all (6), studies. Our results for CIN23 are similar to those reported for one of the two other studies that restricted cases and controls to include only women who were oncogenic HPV DNA positive (8), and our results were slightly lower than those reported in the other study (4). We found a similar association with smoking for both CIN1 and
CIN23. This is in contrast to results reported by Ho et al. (5), who found that various smoking measures were positively associated with CIN2 (smokers of >10 cigarettes per day vs. never/former smokers: OR = 1.38, 95 percent CI: 0.42, 4.54) and CIN3 (smokers of >10 cigarettes per day vs. never/former smokers: OR = 3.35, 95 percent CI: 1.22, 9.15) cases when compared with CIN1 cases. The difference in results between the study by Ho et al. and the present study could be due in part to the fact that 34 percent of subjects with CIN1 in their study were positive only for nononcogenic HPV DNA, whereas in our study, all subjects with CIN1 were positive for oncogenic HPV DNA.
Other studies that assessed more than one type of smoking measure have also found the strongest associations with cigarettes per day (5, 8). Investigators who studied the distribution of Ki-67 staining in the lung found that Ki-67 did not correlate with pack-years or smoke-years, but it did correlate with packs per day (18).
One limitation of this analysis is the potential decreased accuracy of self-reported smoking history. However, there is no reason to suspect that women with CIN would be more likely than women without to report inaccurate smoking histories, since smoking history was obtained prior to biopsy. Patrick et al. (19) found that, compared with biochemical validation, self-report of smoking status had high sensitivity and specificity.
There are several strengths of the current analysis. All three outcome groups, which included a negative group, were diagnosed in the same manner, by cytology and histology. The negative group was required to have negative and/or ASC-US cytology findings at both the screening and colposcopy-biopsy visits to limit misclassification. Other studies have used a negative control group that was determined only by cytology findings and a case group that was determined by histology findings (6, 9), or they used a control group that was not composed of women with negative diagnoses (5, 7). Another strength is that this is not a referral population; the majority of both cases and controls were from the same clinic population presenting for routine annual examinations. If the cases had been selected from only women who were referred for colposcopy and biopsy on the basis of an abnormal Papanicolaou smear, 36 (25 percent) of the CIN23 cases would have been missed. Furthermore, this is the first study to evaluate whether the smoking effect could be explained by an association with repeated detection of oncogenic HPV DNA, detection of infection by more than one oncogenic HPV type, or evidence of proliferation/metaplasia, as measured by Ki-67 staining of the transformation zone epithelium.
A possible mechanism for the relation between smoking and CIN among women positive for oncogenic HPV is that smoking weakens the immune response to HPV infection, allowing the virus to persist for a longer period of time (20). However, previous studies found a significant negative association between smoking and repeated oncogenic HPV DNA positivity, and this association was greater for current versus former smokers and for heavier versus lighter smokers (21, 22). Our stratified analysis also showed a nonsignificant negative association between heavier smoking (>10 cigarettes per day) and repeated oncogenic HPV DNA positivity.
Another possible mechanism for the relation is that smoking alters normal epithelial cell proliferation by increasing cell division and inducing metaplasia (23). This mechanism is supported by studies showing that cigarette smoke induces metaplasia in the lung epithelium and that this metaplasia precedes carcinoma (24, 25). The cigarette smoke components benzo[a]pyrene and N-methyl-N-nitrosourea have been shown to induce epithelial proliferation and metaplasia in vitro and in vivo (2630). Smoking cessation is associated with reversal of metaplasia in the lung (31). The HPV E6 and E7 genes have been found to induce immortalization in bronchial epithelial cells (3234). In addition, nicotine and smoke metabolites (cotinine, benzo[a]pyrene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) have been detected in cervical mucus and cervical epithelium (35, 36). Smoking, therefore, may act by increasing cell turnover in the transformation zone of the cervix. The finding that heavy smoking (>10 cigarettes per day) was associated with Ki-67 staining, which is a marker for proliferation/metaplasia, supports this hypothesis. Because the association was seen regardless of histology findings, smoking may have an early effect on the evolution of HPV-related lesions. Smoking has been associated with the absence of ectopy (37), perhaps, because smoking increases the rate at which columnar epithelium undergoes squamous metaplasia. In our analyses, ectopy confounded the relation between smoking and CIN because ectopy was negatively associated with smoking and positively associated with CIN. A third mechanism for the relation between smoking and CIN is that cigarette smoke by-products may directly transform cells infected by HPV (35, 36, 3842).
This study extended previous findings by showing that, among women with oncogenic HPV infection, the relation between smoking and CIN was present for both CIN1 and CIN23 and that this association appeared to be mediated through the effect of cigarette smoke on cell turnover. On the basis of these findings, women who are HPV positive should be advised not to smoke or to reduce the number of cigarettes that they smoke.
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ACKNOWLEDGMENTS |
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The authors thank Planned Parenthood, Kim Tomlinson, Alison Starling, and Connie Nelson for their work on the Evaluation of Screening Methods for Cervical Cancer Study.
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NOTES |
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REFERENCES |
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