Co-factors related to the causal relationship between human papillomavirus and invasive cervical cancer in Honduras

Annabelle Ferreraa, Johan P Velemab, Manuel Figueroaa, Ricardo Bulnesc, Luis A Toroc, José M Clarosc, Odessa de Barahonad and Willem JG Melcherse

a Department of Microbiology, Universidad Nacional Autónoma de Honduras, Tegucigalpa, Honduras.
b Department of Public Health, Erasmus University, Rotterdam, The Netherlands.
c Department of Oncology and Gynecology, Hospital General San Felipe, Tegucigalpa, Honduras.
d Department of Pathology, Instituto Nacional del Tórax, Tegucigalpa, Honduras.
e Department of Medical Microbiology, University of Nijmegen, The Netherlands.

Reprint requests to: Annabelle Ferrera, PO Box 30078, Tegucigalpa, Honduras. E-mail: anabelle{at}gbm.hn


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background A case-control study was conducted in Honduras to identify co-factors in the carcinogenic pathway by which human papillomavirus (HPV) causes invasive cervical cancer.

Methods Ninety-nine cases aged 23–65 (median 47) years participated. Two controls were matched to each case by age and clinic where they first presented for cytological screening; controls had no cervical abnormalities. Information on risk factors was obtained by personal interviews in the clinics regarding sociodemographic, reproductive and behavourial characteristics. Human papillomavirus was detected in cervical scrapes by general primer-mediated polymerase chain reaction (PCR) followed by sequence analysis to identify the different types present.

Results All cases had squamous cell tumours and most were FIGO (International Federation of Gynecologists and Obstetricians) class II or higher; HPV was strongly associated with cervical cancer (odds ratio [OR] = 7.66, 95% CI : 3.88–15.1). Among HPV-positive women, dose-response relationships were observed for education, age at first intercourse and exposure to wood smoke that persisted after adjustment for previous screening. Among HPV-negative women, the number of sexual partners and parity were associated with cervical cancer. The protective effect of previous cytological screening operated independently of HPV.

Conclusions Our findings speak for the powerful role that both primary and secondary education plays in fostering a lifestyle that reduces the risk of invasive cervical cancer. The data suggest that important elements of such a lifestyle include later age at first sexual intercourse, a limited number of pregnancies, greater likelihood of undergoing cytological screening and reduced exposure to carcinogens in the household environment.

Keywords Human papillomavirus, cervical cancer, risk factors

Accepted 24 March 2000


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The identification of human papillomavirus (HPV) as the cause of cervical cancer1 has given new direction to research on this neoplasia of global importance. Problems of HPV detection and quantification of the viral load, as well as prophylactic and/or therapeutic vaccine development are being addressed.2–5 In addition, known risk factors for cervical cancer are being evaluated to establish whether they are still relevant or have become redundant, as so often happens once the causal agent of a disease has been identified. As Schiffman stated,6 HPV-infection is the unifying, central risk factor for cervical neoplasia throughout the world, but the necessary co-factors for carcinogenesis could vary considerably among different geographical regions, and should therefore be investigated.

Questions in this area have, among others, concerned (1) the effect of early age at first sexual intercourse which is hypothesized to lead to HPV-infection at a time when cells of the cervical epithelium are more susceptible to its effect,7,8 (2) the effect of high parity, which facilitates the carcinogenic process through hormonal and/or immunological changes or increases susceptibility to HPV-infection through the effects of trauma to the uterus during delivery9–11 and (3) the effect of oral contraceptives which might exercise a promoting effect once mutations in the DNA of cervical cells have begun to appear or facilitate the establishment of chronic HPV-infection after exposure.8,10 The role of other carcinogens and protective factors, such as cigarette smoke and dietary factors,12,13 is as yet not well established or adequately understood.

The present case-control study of invasive cervical cancer was undertaken to evaluate the role of known risk factors in residents of Tegucigalpa, Honduras, and to investigate a possible risk factor of local importance: the use of wood as fuel in the kitchen—a still highly prevalent practice among women of low socioeconomic status in Honduras.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Study population
Recruitment of study subjects was conducted from May 1993 to December 1995. Women participating in the study derived from public hospitals, communal health care centres, family planning and gynaecology clinics which provided cytological screening of the cervix and served the population of Tegucigalpa, the capital city of Honduras. Women with cervical lesions were referred or came directly to San Felipe General Hospital where they were enrolled into the study. In total 10 clinics within the city and five within 150 km provided cervical cancer cases. The majority of participating clinics were in the public sector serving people of modest financial means.

Eligible cases were women 20–65 years, with histologically verified invasive cancer, who had had no previous treatment for the disease, and were identified as being resident for at least 6 months in Tegucigalpa or within a 2-hour perimeter (150 km) of the city by car. Women who presented an impaired mental condition inadequate for an interview were excluded.

