1 Program in Epidemlogy, Fred Hutchinson Cancer Research Center, Seattle, WA.
2 Department of Pathology, University of Washington, Seattle, WA.
3 Department of Laboratory Medicine, University of Washington School of Medicine, Seattle, WA.
4 Faculty of Medicine, Mahidol University, Department of Obstetrics and Gynecology, Siriraj Hospital, and Siriraj Family Planning Research Center, Bangkok, Thailand.
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ABSTRACT |
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carcinoma; squamous cell; cervical intraepithelial neoplasia; cervix neoplasms; papillomavirus, human
Abbreviations: CIN-III, cervical intraepithelial neoplasia grade 3; HPV, human papillomavirus
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INTRODUCTION |
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The identification of cofactors for preinvasive disease is best attempted in the context of prospective studies of HPV-infected women. Concurrent prospective studies of untreated women with carcinoma in situ to determine factors associated with progression to invasive diseases would be unethical, and studies of treated women would be uninformative because treatment removes or destroys the in situ lesion. A few historical cohort studies of HPV-infected subjects that included both in situ and invasive cervical carcinoma as endpoints (14
) and case-control studies of invasive disease (1
, 5
9
) have identified a variety of possible cofactors with varying degrees of consistency that could act either before or after the development of in situ disease. These include the following: early age at first sexual intercourse, which may reflect particular vulnerability of the immature cervix to HPV-induced carcinogenesis; hormonal factors such as high parity and use of oral contraceptives; exposures to chemical agents in cigarette smoke; sexually transmitted agents other than HPV; deficiencies in certain micronutrients; an immunocompromised state resulting from human immunodeficiency virus (HIV) infection or immunosuppresion for prevention of organ transplant rejection; and various measures of socioeconomic status, suggesting the presence of additional unrecognized cofactors associated with social class.
Most of these factors, however, have been observed in relation to both in situ and invasive disease (1) and, in studies in which the epidemiologic features of these two stages of cervical carcinoma can be compared, few consistent differences in risk factors have been observed (5
, 10
15
). These observations suggest that most putative cofactors are operative before the development of carcinoma in situ and that factors associated with progression to invasion have yet to be identified.
This is a report of analysis performed to compare risk factors for in situ and invasive disease in order to identify variables associated only with the latter condition, after controlling for the presence of oncogenic HPV types in the tumor tissue. If women with in situ disease are considered the controls and if women with invasive disease are considered the cases in a standard case-control analysis, then elevated odds ratios in relation to a factor represent the risk of invasive disease relative to the risk of in situ disease in women with the factor (16). Because it is reasonable to assume that most or all invasive cervical carcinomas have passed through an in situ stage, an elevated odds ratio would imply that the factor enhances probability of progression from in situ to invasion. To our knowledge, results of direct comparisons of women with in situ and invasive disease have been reported only once previously (10
).
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MATERIALS AND METHODS |
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Two controls for each woman with invasive disease were selected from otolaryngology and general surgery wards of Siriraj Hospital, as previously described (17). The cases of carcinoma in situ were women who had had an abnormal cervical smear during a visit to either the family planning or gynecologic clinic associated with Siriraj Hospital, and an attempt was made to select two controls for each case from the same clinic from which the case came. Cervical smears are taken from many of the women who attend these clinics, and the women selected as controls were the next two women with appointments in the clinic after the appointment for the case, in the same 5-year age group and from the same region of the country as the case, who had returned to the clinic to learn the results of their cervical smears, and whose cervical smears revealed no suspicion of neoplastic change. If a woman refused to participate, the selection process continued until two controls per case were selected.
All cases and hospitalized controls were interviewed while hospitalized; the controls selected for the cases of carcinoma in situ were interviewed in the clinics on the day they were selected. Information was obtained on sexual and reproductive history, prior cervical smears, use of tobacco and alcohol, and indices of prior use of medical resources and socioeconomic status. Dates of all prior cervical smears were obtained, and those taken within the past year were not considered prior screening cervical smears. The presenting symptom of each case was also ascertained and recorded as none (detected by routine cytology only), vaginal bleeding, abnormal vaginal discharge, or other.
