Human Papillomaviruses and Cervical Cancer in Bangkok. I. Risk Factors for Invasive Cervical Carcinomas with Human Papillomavirus Types 16 and 18 DNA

Transcription

1 American Journal of Epidemiology Copyright 2 by The Johns Hopkins University School of Hygiene and Public Health All rights reserved Vol. 5,. 8 Printed in U.S.A. Risk Factors for Cervical Cancers with HPV- and HPV-8 Thomas et al. ORIGINAL CONTRIBUTIONS Human Papillomaviruses and Cervical Cancer in Bangkok. I. Risk Factors for Invasive Cervical Carcinomas with Human Papillomavirus Types and 8 DNA David B. Thomas, Roberta M. Ray, Amorn Koetsawang, 2 Nancy Kiviat, Jane Kuypers, Qin Qin, Rhoda L. Ashley, 4 and Suporn Koetsawang 2 Personal interviews, tests for antibodies to herpes simplex virus type 2, Treponema pallidum, and hepatitis B, tests for hepatitis B surface antigen (HBsAg), and polymerase chain reaction-based assays for human papillomavirus (HPV) DNA in cervical scrapings were obtained from women with squamous cell and 42 women with adenomatous cervical carcinoma and from 2 hospitalized controls diagnosed in Bangkok, Thailand, between September and September. Risk was strongly associated with oncogenic HPV types, with types and 8 predominating in squamous and adenomatous lesions, respectively. The 2 cases with HPV- and the 42 cases with HPV-8 were compared with 25 controls with no evidence of any HPV. The risk of both viral tumor types increased with decreasing age at first intercourse in this predominantly monogamous population, which may be explained by more visits to prostitutes by the husbands of cases with early than late age at first intercourse. HPV- tumors were weakly associated with HBsAg carrier state and smoking. The risk of tumors of both viral types increased with parity and use of oral contraceptives but not with injectable progestogens. Factors that may predispose to persistent, oncogenic HPV- or -8 infection may include estrogens or progestins in the presence of estrogens, immunosuppression, and smoking, but other factors related to low socioeconomic status are also involved. Am J Epidemiol 2;5:2. adenocarcinoma; carcinoma, adenosquamous; carcinoma, squamous cell; cervix neoplasms; papillomavirus, human Received for publication June 28, 2, and accepted for publication December 2, 2. Abbreviations: HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HIV, human immunodeficiency virus; HPV, human papillomavirus; HSV, herpes simplex virus; IUD, intrauterine device. 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. Department of Pathology, University of Washington, Seattle, WA. 4 Department of Laboratory Medicine, University of Washington School of Medicine, Seattle, WA. Correspondence to Dr. David B. Thomas, Program in Epidemiology, Fred Hutchinson Cancer Research Center, Fairview Avenue rth, Seattle, WA 8 ( dbthomas@ fhcrc.org). In 5 a working group of the International Agency for Research on Cancer concluded that human papillomavirus (HPV) types and 8 are carcinogenic to humans, that types and are probably carcinogenic to humans, and that some other types are possibly carcinogenic to humans (). Epidemiologic evidence that these viruses are causes of invasive cervical carcinoma includes the following: the sexual mode of transmission ( 5), which provides an explanation for the sexual risk factors for cervical cancer; casecontrol studies showing strong associations of cervical cancer with specific HPV types based on demonstration of HPV DNA in cervical epithelial cells (, 8) and serologic tests for HPV- antibodies (); and historical prospective studies showing increased risks of subsequent invasive disease in women with antibodies to HPV- ( 2) and HPV-8 () and in women with evidence of HPV DNA types, 8, and others in cervical smears (, 4). In addition, a clear mechanism for the carcinogenic effect of the viruses has been elucidated; the viral genes E and E from oncogenic types of HPV bind to and inactivate the protein products of the tumor suppressor genes p5 and Rb, respectively (5, ). Although cervical carcinoma may arise in the absence of HPV (), the preponderance of evidence suggests that oncogenic types of these viruses are necessary causes of cervical carcinoma (8, ). However, many more women are infected by oncogenic types of HPV sometime during their lives than ultimately develop cervical carcinoma (), 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 ( 8, 2), including both squamous cell (, ) and adenomatous types (, ). Other cofactors that have been Downloaded from at Pennsylvania State University on March 4, 2

