1 Department of Obstetrics and Gynecology, University of New Mexico Health Sciences Center, Albuquerque, NM.
2 Oregon Health Sciences University, Portland, OR.
3 School of Nursing, University of New Mexico Health Sciences Center, Albuquerque, NM.
4 Rehoboth McKinley Christian Health Clinic, Gallup, NM.
5 Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM.
6 New Mexico Tumor Registry, University of New Mexico Health Sciences Center, Albuquerque, NM.
7 Albuquerque Indian Health Service, Albuquerque, NM.
8 Department of Pediatrics, Emory University School of Medicine, Atlanta, GA.
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ABSTRACT |
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cervical intraepithelial neoplasia; cervix dysplasia; Indians, North American; papillomavirus, human; sexually transmitted diseases
Abbreviations: CI, confidence interval; CIN, cervical intraepithelial neoplasia; HPV, human papilloma virus; OR, odds ratio; Pap, Papanicolaou
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INTRODUCTION |
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Despite high rates of cervical neoplasia among American Indian women nationwide and in the Southwest in particular, few published studies have addressed risk factors for CIN in this population. To further characterize risks for CIN among southwestern American Indian women, we carried out a clinic-based case-control study of women with CIN. Our study focused on hypotheses related to sexually transmitted infections and other potential risk factors for CIN.
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MATERIALS AND METHODS |
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The case women in this study were enrolled through the colposcopy clinics at three Indian Health Service facilities before their clinical evaluation for cervical squamous intraepithelial lesions (CIN grade I, II, or III, representing slight dysplasia, moderate dysplasia, and severe dysplasia/carcinoma in situ, respectively), detected on routine Papanicolaou (Pap) tests. Colposcopy clinic visits were scheduled within 1 month of diagnosis of CIN on the routine screening Pap test. Most patients (75 percent) had colposcopy performed within 6 months of their initial diagnosis (range, 062 months). All case women were American Indian (by self-report and validation through medical record reviews, which include tribal enrollment information), aged 1845 years, and not pregnant. Subjects were contacted upon presentation to the colposcopy clinic and invited to participate in the interviews as an additional part of their clinic visit. Informed consent was obtained for subjects to participate in interviews, to provide 20 ml of blood for micronutrient and serologic assays, and to undergo cervical cultures as described below.
Control women with normal cervical epithelium were selected from the same clinics through which case women were referred for their colposcopic examinations. Controls were also American Indian women aged 1845 years who were not pregnant. In addition, controls were required to have histories of all normal Pap tests. We selected control subjects from women appearing at the Indian Health Service hospitals and clinics who required a pelvic examination for any reason, including family planning, an annual checkup, and other reasons (table 1). Besides the age restriction, no matching criteria were employed. Medical records were reviewed to ensure that potential study controls had documented normal Pap tests in addition to meeting the other study enrollment criteria.
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Although case women with CIN were invited into the study on the basis of Pap tests showing squamous epithelial lesions, the presence of CIN upon histologic examination of cervical tissue taken on the day of study entry was necessary in order for subjects to remain in the analysis. Of the case women enrolled on the basis of Pap test results, 57 did not have histologic confirmation of CIN upon biopsy and were dropped from the final analysis. Of those subjects, 56 had normal or nondiagnostic biopsy results and one did not have a biopsy performed. No study subject had invasive cervical carcinoma upon biopsy. Most (94.6 percent) of the cervical smears from the clinic sites were interpreted by a single reference pathology laboratory that was under contract to the Indian Health Service facilities in New Mexico. The remaining 5.4 percent of all study Pap smears were processed by a separate laboratory that was also under contract to the Indian Health Service facility in Albuquerque. To determine the comparability between laboratories in cytologic diagnoses before the study began, Indian Health Service clinicians examined a sample of smears from the alternate laboratory and found that its interpretations correlated highly with those of the main laboratory (Jill Miller, Indian Health Service, personal communication, 1997).
For control women, biopsy specimens were not obtained, and we required that the Pap test on the day of study entry be negative for subjects to remain in the analysis. Women who were selected as controls but who had atypia or dysplasia on the Pap test on the day of study entry were excluded from the analysis (n = 37 (5.1 percent)).
