1 Program in Epidemiology, Fred Hutchinson Cancer Research Center, Seattle, WA.
2 Faculty of Medicine, Mahidol University, Department of Obstetrics and Gynecology, Siriraj Hospital, and Siriraj Family Planning Research Center, Bangkok, Thailand.
3 Department of Pathology, University of Washington, Seattle, WA.
4 Department of Laboratory Medicine, University of Washington School of Medicine, Seattle, WA.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
adenocarcinoma; carcinoma, adenosquamous; carcinoma, squamous cell; cervix neoplasms; papillomavirus, human
Abbreviations: HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HIV, human immunodeficiency virus; HPV, human papillomavirus; HSV, herpes simplex virus; IUD, intrauterine device
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although cervical carcinoma may arise in the absence of HPV (17), the preponderance of evidence suggests that oncogenic types of these viruses are necessary causes of cervical carcinoma (18
, 19
). However, many more women are infected by oncogenic types of HPV sometime during their lives than ultimately develop cervical carcinoma (1
), suggesting an important role for cofactors. Case-control studies have consistently shown, in HPV-positive women, associations of various measures of socioeconomic status and use of oral contraceptives with risk of invasive cervical carcinoma (6
8
, 20
), including both squamous cell (6
, 7
) and adenomatous types (6
, 7
). Other cofactors that have been investigated have only inconsistently been associated with risk of invasive disease, possibly because of variations in the sensitivity of the polymerase chain reaction-based assays used to detect oncogenic HPV DNA in normal exfoliated cells of controls, variations in expression of HPV in controls due to variation in the prevalence of factors that alter this expression, and differences in the proportion of positive assays that represent persistent and transient infections (2
, 21
).
In this first of three papers, we present results of an investigation of possible differences in the epidemiologic features of HPV-16- and HPV-18-associated carcinomas. To maximize the chances of identifying variables associated with any phase of the carcinogenic process, cases were compared with controls with no evidence of HPV in cervical scrapings. In the second paper (22), we present results of an attempt to identify cofactors associated with progression from in situ to invasive disease. The third paper (23
) addresses the role of male sexual behavior in the transmission of oncogenic types of HPV, the role of commercial sex workers as reservoirs of these viruses, risk factors for HPV infection in this heavily exposed group, and the role of the human immunodeficiency virus (HIV) and other variables as possible cofactors with HPV in the early stages of the carcinogenic process.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An attempt was made to select two controls for each woman with invasive disease from one otolaryngology and two general surgery wards. Wards were visited on a rotating basis to select the first two women who were admitted during the previous 24 hours, who were in the same 5-year age group, and who resided in the same region of the country as the corresponding case. Women admitted for treatment of conditions that have been associated with the use of steroid contraceptives, including circulatory or cardiovascular diseases, diabetes, chronic renal disease, benign breast disease, a previously diagnosed cancer, chronic liver disease, or any obstetric or gynecologic condition, were not selected, although women with a history of these conditions were eligible. In most instances, if an eligible woman refused to participate, the selection process was continued until two controls were found for each case. For the first 50 cases of invasive cervical cancer, one control woman admitted for a hysterectomy for a noncancerous condition (hysterectomy control) was also selected and matched to the corresponding case on 5-year age group and region of residence.
All cases and controls were interviewed in the hospital to obtain information on sexual and contraceptive practices, reproductive history, prior cervical smears, use of tobacco and alcohol, and indices of socioeconomic status and use of medical services. As detailed elsewhere (23), attempts were also made to interview the husbands of currently married cases and controls. All interviewed women were requested to donate a 15-ml blood specimen. The resultant serum was stored in four aliquots at -70°C; two aliquots were retained locally, and two were shipped to Seattle, Washington, on dry ice.
Cervical scrapings for HPV DNA assays were obtained from the hysterectomy controls and the cases prior to surgery or radiotherapy by the patient's surgeon. 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 the swab was broken off into a vial containing 2 ml of specimen transport medium (Digene Diagnostics, Inc., Beltsville, Maryland) and frozen at -70°C. Tumor tissue specimens were also obtained from each case at the time of surgery (hysterectomy or conization) or by punch biopsy prior to radiation treatment for women with inoperable disease. Normal cervical tissue was similarly obtained at surgery from the control women having hysterectomies. All scrapings and tissue specimens were frozen at -70°C and shipped to Seattle on dry ice.