2 Thomas et al. 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, 2). In this first of three papers, we present results of an investigation of possible differences in the epidemiologic features of HPV-- and HPV-8-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 (2) 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 Cases eligible for these studies were women who were admitted to public wards of Siriraj Hospital in Bangkok, Thailand, with a new histologically confirmed diagnosis of either in situ or invasive cervical carcinoma between September and September, who were born in or later, and who resided for at least the past year in Thailand. Older women were not included because they would have been unlikely to have used steroid contraceptives, a possible cofactor of interest. 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 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 5 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 (2), attempts were also made to interview the husbands of currently married cases and controls. All interviewed women were requested to donate a 5-ml blood specimen. The resultant serum was stored in four aliquots at 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 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. rmal cervical tissue was similarly obtained at surgery from the control women having hysterectomies. All scrapings and tissue specimens were frozen at 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 () 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) (2). 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 (2). Antibodies to herpes simplex virus types and 2 (HSV- and HSV-2) were confirmed by Western blot assay (28, 2). 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 2 µl of the original sample was analyzed in duplicate by polymerase chain reaction amplification using two sets of primers. Consensus primers MY and MY target an approximately 45-base pair region of the HPV L gene, and PC4 and GH2 target a 28-base pair region of the human β-globin gene (). The presence Am J Epidemiol Vol. 5,. 8, 2 Downloaded from at Pennsylvania State University on March 4,

3 Risk Factors for Cervical Cancers with HPV- and HPV-8 25 of the β-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 β- globin fragment were considered unsatisfactory. After amplification, 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 and, type, type 8, type,, 5, and, 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 4 SiHa cells, an HPV type - positive cell line; DNA from K52 cells, an HPV-negative human cell line; K52 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 (). RESULTS Adenocarcinomas and adenosquamous carcinomas have been combined into a single group, hereafter referred to as adenomatous. Slides from 4 cases diagnosed in Bangkok as invasive cervical carcinoma were read again in Seattle. Of 8 cases originally diagnosed as squamous cell, (2.8 percent) were so diagnosed in Seattle, and of 2 cases diagnosed in Bangkok as adenomatous, (. percent) were similarly confirmed. Because of this satisfactory level of concordance between the two pathologists, slides from some cases were not read in Seattle, and for the purposes of this report, the diagnosis made in Bangkok was used to classify tumors as to histologic type. Of the 42 eligible cases identified, 8 (8.5 percent) were interviewed. Cervical scrapings were obtained from (. percent) of those interviewed, 2 of whom had invasive squamous and 4 of whom had invasive adenomatous cervical carcinoma. The remaining 5 cases had carcinoma in situ. The cervical samples from 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 women with invasive squamous cell and 42 women with adenomatous cervical carcinoma in the analyses for this report. Of the 4 women selected as controls, 4 (.8 percent) were interviewed. Cervical scrapings were obtained from (2.4 percent) of these 4 women, 28 (.