A 15-ml blood specimen was obtained from all interviewed women. Four aliquots of serum were stored at -70°C, two of which were retained in Bangkok and two of which were shipped to Seattle, Washington, on dry ice.
Cervical scrapings for HPV DNA assays were obtained from cases by the patient's surgeon prior to treatment. Cervical smears and cervical scrapings were taken by these same surgeons from the hospital controls while they were hospitalized and from the clinic controls in the clinic on the same day they were selected and interviewed. The cervix, including the cervical os, was scraped with a Teflon (E. I. du Pont de Nemours and Company, Wilmington, Delaware)-coated swab, the end of which was then broken off into a vial containing 2 ml of specimen transport medium (Digene Diagnostics, Inc., Beltsville, Maryland). The specimens were stored at -70°C and periodically shipped on dry ice to Seattle.
Histologic slides from the blocks that were used to make the diagnosis in the cases were read by a single collaborating pathologist in Bangkok. Information was recorded on source of specimen, tumor size, stage, and histologic diagnosis. A pathologist in Seattle reread the slides from 174 of the cases included in this report. Of 23 in situ cases, only one (4.3 percent) was considered invasive by the reference pathologist, and only eight (5.3 percent) of 151 cases considered invasive in Bangkok were read in Seattle as in situ with no evidence of microinvasion. The diagnosis made in Bangkok was thus used in the analysis in this report.
As detailed elsewhere (17), serologic assays were performed for antibodies to human immunodeficiency virus, hepatitis B, Treponema pallidum, and herpes simplex virus types 1 and 2, as well as for hepatitis B surface antigen. Cervical scrapings were assayed for evidence of any HPV DNA and for type-specific DNA of types 6 and 11, type 16, type 18, types 31/33/35/39, and type 45 using polymerase chain reaction-based technology (17
). Samples that hybridized with a generic probe but not with any of the type-specific probes were considered positive for untyped HPV.
Cases and controls were compared, and odds ratios as estimates of relative risks were calculated using unconditional logistic regression (18) when comparing invasive cases with their unmatched controls and using conditional logistic regression (18
) when comparing in situ cases with their matched controls. In addition, to directly compare risk factors for invasive and in situ disease, we performed unconditional logistic regression analyses using the women with invasive disease as "cases" and women with carcinoma in situ as "controls." Because the purpose of these analyses was to identify any factors that distinguish invasive from in situ disease, and because no such factors were identified other than HPV status, no attempts were made to adjust odds ratios for factors other than age and (for the case-case comparisons) HPV status.
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RESULTS |
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Of the 614 women selected as controls for the cases of invasive disease, 490 (79.8 percent) were interviewed. Cervical scrapings were obtained from 306 (62.4 percent) of these 490 women, 298 (97.4 percent) of which yielded adequate DNA samples. Three women with no history of sexual intercourse, one with a subtotal hysterectomy, and three with ineligible diagnoses were excluded, leaving 291 controls for analysis.
Of the 161 women selected as controls for the in situ cases, 160 (99.4 percent) were interviewed, 144 of whom were subsequently confirmed as meeting the eligibility criteria for inclusion in the study. Cervical scrapings were obtained from 133 (92.4 percent) of these 144 women, 128 of which contained adequate amounts of DNA. Four of the 128 women from whom these samples were taken had been matched to excluded cases and were omitted, leaving 124 women as controls for the in situ cases in the analyses.
As shown in table 1, women with invasive disease were slightly older than their controls. The women with carcinoma in situ and their controls were comparable in age and younger than the women with invasive carcinoma and their controls. The 10 additional cases of carcinoma in situ used in the comparisons of in situ and invasive disease are not appreciably different in age from the 65 cases shown in the table. All odds ratio estimates presented were adjusted for age using the age categories shown in the table.