The same surgeons who treated the cases took cervical smears and cervical scrapings for HPV DNA assays from the control women while they were hospitalized. The cervical smears were read locally, and any woman found to have a high-grade cervical intraepithelial lesion was considered ineligible for inclusion in this study as a control.
Histologic slides from the blocks that were used to make the diagnosis in the cases and comparable sections from two blocks from the hysterectomy controls were stained with hematoxylin and eosin and read by a collaborating pathologist in Bangkok who also provided information on the source of the specimen, tumor size, and stage. Invasive carcinomas were coded according to the World Health Organization's "Histological Typing of Female Genital Tract Tumours" (24) as squamous cell carcinoma, adenocarcinoma, or adenosquamous carcinoma.
The enzyme-linked immunosorbent assay procedure was used to test for antibodies to HIV, and those that tested positive were confirmed by Western blot (25). All samples were assayed for anti-hepatitis B core antibodies, anti-hepatitis B surface antibody, and hepatitis B surface antigen (HBsAg) (26
). The standard venereal disease research laboratory test for syphilis was performed on all samples, and those that tested positive were confirmed using the microhemagglutination assay for antibodies to Treponema pallidum (27
). Antibodies to herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) were confirmed by Western blot assay (28
, 29
).
The samples for HPV DNA assays were digested with proteinase K, and the DNA was precipitated by ethanol and suspended in Tris-ethylenediaminetetraacetic acid (EDTA) buffer. The DNA from 20 µl of the original sample was analyzed in duplicate by polymerase chain reaction amplification using two sets of primers. Consensus primers MY09 and MY11 target an approximately 450-base pair region of the HPV L1 gene, and PC04 and GH20 target a 268-base pair region of the human ß-globin gene (30). The presence of the ß-globin fragment on ethidium bromide-stained gels after electrophoresis of the polymerase chain reaction amplicons was used to assess whether the samples contained adequate DNA and whether polymerase chain reaction inhibitors were present. Samples negative for the ß- globin fragment were considered unsatisfactory. After amplification, 3 µl of each amplicon were spotted onto nylon filters and hybridized with a biotin-labeled generic HPV probe and with five mixtures of biotin-labeled, type-specific oligonucleotide probes for HPV types 6 and 11, type 16, type 18, type 31, 33, 35, and 39, and type 45. Samples hybridizing with the generic probe but not with any of the type-specific probes were considered positive for unclassified HPV.
Each polymerase chain reaction run included the following controls: DNA from 400 SiHa cells, an HPV type 16-positive cell line; DNA from K562 cells, an HPV-negative human cell line; K562 cells in specimen transport medium, handled concurrently with the patient samples; and a reagent blank (no DNA). All negative controls were negative for HPV DNA by polymerase chain reaction.
For this report, women with invasive disease were compared with the hospital controls, excluding the hysterectomy controls. Odds ratios, as estimates of relative risks, were calculated using unconditional logistic regression models (31).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Of the 420 eligible cases identified, 338 (80.5 percent) were interviewed. Cervical scrapings were obtained from 337 (99.7 percent) of those interviewed, 216 of whom had invasive squamous and 46 of whom had invasive adenomatous cervical carcinoma. The remaining 75 cases had carcinoma in situ. The cervical samples from 191 and 42 of the invasive squamous and adenomatous cases, respectively, contained adequate amounts of DNA for assay. One woman who claimed no history of sexual intercourse was omitted from the analysis, leaving 190 women with invasive squamous cell and 42 women with adenomatous cervical carcinoma in the analyses for this report.