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 2 controls in the analyses for this report. The age distributions of the cases and controls are shown in table. The controls with cervical scrapings and the 84 interviewed control women without cervical scrapings did not differ appreciably by age or number of sexual partners. A higher proportion of women with scrapings had used oral contraceptives (4.4 percent vs. 8. percent), injectable contraceptives (2. percent vs.. percent), and an intrauterine device (IUD) (. percent vs.. percent) and had had at least one cervical smear (4. percent vs. 2. percent), which would result in spuriosly low odds ratios in relation to these factors. Fewer women with scrapings had their first sexual experience before age 8 (.8 percent vs. 4.2 percent), which would result in an overestimate of the strength of the association with this variable. Results of HPV DNA assays on a sample of the tissue specimens and scrapings from the same individuals were compared. Of the paired samples from cases, seven were positive for HPV DNA type or 8 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 ) and only in the scraping. Results of assays of exfoliated cells TABLE. Age distributions of invasive squamous cell and adenomatous cervical carcinoma cases, cases with HPV-* and HPV-8* DNA, and controls, Bangkok, Thailand, Downloaded from at Pennsylvania State University on March 4, Age (years) Squamous Adenomatous HPV- HPV-8 Controls. %. %. %. %. % > Total. Am J Epidemiol Vol. 5,. 8, * HPV- and HPV-8, human papillomavirus types and 8, respectively. The mean and median ages (years) are the following: squamous cell carcinoma, 45.5 and 45.5; adenomatous cervical carcinoma, 42.4 and 42.5; HPV-, 44. and 4.; HPV-8, 44. and 44.; controls, 4. and 4., respectively

4 2 Thomas et al. were therefore the more sensitive indicators of the presence of oncogenic HPV DNA and were used in the analyses for this report. study subjects were positive for HPV type or type. As shown in table 2, percent of the scrapings from the women with squamous cell carcinomas and percent of the scrapings from the women with adenomatous carcinomas contained known oncogenic HPV DNA (types, 8,,, 5,, or 45), compared with only 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 (8 percent), 5 (8. percent), and 4 (4. percent), respectively. HPV- is most common in squamous cell tumors, and HPV-8 is the most frequent type in adenomatous carcinomas. These differences are unlikely due to chance (p <.). The two tumor types did not differ significantly in the proportions with HPV types //5/ (p.), untyped HPV DNA (p.4), any HPV type (p.), or any oncogenic type (p.52). The odds ratios for the oncogenic types are all very large with lower 5 percent confidence limits well above unity. The presence of untyped HPV DNA is not significantly associated with the risk of either squamous or adenomatous carcinoma. 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 and 8. Two cases were infected by both types and 8 and have been eliminated from these analyses, leaving 2 cases with type and 42 cases with type 8. Of the 2 cases with HPV-, had squamous and had adenomatous carcinomas, and of the 42 tumors with HPV-8, 25 were squamous and were adenomatous carcinomas. The age distributions of the women with HPV-- and HPV-8-associated tumors are shown in table. These women were compared with the 25 controls with no evidence of any types of HPV; that is, the 4 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 25 controls did not differ appreciably from that of all controls shown in table. As shown in table, the risk of both a carcinoma with HPV- and a carcinoma with HPV-8 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- whose husbands were interviewed, (58.5 percent) of women who first had sexual intercourse before age had husbands who had visited prostitutes over 8 times, compared with eight (.4 percent) of 22 women who became sexually active at a later age (chi-square.4; p.). The relation of the risk of HPV--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-8-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- in women with HSV-2 antibodies, no significant associations were observed with serologic evidence of prior infection with HSV- (not shown), genital herpes simplex, syphilis, or hepatitis B, and the associations with the sexual variables shown were