We also excluded seven women from the final analysis who reported no sexual activity with a male partner, as these subjects could not have been exposed to the sexually transmitted disease risk factors of interest in this study. We recognize that virginal women can still develop Pap smear abnormalities related to human papillomavirus (HPV) infection; HPV in these women is likely to be acquired via fomites or other means not related to intercourse with males (9). However, the major hypotheses of interest in this research also related to risks associated with sexually transmitted diseases other than HPV infection, and transmission risks for these other sexually transmitted diseases are related almost exclusively to heterosexual intercourse.
All study subjects were paid $20.00 for their participation in this investigation.
Interviews
All study subjects were interviewed by trained interviewers who focused on relevant risk factors for the development of CIN as suggested in previous research. After informed consent was obtained, the interviewers asked participants about sexual histories, sexually transmitted diseases, cervical cancer screening practices, and cigarette smoking. Demographic data were also collected. All interviews were carried out in English and lasted 6090 minutes. The colposcopy clinics were scheduled to be open on specific days of the week; thus, because of logistical problems associated with the clinic schedules, the interviewers were not blinded to the case/control status of the study subjects. Medical records were examined to validate responses about episodes of sexually transmitted disease, Pap test screening, and contraceptive use. Interviewers were trained to examine all laboratory reports, in addition to charted comments by clinicians, to verify subjects' responses.
Pelvic examination and specimen collection
Pelvic examinations and cervical specimen collection preceded colposcopic examination of the cervix for case women. Control women had cervical specimens taken at the time of pelvic examination. Specimens were collected from all women in the following order: 1) a Pap test, fixed and air-dried; 2) a Dacron cervical swab (E. I. Du Pont de Nemours and Company, Wilmington, Delaware) of the endocervix and ectocervix, placed in standard transport medium (Digene Diagnostics, Silver Spring, Maryland) for later identification of the HPV genome; 3) an endocervical swab for identification of Chlamydia trachomatis and Neisseria gonorrhoeae using Gen-Probe assays (Gen-Probe, Inc., San Diego, California); 4) a vaginal pool swab for wet mount identification of trichomonads, yeast, and clue cells under light microscopy; 5) a vaginal pool swab placed on a glass slide and air-dried for later microscopic identification of organisms that are associated with bacterial vaginosis (10); and 6) cervical-vaginal lavage using 3 ml of normal saline solution.
Laboratory testing included identification of antibodies to herpes simplex virus types 1 and 2 using purified glycoprotein assays (11, 12
). We also carried out microhemagglutination assays to identify antibodies to Treponema pallidum (Sera-tek treponemal antibody test; Fujirebio, Inc., Tokyo, Japan) and commercial assays to identify antibodies to hepatitis B virus (Corzyme; Abbott Laboratories, Abbott Park, Illinois) and hepatitis C virus (Abbott HCV EIA2.0; Abbott Laboratories). Laboratory personnel were not informed as to the case/control status of the subjects. Because of the very low prevalence of human immunodeficiency virus infection in American Indian communities in the Southwest (13
), we did not test for antibodies to human immunodeficiency virus.
Following cervical and blood specimen collection, case subjects underwent colposcopic examination of the cervix using application of 5 percent acetic acid with cotton swabs. All case women underwent cervical biopsy and endocervical curettage. Treatment for any abnormal conditions was provided by gynecologists and staff at the Indian Health Service.
Identification of HPV type
HPV genotype was identified from cervical specimens using a newly developed reverse blot method that employs a biotin-labeled polymerase chain reaction product hybridized to an array of immobilized oligonucleotide probes. Using the reverse blot "strip" test, genotype discrimination of multiple types of HPV can be accomplished in a single hybridization and wash cycle (14). The genotypes distinguished on this strip included types 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 5159, 66, and 68 (ME180), MM4 (W13B), MM7 (P291), MM8 (P155), and MM9 (PAP 238A). Two concentrations of ß-globin probes allowed for assessment of individual specimen adequacy following amplification. This method of HPV detection has shown high accuracy in comparison with other dot blot methods (15
). HPV types were classified as high risk versus low risk on the basis of previously reported cancer associations (14
, 16
). High risk types included types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 55, 56, 58, 59, 68, MM4, MM7, and MM9. Low risk types included types 6, 11, 40, 42, 53, 54, 57, 66, and MM8.