Of the 614 women selected as controls, 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 samples of DNA. Three additional women with no history of sexual intercourse, one woman with a subtotal hysterectomy, and three with ineligible diagnoses were subsequently excluded, leaving 291 controls in the analyses for this report. The age distributions of the cases and controls are shown in table 1.
|
Results of HPV DNA assays on a sample of the tissue specimens and scrapings from the same individuals were compared. Of the 30 paired samples from cases, seven were positive for HPV DNA type 16 or 18 in scrapings but negative in the corresponding tumor tissue, whereas none that tested negative in scrapings were positive in tumor tissue. Of 45 tested hysterectomy controls with normal cervixes, only one tested positive for oncogenic HPV DNA (type 16) and only in the scraping. Results of assays of exfoliated cells were therefore the more sensitive indicators of the presence of oncogenic HPV DNA and were used in the analyses for this report.
No study subjects were positive for HPV type 6 or type 11. As shown in table 2, 79 percent of the scrapings from the women with squamous cell carcinomas and 76 percent of the scrapings from the women with adenomatous carcinomas contained known oncogenic HPV DNA (types 16, 18, 31, 33, 35, 39, or 45), compared with only 3 percent of the scrapings from the controls. Additional samples contained untyped HPV DNA, and those with strong reactions in the generic probe are shown in the sixth line of the table. If weakly reactive tests in the generic probe are considered positive, then the numbers (and percentages) of women with squamous tumors, adenomatous tumors, and no tumors (controls) with any type of HPV DNA are 169 (89 percent), 35 (83.3 percent), and 41 (14.1 percent), respectively.
|
No other risk factor considered in this investigation was found to significantly distinguish adenomatous and squamous cell carcinomas (not shown), so the two histologic types were combined for the purpose of comparing risk factors for tumors with HPV types 16 and 18. Two cases were infected by both types 16 and 18 and have been eliminated from these analyses, leaving 126 cases with type 16 and 42 cases with type 18. Of the 126 cases with HPV-16, 113 had squamous and 13 had adenomatous carcinomas, and of the 42 tumors with HPV-18, 25 were squamous and 17 were adenomatous carcinomas. The age distributions of the women with HPV-16- and HPV-18-associated tumors are shown in table 1. These women were compared with the 250 controls with no evidence of any types of HPV; that is, the 41 controls with either any type of HPV DNA or a weakly reactive test in the generic probe were omitted from the analyses. The age distribution of these 250 controls did not differ appreciably from that of all controls shown in table 1.
As shown in table 3, the risk of both a carcinoma with HPV-16 and a carcinoma with HPV-18 was weakly associated with having more than one sexual partner. The risk also tended to increase with decreasing age at first intercourse. However, of the cases with HPV-16 whose husbands were interviewed, 10 (58.5 percent) of 17 women who first had sexual intercourse before age 19 had husbands who had visited prostitutes over 80 times, compared with eight (36.4 percent) of 22 women who became sexually active at a later age (chi-square = 1.4; p = 0.24). The relation of the risk of HPV-16-associated tumors to age at first intercourse was not altered by controlling for months since the last cervical smear, number of pregnancies, use of oral contraceptives, and use of an IUD (not shown). Similar adjustments could not be made in the analyses of the HPV-18-associated tumors because of the small numbers of cases. The odds ratios adjusted only for age are therefore presented in this and subsequent tables to provide comparable estimates for both tumor types. With the possible exception of an increase in risk of a carcinoma with HPV-16 in women with HSV-2 antibodies, no significant associations were observed with serologic evidence of prior infection with HSV-1 (not shown), genital herpes simplex, syphilis, or hepatitis B, and the associations with the sexual variables shown were generally similar for carcinomas with HPV-16 and -18. Only one case and no controls were HIV positive. Although quite possibly due to chance, the odds ratio of tumors with HPV-16 is higher in women with HBsAg than in women who have anti-HBV antibodies.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although having more than one sexual partner was a risk factor for both HPV-16 and HPV-18 tumor types, the association was not strong and few women were not monogamous. These observations are consistent with the demonstrated importance of the husband's behavior in the development of cervical cancer in this population (23, 37
). Although the risk of both tumor types was increased in women with an early age at first sexual intercourse, such women tended to be married to men who frequently visited prostitutes, which provides an alternative to the hypothesis that HPV infection at an early age is more likely to lead to persistence of infection and the development of cervical carcinoma than infection later in life.