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Oncogenic types of HPV were strongly associated with both invasive and in situ disease (table 2). Type 16 DNA was the most frequently identified type in both invasive and in situ tumors. Both types 16 and 18 were found in a higher proportion of invasive than in situ carcinomas. Types 31/33/35/39 occurred with nearly equal frequency in both tumor types, and untyped HPV DNA was found in a higher proportion of the in situ than invasive lesions. Few women in either control group had any type of HPV in their cervical scrapings, although the frequency of a positive test was slightly higher in the controls for the in situ cases. The estimates of the odds ratio of both invasive and in situ carcinomas are large, with those in relation to types 16 and 18, any oncogenic type, and any HPV type being stronger for invasive disease; those in relation to types 31/33/35/39 being similar for both tumor types; but those in relation to untyped HPV being greater for in situ disease. As shown in table 3, after controlling for age and other HPV types, the odds ratio of invasive disease is four times greater than the risk of in situ disease in women with HPV types 16 and 18, twice as high in women with HPV types 31/33/35/39 (although possibly due to chance), and also significantly higher in relation to any oncogenic type and any type of HPV. The risk is not greater for invasive than in situ disease in women with untyped HPV DNA.
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The risk of both in situ and invasive disease increased with increasing number of pregnancies (table 5). The risk of neither tumor type was significantly associated with the other hormonal factors considered, except for an increase in risk of in situ carcinoma in users of depot-medroxyprogesterone acetate for contraception. Use of oral contraceptives was weakly associated with both tumor types. None of the hormonal factors shown in table 5 was significantly more strongly associated with invasive than in situ disease. Ages at menarche and first livebirth were also not associated with either tumor type (not shown).
Women who had used an intrauterine device and who had a history of prior cervical smears were at reduced risk of both invasive and in situ disease, and the risk of both types was slightly but not significantly increased in smokers (table 6). The risk of invasive but not of in situ carcinoma was lower in women with than without a history of a screening chest radiographic examination and formal schooling. The risk of invasive diseases was low relative to the risk of in situ disease in women with a tubal ligation, prior cervical smears, and a prior chest radiographic examination, and possibly in women who had attended school.
When the analyses for the last columns in tables 46 were restricted to data on study subjects who had never had a cervical smear, to eliminate differences between women with invasive and in situ disease that may be due to prior screening behavior, the results were not appreciably different from those presented. Moreover, the same analyses were performed separately for invasive and in situ cases with an oncogenic HPV type (150 invasive and 42 in situ cases) and without an oncogenic HPV type (26 invasive and 18 in situ cases), and no significant differences (p > 0.05) were observed in the comparable odds ratio estimates from the two sets of analyses.
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DISCUSSION |
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There is little reason to suggest that the odds ratio estimates in this study in relation to HPV type are biased: cervical scrapings were obtained from a high proportion of both the invasive and in situ cases; both groups of women resided in the same areas of Thailand; and the prevalence of the HPV types that were assayed is similar to that observed by others (1). The odds ratio estimates shown in table 2 thus provide evidence that HPV types 16 and 18 are associated with a fourfold increase in the risk of progression to invasion, that types 31/33/35/39 are less strongly predictive of progression, and that the other (untyped) HPV types are not indicative of subsequent invasion. This interpretation is consistent with the observation (27
) that squamous intraepithelial lesions with oncogenic HPV DNA tend to be monoclonal, and those with other HPV DNA types tend to be polyclonal.
Although assays for types of HPV other than those that were investigated in this study are now available, and although some of these more recently identified types may be oncogenic, it is unlikely that their omission from this study influenced the results that pertain to types 16, 18, and 31/33/35/39, because only 14 women with invasive disease and 12 with in situ tumors had untyped HPV DNA in their cervical scrapings. The observation that untyped HPV types are more strongly associated with in situ than invasive disease would probably be strengthened if we had tested for the more recently recognized oncogenic types and removed them from the group with untyped DNA; our conclusion that there are HPV types associated with in situ disease that do not progress to invasion would not change.
After controlling for age and HPV type, the epidemiologic features of in situ and invasive cervical carcinomas were found to be quite similar. The age at first intercourse was a strong risk factor for both tumor types in this largely monogamous population, suggesting either that early exposure to oncogenic types of HPV may enhance the risk of persistent infection and carcinogenesis, or that the sexual behavior of the husbands of women with early and late onset of sexual activity may differ (17). Serologic indices of exposure to sexually transmitted agents other than HPV were generally not strongly related to either invasive or in situ disease, and none was significantly more strongly related to one tumor type or the other. This is as expected, because these factors are more likely indicators of risk of initial infection with HPV rather than cofactors operative after neoplastic changes. Although quite possibly due to chance, the risk in relation to serologic evidence of hepatitis B surface antigen (table 4) was slightly greater for invasive than in situ disease. Because persistent hepatitis B virus antigenemia may suggest a deficient immune response, this observation is compatible with a role for immunodeficiency in the persistence of HPV infection that can lead to invasive carcinoma.