Statistical analysis
We used logistic regression analysis to model case/control status and to estimate odds ratios. We delineated case groups according to level of severity of the histologic findings (CIN I and CIN II/III). Adjusted odds ratios were obtained by including a set of adjustment factors in the model. We calculated 95 percent confidence intervals about the point estimates of effect for each of the variables. Although crude odds ratios are reported in the tables, we report only adjusted odds ratios in the text. Most of the predictor variables were structured as categorical variables; other variables were recoded into categories for ease of interpretation or to diminish the effects of outliers. Analysis of specific HPV genotypes was accomplished by comparing subjects with a specific type of HPV to subjects with no HPV infection. In our first multiple logistic regression model, we included variables that were biologically plausible and variables that were statistically significant in our initial analyses after adjustment for age, age at first sexual intercourse, and lifetime number of sex partners. The final model included variables that were biologically plausible and statistically significant in the stepwise regression analysis. We used standard SAS software for all of these analyses (version 6.12; SAS Institute, Cary, North Carolina).
The study protocol was approved by the National Indian Health Service Human Research Review Board, as well as by the appropriate Indian Health Service hospital health boards.
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RESULTS |
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Demographic data for controls and CIN II/III cases are presented in table 1. Most of the American Indian women enrolled in this study were from southwestern tribes and had full Indian blood quantum. The majority were married or living with a partner and had completed some education beyond high school. The mean age for the controls was 28.6 years, while the mean age for CIN II/III cases was 27.6 years. Women with the lowest levels of education (less than high school) and income (<$10,000/year) had significantly increased risks of CIN compared with those with the highest levels of education and income. The adjusted (for age, age at first intercourse, and lifetime number of sex partners) odds ratio for women with less than a high school education compared with those with an educational level exceeding high school was 2.1 (95 percent CI: 1.0, 4.3). Having an income of less than $10,000 per year, compared with an annual family income of $20,000 or more, was associated with CIN II/III (adjusted OR = 3.1; 95 percent CI: 1.7, 5.3).
Mean age at first intercourse was 17.4 years (median, 17; range, 532) for control subjects and 16.9 years (median, 17; range, 525) for case women with CIN II/III. Mean lifetime number of sex partners was 5.8 (median, 3; range, 1100) for control subjects and 8.7 (median, 4; range, 1222) for women with CIN II/III. Mean number of sex partners before age 20 years was 2.6 (median, 1; range, 055) for controls and 4.8 (median, 1; range, 0200) for subjects with CIN II/III. Neither lifetime number of sex partners, age at first intercourse, nor number of sex partners before age 20 was a significant risk factor for CIN II/III (table 2).
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Cigarette smoking, as measured by ever use (adjusted OR = 0.7; 95 percent CI: 0.4, 1.3) or current use (adjusted OR = 0.6; 95 percent CI: 0.3, 1.3), was not associated with CIN II/III. Levels of smoking, as measured by number of cigarettes smoked per day or pack-years of use, also were not associated with CIN (data not shown). Using regression modeling, we examined the data for a possible interaction between cigarette smoking and HPV infection among cases and controls. We did not find any interaction effects that showed increased risks for CIN.
Multiple logistic regression modeling was performed to simultaneously control for confounding variables in a final regression model (table 6). For CIN II/III, our final model included age, income, history of any sexually transmitted disease, any type of HPV, and HPV type 16. HPV type 16 emerged as the strongest risk factor for CIN II/III in the final logistic regression model (OR = 7.6; 95 percent CI: 2.4, 23.2).
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DISCUSSION |
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Previous research in other populations has documented an increased risk of cervical cancer among women of lower socioeconomic status (1719
). In our study, women with less than a high school education and an income less than $10,000 per year were at increased risk of developing CIN. In New Mexico, over 40 percent of American Indians live below the poverty level, and only 60 percent are high school graduates (20
). In a similarly designed previous study of risk factors for CIN among New Mexico Hispanics and non-Hispanic Whites, Becker et al. (21
) also found strong risks associated with low economic status and low education levels. Brinton and Fraumeni (17
) reported an inverse relation between socioeconomic indicators of income and education and risk of invasive cervical neoplasia for both Black and White women.