Although the odds ratio of 1.9 for tumors with HPV-16 in women who are positive for HBsAg could readily be due to chance, it is higher than that in women who developed HBV antibodies, suggesting a role for relative immunoincompetence in the genesis of persistent HPV-16 infection and cervical carcinoma. An association was also observed between the prevalence of HPV-16 and HBsAg in prostitutes in Bangkok (23).
The absence of appreciable associations of either HPV-16 or HPV-18 tumor type with antibodies to HSV-2, syphilis, and hepatitis B suggests that these serologic tests are not good indices of behavior that leads to acquisition or persistence of HPV infection and that these organisms do not act as cofactors in the genesis of cervical cancer. Consistent with these conclusions, associations with various sexual practices and sexually transmitted agents have only inconsistently been observed among studies based on HPV-positive cases and controls (6, 8
).
The increase in risk of both HPV-16 and HPV-18 tumor types with number of pregnancies observed in this study is consistent with evidence from some (7, 8
), although not other (6
, 20
), recent investigations and suggests that hormonal changes associated with pregnancy may enhance the likelihood that an infection with either HPV-16 or HPV-18 will lead to cervical cancer. Similarly, most previous studies (38
), including all those that have compared cases and controls with oncogenic HPV types (6
8
, 20
), have shown the risk of cervical carcinoma to be weakly associated with the use of oral contraceptives, and the modest associations observed in this study suggest that the use of oral contraceptives may enhance the likelihood that infection with HPV-16 and HPV-18 will result in carcinogenesis. The absence of associations with the progestational contraceptive depot-medroxyprogesterone acetate in this study and a prior study (39
) suggests that the possible effect of oral contraceptives and pregnancies on risk is due to the action of either estrogens or progestins in the presence of estrogens on the cervical epithelium. A role for progestins in the presence of estrogens is suggested by prior observations of a stronger association of adenomatous cervical carcinomas with combined oral contraceptives of high than low progestin potency (40
) and enhancement of in vitro oncogenic transformation by HPV-16 in the presence of progestins from oral contraceptives (41
).
The consistently observed relation of cervical cancer to some measure of socioeconomic status among studies in which cases were compared with HPV-infected controls (68
, 20
) suggests that other unidentified cofactors associated with social class remain to be identified. The association of risk with absence of schooling and prior chest radiographic examinations in this study provides evidence that such cofactors interact with both HPV-16 and HPV-18. The low level of smoking in the women in this study suggests that other factors related to socioeconomic status must be operating. Although smoking does not play an important role in the genesis of cervical cancer in Thai women, a group at high risk of cervical cancer (42
), the association of smoking with HPV-16, the viral type that predominates in squamous cell carcinomas, but not with HPV-18, which is the main virus type in adenomatous tumors, although possibly due to chance, is consistent with observations by others (32
, 33
, 43
) that smoking is related to squamous, but not adenomatous, carcinomas.
As expected, cervical smear screening was shown in this study to reduce the risk of both HPV-16 and HPV-18 tumor types. The reduced risk in women who had used an IUD likely represents an effect of screening at the time of IUD insertion or removal, a phenomenon also reported from China (44).
The similarity in risk factors for cervical carcinomas with HPV-16 and HPV-18 DNA provides strong evidence that the same cofactors operate to enhance the carcinogenicity of these two viral types. The preponderance of HPV-16 in squamous cell carcinomas and the more frequent occurrence of HPV-18 in adenomatous carcinomas are thus a reflection of inherent differences in the viruses themselves. HPV-16 is the more common of the two viruses in exfoliated cells from both cervical and penile scrapings (1), perhaps because it is better adapted to survival in squamous epithelial tissue than is HPV-18. HPV-18 may be more dependent on reaching the glandular epithelium of the endocervical canal for survival.
![]() |
ACKNOWLEDGMENTS |
---|
The assistance of Drs. Anna Marie Beckmann and Larry Corey is gratefully acknowledged.
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|