Controls, two per case whenever possible, were matched to cases according to age (±5 years) and clinic. They had a normal cervix (i.e. a negative Pap smear or inflammatory changes only), did not have a history of hysterectomy or conization and were recruited within one month from the date of case diagnosis. If a case had been referred because of abnormal findings during cervical screening, interviewers went to the clinic where original screening took place. They selected all possible controls from the list of women waiting to be screened at the time of their visit and randomly selected two for participation in the study. Controls whose cytology turned out to be positive were retained in the study as cases if they met the criteria.

Data collection
Trained women interviewers, one nurse and one social worker, conducted a standard personal interview at the hospital or clinic from where the controls were recruited, and at the San Felipe General Hospital for cases. All women invited to participate gave informed consent. Interviews focused on socio-demographic factors, education, reproductive and sexual history, contraceptive use, smoking history, previous cervical smear history and exposure to wood smoke while cooking. Women answered three questions about the use of wood as fuel in the kitchen: whether this was a habit in their childhood home, how many years they used wood in their own kitchen, and how many years since they had used wood. The number of years of exposure to wood smoke after childhood was estimated as the number of years lived after age 14 minus the number of years since the use of wood had been abandoned. Since virtually all women had been exposed to wood smoke during childhood, this information was not taken into account. All women who were not exposed during childhood did not use wood in the kitchen as adults or used it only for a very short time and were, therefore, classified in the low exposure category.

Detection of human papillomavirus-DNA
The detection techniques for HPV-DNA have been described in detail elsewhere.14 Cervical scrapes were taken from the transformation zone of all cases and controls and DNA extracted according to the standard SDS-proteinase K-phenol-chloroform method.14 Amplification of a 110-bp region of the beta-globin gene15 was used to verify sample integrity.

We assessed HPV-DNA status by a general primer-mediated polymerase chain reaction (PCR) using HPV-L1 consensus primers16 which amplify a fragment of about 450 base-pairs of many HPV types as well as unidentified HPV (HPV X). Negative (distilled water) and positive (CaSki cells) controls were included. All HPV-DNA that amplified with the consensus primers were PCR sequenced using the Perkin Elmer AmpliCycle sequencing kit (Roche Molecular Systems, Inc., Branchburg, New Jersey). Sequenced HPV-DNA fragments were compared with the GenBank to determine the HPV type.

Statistical analysis
Ninety-nine cases with squamous cervical carcinoma and 197 controls were included in the analysis. Five cases with adenocarcinomas were excluded. Approximately five women who met the criteria for enrolment were not included for logistic reasons, e.g. inability to recruit controls soon enough or rapid deterioration in the condition of the case.

Matched odds ratios (OR) and 95% CI were calculated by means of conditional logistic regression.17 Most variables were categorical. Dummy variables were used to indicate categories and the significance of adding this set of dummy variables to the model was compared to the significance of adding a linear variable to the model which simply numbered categories as 1, 2, 3, etc. The likelihood ratio test was used, which for a single linear variable represents a test for trend and has a {chi}2 distribution with one degree of freedom. If comparison of the goodness-of-fit of models with a linear variable and with dummy variables suggested departure from linear trend, significance was assessed by adding the full set of dummy variables to the model. In this situation the likelihood ratio test has a {chi}2 distribution with the number of degrees of freedom equal to the number of categories minus one. Unless otherwise indicated in the Tables, {chi}2 statistics were of the former type with one degree of freedom.

Splitting the dataset into HPV-positives and -negatives drastically reduced the number of matched sets with at least one case and one control in the same HPV category. To make maximum use of the information available, unconditional logistic regression was used for the separate analyses of HPV-positives and -negatives. Since conditioning on clinic and age was no longer possible, these variables were now included in all logistic models. For this purpose, clinics were categorized as follows: the hospital of the medical school (38 cases), the San Felipe hospital (26 cases), other clinics within the city (16 cases) and clinics outside the city (19 cases).

Parity was computed as the sum of completed pregnancies and abortions, whether spontaneous or induced. There was a suspicion that women included one or both of the screening events related to their enrolment into the study when reporting the number of previous cytological screens. In the analysis, the number of previous screens were therefore reduced by one if the most recent event was reported to have taken place within 12 months prior to the interview.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The mean age (±SE) of both cases and controls was similar, being 45.8 ± 0.95 and 45.2 ± 0.98, respectively. The youngest case was 23 years and seven cases were younger than 30 years. At the time of diagnosis, five cases were classified according to the International Federation of Gynecologists and Obstetricians (FIGO) criteria in class I, 35 in class II, 52 in class III and 3 women in class IV; the information was missing for 4 women. There were no substantial differences between cases and controls with regard to region of birth, income or home ownership. Seventy per cent of women reported monthly family incomes of <US$50 and not one exceeded US$375. However, 73% of women reported that their family owned the home they lived in. Among controls, 23% of women had never been to school and only 17% had continued beyond primary school. Only three women went to university but none completed it. All women were of Mestizo descent (an admixture of the Caucasian, Black and Oriental gene-pool), except one woman who was black.