Several investigators have documented higher rates of sexually transmitted disease in American Indian and Alaska Native women (2224
) compared with White women in the United States. In our study, 57 percent of women reported a history of sexually transmitted disease and 62 percent had a sexually transmitted disease documented by laboratory evaluation at the time of enrollment. Our data showed that a history of any sexually transmitted disease was a significant risk factor for CIN in our study population. For individual infections/diseases, a history of genital warts for women with CIN I and histories of gonorrhea and chlamydia for women with CIN II/III were associated with disease. We did not find significant associations for histories of genital herpes and several other sexually transmitted infections (table 3). Genital herpes infection, in particular, has been implicated as an etiologic factor in the development of cervical neoplasia in research carried out over the past 25 years (25
, 26
), and the presence of serum antibodies to herpes simplex virus type 2 has been shown to be a significant risk factor for high grade dysplasia in southwestern non-Hispanic White women, even after adjustment for HPV infection and other risk factors (21
).
In our study, infection with HPV of any type was a strong risk factor for both CIN I and CIN II/III (OR = 3.2 (95 percent CI: 2.1, 4.8) and OR = 7.9 (95 percent CI: 4.7, 13.2), respectively). Davidson et al. (27) reported increasing risk of cervical cytologic abnormalities among Alaska Native women with documented HPV infection in comparison with women with no HPV infection. Our pilot study (8
) of risk factors for CIN among southwestern American Indian women also showed HPV infection (as measured by polymerase chain reaction) to be a strong risk factor for CIN (OR = 19.4; 95 percent CI: 10.1, 37.9). A small case-control study of Alaska Native women suggested an 8.4-fold increased risk of CIN among women with documented HPV infection compared with women with no HPV infection (28
), with a particularly high risk being associated with HPV type 16 (OR = 40.8; 95 percent CI: 9.4, 17.4). While the common high risk HPV genotypes were evaluated in our study population, we found several other types of HPVusually referred to as low risk typesto be strongly associated with CIN. HPV type 54 has not been reported to be associated with cervical neoplasia and is a type most commonly found on the vulva (29
). In our study, HPV type 54 was associated with a 40-fold increase in risk among subjects with CIN II/III. Only HPV type 16 had a higher risk than type 54. In a review by Wheeler (30
), HPV type 54 was not found to be prevalent in high grade dysplasia cases in studies carried out in Taiwan, Brazil, the Netherlands, Portland, Oregon, and Albuquerque, New Mexico.
Bacterial vaginosis, as assessed by the presence of clue cells upon light microscopic examination, was a risk factor for CIN in our study. Our data showed clue cells on wet mount to be associated with both CIN I and CIN II/III. However, we did not find history of bacterial vaginosis, or BV score, to be related to CIN in our study subjects. In several other studies (8, 28
) conducted in American Indian and Alaska Native populations, as well as in other non-Native populations (21
, 31
), bacterial vaginosis has not been found to be a risk factor for CIN. We are not convinced that the organisms that are associated with bacterial vaginosis and that affect the morphology of squamous epithelial cells under light microscopy (as seen with clue cells) are an important contributor to the development of cervical neoplasia in this population.
Previous research among southwestern Hispanic and non-Hispanic White women found risk factors for CIN similar to those found in our population of southwestern American Indian women (21). In addition to low income and low educational level, history of any sexually transmitted disease and infection with HPV were risk factors for CIN among southwestern Hispanic and non-Hispanic White women in New Mexico. HPV types 16/18 and 31/33/35 were strongly associated with CIN in these two ethnic groups as well. Unlike the previous study of New Mexico Hispanic and non-Hispanic White women (21
), cigarette smoking and hepatitis B virus antibodies were not significant risk factors for CIN in this American Indian population. These findings may be explained by low levels of cigarette smoking and intravenous drug use in this population.