An increase in the risk of both in situ and invasive disease with parity and use of oral contraceptives was observed, but the associations were not significantly stronger for invasive disease, suggesting that these hormonal factors operate prior to the development of in situ lesions.
The risk of carcinoma in situ, but not of invasive disease, was elevated in users of depot-medroxyprogesterone acetate. This same observation was previously observed in a larger study conducted in Thailand and elsewhere (28, 29
) and is compatible with the interpretation that any effect of depot-medroxyprogesterone acetate on the risk of cervical carcinoma in situ is reversible or that the lesions induced by this product have a low invasive potential.
Smoking was weakly associated with the risk of both invasive and in situ lesions, but the prevalence of smoking among the study subjects was low and the odds ratio estimate had wide confidence limits. If smoking is a cofactor, it operates prior to the development of in situ disease and is not an important determinant of risk in Thailand.
Having had a screening chest radiographic examination and having ever attended school were both associated with a reduced risk of invasive disease but not of in situ disease. These associations persisted after controlling for the frequency of prior cervical smear screening, suggesting that these associations are not due to more cervical cancer screening in the more educated women and in women who are screened for tuberculosis. Another possible explanation is that women of higher socioeconomic status may tend to seek care earlier than women of lower status and, hence, are more likely to have their cervical neoplasia diagnosed when still in a preinvasive stage. However, the observation that the presenting symptoms of women with in situ and invasive disease did not differ is not supportive of this interpretation. Other studies have also shown odds ratios in relation to school attendance to be less than unity for invasive but not in situ disease (10, 14
, 15
, 30
), and the consistency of these results and ours suggests that factors associated with education or other measures of socioeconomic status, such as nutritional factors, may inhibit invasion.
Prior cervical smears were shown to be related to a reduced risk of cervical carcinoma and, as expected, to be more strongly protective against invasive than in situ disease. As has also been observed elsewhere (31), reduced risks in relation to use of an intrauterine device and a tubal ligation most likely reflect screening for cervical cancer at the time of intrauterine device insertion or removal and at the time of tubal surgery.
Although there has been only one prior report of studies in which direct comparisons of risk factors for in situ and invasive carcinomas were made after controlling for HPV type in the tumor (10), there have been other investigations in which women with both in situ and invasive disease have been studied using comparable methodology but without inclusion of HPV testing (13
15
, 30
). The results of all these studies are generally consistent with our findings in showing no sexual or hormonal factors, or smoking, to be more strongly associated with invasive than in situ carcinomas. Additional studies of the role of these factors in the genesis of cervical carcinoma should focus on the more proximal preinvasive stages of the carcinogenic process.
Several possible sources of bias could have influenced the odds ratio estimates in this study. If women with carcinoma in situ were more likely than women with invasive carcinoma to have been screened for cervical cancer, then observed differences between women with these two tumor types could reflect factors associated with screening behavior rather than with development of invasive disease. This is an unlikely explanation for our results. There is no mass screening of asymptomatic women in Thailand, and cases of in situ disease in this study did not differ from women with invasive disease with respect to the presenting symptoms that lead to diagnosis. In addition, controlling odds ratio estimates for history of prior cervical smears had no appreciable effect on the results.
Confounding by HPV status could have influenced the odds ratio estimates based on comparisons of cases and controls, although the consistency of our results with those of others suggests that this is not a likely explanation for the findings. More importantly, the direct comparisons of women with invasive and in situ carcinomas were controlled for HPV type, thus eliminating confounding by these viruses as an explanation for the few associations observed in those analyses.
The comparison of invasive with in situ disease is an investigative approach that other investigators should consider in an attempt to identify factors that may enhance or prevent progression of intraepithelial lesions to invasive disease. Because HPV type was the only predictor of progression identified, other studies should focus on additional factors not adequately considered in this and other investigations.
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ACKNOWLEDGMENTS |
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The assistance of Drs. Anna Marie Beckmann and Larry Corey is gratefully acknowledged.
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NOTES |
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REFERENCES |
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