generally similar for carcinomas with HPV- and -8. Only one case and no controls were HIV positive. Although quite possibly due to chance, the odds ratio of tumors with HPV- is higher in women with HBsAg than in women who have anti-hbv antibodies. The risk of both types of carcinomas increased with the number of pregnancies (table 4). This association with HPV- tumors remained after adjustment for months since last cervical smear, age at first intercourse, use of oral contraceptives, and use of an IUD. Weak positive associations with both types were found with ever having had a spontaneous abortion and having used oral contraceptives, and a TABLE 2. Age-adjusted odds ratios of invasive squamous cell and adenomatous cervical carcinoma in relation to specific types of human papillomaviruses, Bangkok, Thailand, Downloaded from at Pennsylvania State University on March 4, HPV* type Squamous (n = ) cases Adenomatous (n = 42) controls (n = 2) Squamous Adenomatous. %. %. % OR* 5% CI* OR 5% CI 8 //5/ 45 Any oncogenic type Untyped Any type , 22 22, 4., 2, 85., 4. 52,.. 4 * HPV, human papillomavirus; OR, odds ratio in relation to women without the specific HPV type; CI, confidence interval. study subjects tested positive for HPV type or. Includes types /8///5//45. Weakly reactive tests considered negative. 8., 5,.2, 44 4,.,.4 2, 25 Am J Epidemiol Vol. 5,. 8, 2

5 Risk Factors for Cervical Cancers with HPV- and HPV-8 2 TABLE. Age-adjusted odds ratios of HPV-*- and HPV-8*-associated invasive cervical carcinoma in relation to sexual factors and infectious agents, Bangkok, Thailand,. of sexual partners > Age (years) at first intercourse HSV-2* antibodies Syphilis serology HBsAg*, Factor/agent Anti-HBs antibodies*, Anti-HBc antibodies*, HPV Cases HPV-8 Controls (no HPV*) slight reduction in the risk of both types of tumors in users of depot-medroxyprogesterone acetate for contraception was seen, although all of these possible associations could readily have occurred by chance. trends of increasing risk with duration of use of oral contraceptives were observed (not shown). The weak association between HPV- -associated tumors and oral contraceptive use was not further attenuated by controlling for months since the last cervical smear, age at first sexual intercourse, and number of pregnancies. ne of the odds ratios for any of the hormonal variables shown differed significantly for tumors associated with HPV types and 8. The risks of both HPV- and HPV-8 tumor types were reduced in users of an IUD and in women who had had cervical smears, had ever had a chest radiographic examination, and had attended school (table 5). The reduced risk in relation to cervical smears was greatest in women whose last smear was within the past year. These relations with the HPV- tumor type were not altered by controlling for the other variables in the table or by the use of oral contraceptives, number of pregnancies, or age at first intercourse. significant associations with tubal ligation or alcohol consumption were observed. Cigarette smoking was weakly associated with HPV type tumors only, but the number of smokers in this study population was too small to allow control for other variables or for the absence of smokers among women with HPV-8 tumors to be very meaningful. With this possible exception, the tumors with HPV- and HPV- 8 did not differ appreciably by any of the factors shown. DISCUSSION HPV- OR* 5% CI* OR 5% CI ,. 2.,..,.., 4..8,., 2.5.,.5.8, 4..,.8., , 5.5.,..,., 8.2.,.4.5,..4, 5.8.,..5, 2..4,. * HPV- and HPV-8, human papillomavirus types and 8, respectively; HPV, human papillomavirus; OR, odds ratio; CI, confidence interval; HSV-2, herpes simplex virus type 2; HBsAg, hepatitis B surface antigen; anti-hbs antibodies, anti-hepatitis B surface antibodies; anti-hbc antibodies, anti-hepatitis B core antibodies. Two cases with both HPV- and HPV-8 were omitted from analyses. Study subjects with no serologic test results were omitted from analyses. HPV-8 As with previous studies (8), oncogenic types of HPV DNA were demonstrated in a high proportion of the cases, with HPV- and HPV-8 predominating in squamous cell and adenomatous carcinomas, respectively, and with other oncogenic types (i.e.,,, 5,, and 45) occurring in smaller and similar proportions of the tumors of both histologic types. Use of additional probes would not have altered these results, since only 5 cases and controls were found to have untyped HPV DNA, and some of these are probably nononcogenic types since they occurred nearly as frequently in controls as in cases. The high proportion of tumors with HPV DNA supports the hypothesis that HPV infection is Downloaded from at Pennsylvania State University on March 4, Am J Epidemiol Vol. 5,. 8, 2