This study had potential limitations that must be considered in interpretation of our results. Some of these limitations include the lack of blinding of the interviewers to the case/control status of the subjects and the lack of validation of interview data. In addition, recall bias is always a potential problem in case-control research. To address some of these potential problems, we used a structured interview and a standard protocol for cases and controls. We also validated a sample of nurse practitioners' study interviews through reinterviews of study subjects, and examined medical records for possible discrepancies between reported and charted information. For the few inconsistencies we found between interview and medical record data, we used charted information in the analysis. To increase the likelihood of obtaining accurate responses regarding sexual behavior, we reminded study subjects at several points in the interview about the confidential nature of all data collected.
We would have preferred random sampling of controls from tribal rosters or other Indian Health Service rosters, instead of selection of controls from the clinic patients who appeared for routine care and other reasons (table 1). Bias due to the use of a clinic-based comparison group is possible, and it may explain the lack of associations between CIN and cigarette smoking and sexual history measures; the magnitude of effect of this source of bias is not easily estimated. Another limitation was the long time period between initial abnormal Pap smear and colposcopy for the case women. Some of the potential cases became ineligible because their CIN was not confirmed at the time of biopsy. Our HPV data were limited by the collection of a single specimen at study entry; additional sampling would likely have resulted in a higher proportion of control women with HPV infection (32).
We did not collect information on the sexual behavior of male partners of the study subjects. Thus, we cannot evaluate the possible effects of male behavior as a risk factor for CIN among females. We included American Indian women from tribes outside of the Southwest (approximately 10 percent of all study subjects). Although this feature of the study is not likely to have introduced bias, the results are not strictly focused on risk factors for CIN among southwestern American Indian women. The proportion of nonsouthwestern tribal subjects was not large enough to allow broad generalizability of our findings to all American Indian groups. Lastly, we had limited statistical power related to the numbers of cases of CIN II/III in our study. However, since CIN I and CIN II/III may represent different disease processes (33), we determined that the appropriate strategy was to analyze and present the study findings stratified according to these two groups, even though study power would have been increased by combining them.
Despite these potential limitations, our data suggest that many risk factors for CIN among American Indian women are similar to those found among southwestern Hispanic and non-Hispanic White women, as assessed by a similar case-control approach in an earlier study (21). The strengths of the risks associated with sexual behavior variables, sexually transmitted disease histories, and several sexually transmitted diseases that could be identified at the time of study entry were also comparable among these racial/ethnic groups. We will continue to evaluate the roles of dietary micronutrients and human lymphocyte antigen haplotypes in an attempt to further understand etiologic factors that may influence the development of cervical disease in this population. However, we predict that HPV vaccines currently under development will be the most effective strategy for prevention of cervical neoplasia in this population.
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ACKNOWLEDGMENTS |
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The investigators thank all of the Indian Health Service personnel who helped to facilitate this study at the clinic sites. Dr. Eve Espey, Dr. Tony Ogburn, Dr. Alan Waxman, and Rose Rowan were particularly valuable to the study's success. Dr. Jill Miller carried out most of the colposcopic examinations at the Albuquerque clinical site. Drs. Gary Escudera, William Freeman, and Tim Fleming assisted the study team with protocol approvals. Drs. Jennie Joe and Linda Burhanisstapanov provided expertise in the design of the questionnaire. Dee Comiskey and Tresia Denetclaw assisted with data entry. Dawn Hamilton and Barbara Evans assisted with programming of data entry screens. George Montoya, Pat Stauber, and Dr. Phil Garry provided valuable assistance and advice with regard to the clinical nutrition specimens. Darren Schaffer, Helena Frank, and Evelyn Hood were very valuable as laboratory assistants. Gary Oty, Kerry Hatch, Linda Nims, Dr. David Mills, and the support staff of the New Mexico Department of Health, Scientific Laboratory Division, provided laboratory expertise for analyses of study specimens. The laboratory of Drs. Francis Lee and Andre Nahmias performed serum antibody assays for herpes simplex virus. The investigators recognize the valuable contributions of Fred Ettcity and Ray Rodgers of the Indian Health Service. The guidance provided by the Service Unit Health Boards of the Indian Health Service was also very helpful to the project.
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NOTES |
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REFERENCES |
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