6 28 Thomas et al. TABLE 4. Age-adjusted odds ratios of HPV-*- and HPV-8*-associated invasive cervical carcinoma in relation to hormonal factors, Bangkok, Thailand, Factor. of pregnancies Ever had stillbirth Ever had spontaneous abortion Ever had induced abortion Ever used oral contraceptives Ever used DMPA* HPV Cases HPV-8 Controls (no HPV*) HPV- OR* 5% CI* OR 5% CI , 2..,..5, 5..8,..5,.8., 2.2.,..8, 2..5, ,.., 8.2.2, 2.2., 4.5., 4..,.., 2..,. * HPV- and HPV-8, human papillomavirus types and 8, respectively; HPV, human papillomavirus; OR, odds ratio; CI, confidence interval; DMPA, depot-medroxyprogesterone acetate. p value of test for trend <.. p value of test for trend =.. Analyses restricted to parous women. HPV-8 necessary for the development of cervical carcinoma. In accordance with prior investigations (,, 8), the odds ratios for both squamous and adenomatous carcinomas, in relation to HPV types, 8, and //5/, were very large. Moreover, in accordance with prior studies (2 ), the epidemiologic features of squamous and adenomatous carcinomas were similar, justifying our decision to combine the two types in the analyses. Although having more than one sexual partner was a risk factor for both HPV- and HPV-8 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 (2, ). 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. for tumors with HPV- 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- infection and cervical carcinoma. An association was also observed between the prevalence of HPV- and HBsAg in prostitutes in Bangkok (2). The absence of appreciable associations of either HPV- or HPV-8 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 (, 8). The increase in risk of both HPV- and HPV-8 tumor types with number of pregnancies observed in this study is consistent with evidence from some (, 8), although not other (, 2), recent investigations and suggests that hormonal changes associated with pregnancy may enhance the likelihood that an infection with either HPV- or HPV-8 will lead to cervical cancer. Similarly, most previous studies (8), including all those that have compared cases and controls with oncogenic HPV types ( 8, 2), 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 Downloaded from at Pennsylvania State University on March 4, Am J Epidemiol Vol. 5,. 8, 2

7 Risk Factors for Cervical Cancers with HPV- and HPV-8 2 TABLE 5. Age-adjusted odds ratios of HPV-*- and HPV-8*-associated invasive cervical carcinoma in relation to nonsexual and nonhormonal factors, Bangkok, Thailand, Ever used an IUD* Tubal ligation Factor Months since last cervical smear cervical smear 2 >2 Unknown months Ever smoked cigarettes Ever drank alcoholic beverages Ever had chest radiographic examination Ever attended school HPV Cases HPV-8 Controls (no HPV*) HPV- and HPV-8 will result in carcinogenesis. The absence of associations with the progestational contraceptive depot-medroxyprogesterone acetate in this study and a prior study () 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 (4) and enhancement of in vitro oncogenic transformation by HPV- in the presence of progestins from oral contraceptives (4). The consistently observed relation of cervical cancer to some measure of socioeconomic status among studies in which cases were compared with HPV-infected controls ( 8, 2) 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- and HPV-8. The low level of smoking in the women in this study suggests HPV- OR* 5% CI* OR 5% CI ,..5,.2.,.4.,.8.,.,.8.,..4,., 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-, the viral type that predominates in squamous cell carcinomas, but not with HPV-8, which is the main virus type in adenomatous tumors, although possibly due to chance, is consistent with observations by others (2,, 4) 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- and HPV-8 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- and HPV-8 DNA provides strong evidence that the same cofactors operate to enhance the carcinogenicity of these two viral types. The preponderance of HPV- in squamous cell carcinomas and the more frequent occurrence of HPV-8 in adenomatous carcinomas are thus a reflection ,.2., 2..,..,.8., 2..8, 2..,..2,.5 * HPV- and HPV-8, human papillomavirus types and 8, respectively; HPV, human papillomavirus; OR, odds ratio; CI, confidence interval; IUD, intrauterine device. One case with unknown smoking history was excluded. HPV-8 Downloaded from at Pennsylvania State University on March 4, Am J Epidemiol Vol. 5,. 8, 2

8 Thomas et al. of inherent differences in the viruses themselves. HPV- is the more common of the two viruses in exfoliated cells from both cervical and penile scrapings (), perhaps because it is better adapted to survival in squamous epithelial tissue than is HPV-8. HPV-8 may be more dependent on reaching the glandular epithelium of the endocervical canal for survival. ACKNOWLEDGMENTS This study was supported by grant CA444 from the US National Institutes of Health. The assistance of Drs. Anna Marie Beckmann and Larry Corey is gratefully acknowledged. REFERENCES. International Agency for Research on Cancer. Human papillomaviruses. IARC Monogr Eval Carcinog Risks Hum 5;4: Ho GY, Bierman R, Beardsley L, et al. Natural history of cervicovaginal papillomavirus infection in young women. N Engl J Med 8;8: Svare EI, Kjaer SK, Worm AM, et al. Risk factors for HPV infection in women from sexually transmitted disease clinics: comparison between two areas with different cervical cancer incidence. Int J Cancer 8;5: Young TK, McNicol P, Beauvais J. Factors associated with human papillomavirus infection detected by polymerase chain reaction among urban Canadian aboriginal and non-aboriginal women. Sex Transm Dis ;: Kjaer SK, van den Brule AJ, Bock JE, et al. Determinants for genital human papillomavirus (HPV) infection in randomly chosen young Danish women with normal Pap smear: are there different risk profiles for oncogenic and nononcogenic HPV types? Cancer Epidemiol Biomarkers Prev ;: 85.. Chichareon S, Herrero R, Muñoz N, et al. Risk factors for cervical cancer in Thailand: a case-control study. J Natl Cancer Inst 8;:5.. Ngelangel C, Muñoz N, Bosch FX, et al. Causes of cervical cancer in the Philippines: a case-control study. J Natl Cancer Inst 8;:4. 8. Chaouk N, Bosch FX, Muñoz N, et al. The viral origin of cervical cancer in Rabat, Morocco. Int J Cancer 8;5: Ferrera A, Baay MF, Herbrink P, et al. A sero-epidemiological study of the relationship between sexually transmitted agents and cervical cancer in Honduras. Int J Cancer ;:8 5.. Dillner J, Lehtinen M, Bjorge T, et al. Prospective seroepidemiologic study of human papillomavirus infection as a risk factor for invasive cervical cancer. J Natl Cancer Inst ;8: 2.. Lehtinen M, Dillner J, Knekt P, et al. Serologically diagnosed infection with human papillomavirus type and risk for subsequent development of cervical carcinoma: nested casecontrol study. BMJ ;2:5. 2. Shah KV, Viscidi RP, Alberg AJ, et al. Antibodies to human papillomavirus and subsequent in situ or invasive cancer of the cervix. Cancer Epidemiol Biomarkers Prev ;:2.. Cavalcanti SM, Deus FC, Zardo LG, et al. Human papillomavirus infection and cervical cancer in Brazil: a retrospective study. Mem Inst Oswaldo Cruz ;: Chua KL, Hjerpe A. Persistence of human papillomavirus (HPV) infections preceding cervical carcinoma. Cancer ;:2. 5. Lowy DR, Schiller JT. Human papillomavirus biology. J Natl Cancer Inst Monogr ;2:4.. Pillai MR, Halabi S, McKalip A, et al. The presence of human papillomavirus-/-8 E, p5, and Bcl-2 protein in cervicovaginal smears from patients with invasive cervical cancer. Cancer Epidemiol Biomarkers Prev ;5:2 5.. Scheffner M, Munger K, Byrne JC, et al. The state of the p5 and retinoblastoma genes in human cervical carcinoma cell lines. Proc Natl Acad Sci U S A ;88: Bosch FX, Manos MM, Muñoz N, et al. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. J Natl Cancer Inst 5;8: 82.. Franco EL, Rohan TE, Villa LL. Epidemiologic evidence and human papillomavirus infection as a necessary cause of cervical cancer. J Natl Cancer Inst ;:5. 2. Bosch FX, Muñoz N, de Sanjosé S, et al. Risk factors for cervical cancer in Colombia and Spain. Int J Cancer 2;52: Wheeler CM, Greer CE, Becker TM, et al. Short-term fluctuations in the detection of cervical human papillomavirus DNA. Obstet Gynecol ;88: Thomas DB, Qin Q, Kuypers J, et al. Human papillomaviruses and cervical cancer in Bangkok. II. Risk factors for in situ and invasive squamous cell cervical carcinomas. Am J Epidemiol 2;5:2. 2. Thomas DB, Ray RM, Kuypers J, et al. Human papillomaviruses and cervical cancer in Bangkok. III. The role of husbands and commercial sex workers. Am J Epidemiol 2; 5:4 8.. Histological typing of female genital tract tumours. In: Poulsen HE, Taylor CW, Sobin LH, eds. International histological classification of tumours. Geneva, Switzerland: World Health Organization, Sarngadharan MG, Popovic M, Bruch L, et al. Antibodies reactive with human T-lymphotropic retroviruses (HTLV-III) in the serum of patients with AIDS. Science 84;2: Hollinger FB, Dienstag JL. Hepatitis B and D viruses. In: Murray PR, Baron EJ, Pfaller MA, et al, eds. Manual of clinical microbiology. th ed. Washington, DC: American Society for Microbiology, : Larsen SA, Hunter EF, Kraus SJ, eds. A manual of tests for syphilis. 8th ed. Washington, DC: American Public Health Association, : Ashley RL, Militoni J. Use of densitometric analysis for interpreting HSV serologies based on Western blot. J Virol Methods 8;8: Ashley RL, Militoni J, Lee F, et al. Comparison of Western blot (Immunoblot) and glycoprotein G-specific immunodot enzyme assay for detecting antibodies to herpes simplex virus types and 2 in human sera. J Clin Microbiol 88;2:2.. Bauer HM, Ting Y, Greer CE, et al. Genital human papillomavirus infection in female university students as determined by a PCR-based method. JAMA ;25:42.. Breslow NE, Day NE, eds. Statistical methods in cancer research. Vol I. The analysis of case-control studies. Lyon, France: International Agency for Research on Cancer, 8:25 8. (IARC scientific publication no. 2). 2. Honore LH, Koch M, Brown LB. Comparison of oral contraceptive use in women with adenocarcinoma and squamous cell carcinoma of the uterine cervix. Gynecol Obstet Invest ; 2:8.. Hopkins MP, Morley GW. A comparison of adenocarcinoma and squamous cell carcinoma of the cervix. Obstet Gynecol ;:2. 4. Brinton LA, Herrero R, Reeves WC, et al. Risk factors for cervical cancer by histology. Gynecol Oncol ;5:. 5. Brinton LA, Tashima KT, Lehman HF, et al. Epidemiology of cervical cancer by cell type. Cancer Res 8;4:.. Bjorge T, Kravdal O. Reproductive variables and risk of uterine cervical cancer in rwegian registry data. Cancer Causes Control ;:5.. Thomas DB, Ray RM, Pardthaisong T, et al. Prostitution, condom use, and invasive squamous cell cervical cancer in Thailand. Am J Epidemiol ;4: 8. Am J Epidemiol Vol. 5,. 8, 2 Downloaded from at Pennsylvania State University on March 4,

9 Risk Factors for Cervical Cancers with HPV- and HPV-8 8. International Agency for Research on Cancer. Hormonal contraception and post-menopausal hormonal therapy. IARC Monogr Eval Carcinog Risks Hum ;2:.. The WHO Collaborative Study of Neoplasia and Steroid Contraceptives. Depot-medroxyprogesterone acetate (DMPA) and risk of invasive squamous cell cervical cancer. Contraception 2;45: Thomas DB, Ray RM, WHO Collaborative Study of Neoplasia and Steroid Contraceptives. Oral contraceptives and invasive adenocarcinomas and adenosquamous carcinomas of the uterine cervix. Am J Epidemiol ;44: Pater A, Bayatpur M, Pater MM. Oncogenic transformation by human papilloma viruses type desoxyribonucleic acid in the presence of progesterone or progestins from oral contraceptives. Am J Obstet Gynecol ;2:. 42. International Agency for Research on Cancer. Cancer incidence in five continents. Vol VII. Lyon, France: International Agency for Research on Cancer, :44. (IARC scientific publication no. 4). 4. Ursin G, Pike MC, Preston-Martin S, et al. Sexual, reproductive, and other risk factors for adenocarcinoma of the cervix: results from a population-based case-control study (California, United States). Cancer Causes Control ;: Li HQ, Thomas DB, Jin SK, et al. Tubal sterilization and use of an IUD and risk of cervical cancer. J Womens Health Gend Based Med 2;:. Downloaded from at Pennsylvania State University on March 4, Am J Epidemiol Vol. 5,. 8, 2