Univariate analysis
In matched univariate analysis (Table 1Go), HPV-DNA was significantly more often identified among cases (80%) than among controls (36%) suggesting a high risk of cervical cancer (OR = 7.66, 95% CI : 3.88–15.1).


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Table 1 Risk factors and associated odds ratios (OR) for invasive cervical cancer in Tegucigalpa, Honduras
 
The number of lifetime sex partners varied little. Forty-one per cent of controls and 26% of cases were monogamous and 56% of cases reported two or three lifetime sex partners; only four women reported more than five partners. Women who reported more than three partners had a threefold increase in risk compared to monogamous women.

Fifty per cent of control women had begun sexual activity between the ages of 16 and 19 years; 24% had their first sexual intercourse earlier and 26% at age 20 or later with a maximum of 38 years. Cases had more often started sexual activity at an earlier age resulting in a statistically significant association: when age at first intercourse was entered into the logistic model as a continuous variable, the risk was reduced by 11% for every year that sexual activity was postponed. The goodness-of-fit between a model using categories and a model using the continuous variable was not significantly different.

The number of pregnancies varied from one to 17. Sixty-five per cent of controls and 83% of cases had more than four pregnancies. Again, a linear dose-response relationship was observed. The risk increased by 13% for each successive pregnancy.

Of cases, 41% had their first pregnancy before age 17 while 42% of controls had the first pregnancy at aged at least 20. The OR was 0.53 for a first pregnancy at 20+ years compared to women who had their first pregnancy before the age of 17. Risk was reduced by 7.6% for every year that the first pregnancy was delayed.

Only 4% of cases had continued education beyond primary school versus 17% of controls. The matched OR for women who had completed secondary education compared to women without any formal education was 0.08 (95% CI : 0.02–0.30).

Virtually all women had grown up in a home where wood was used as fuel in the kitchen. The number of years that a woman had been exposed to wood smoke in the kitchen after the age of 14 years was higher among cases than controls. For 67% of cases and 50% of controls, exposure had continued for at least 25 years. The OR associated with >=35 years of exposure was 6.35 (95% CI : 2.1–19.2). The estimated regression coefficient indicated that the OR increased by 6.3% for every year of exposure to wood smoke.

In the present study, only 25 women were current smokers while 27 were ex-smokers; the remaining 82% of study subjects had never smoked and this percentage hardly differed between cases and controls.

Of controls, 92% had undergone cytological screening at least 12 months prior to the interview; 53 women (28%) reported 10 or more occasions where they were screened. In sharp contrast, 49% of cases had never been screened before they were diagnosed. As a result, even women in the lowest exposure group, i.e. screened 1–4 times previously, reduced their risk of invasive cervical cancer to only 14% of the risk of women who had never been screened.

Bivariate analysis
The dataset was split according to the presence or absence of HPV-DNA in the cervical scrapes. The distribution of cases and controls and risk estimates on relevant identified risk factors, adjusted for age and clinic, are presented in Table 2Go.


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Table 2 Risk factors for invasive carcinoma of the cervix among human papillomavirus (HPV)-positive and HPV-negative women in Tegucigalpa, Honduras
 
It is interesting to note that the number of sexual partners was no longer a determinant of cervical cancer risk among HPV-positives although the association remained among HPV-negatives. By contrast, the age at which women began sexual activity was not associated with cervical cancer risk among HPV-negative women but continued to be a risk factor among HPV-positives after adjusting for age, centre and previous screening: the risk being only one quarter for women who had first sexual intercourse aged >=20.

High parity continued to be associated with risk; the effect was stronger among HPV-negatives but a significant departure from linear trend was observed in this group. A later age at first pregnancy was associated with a reduced risk of borderline significance among HPV-positives but there was no evidence of a similar association among HPV-negatives. Age at first intercourse and age at first pregnancy were strongly associated.

The protective effect of previous cytological screening operated independently of HPV status and remained significant in both groups.

Education was strongly associated with risk among HPV-positives, but the observed risk reduction of 56% among HPV-negatives was not statistically significant.

A dose-response relationship was observed for use of wood in the kitchen regardless of whether HPV was present or absent; the risk increased more steeply with increasing exposure among HPV-positives, however.

Since previous cytological screening was the most import-ant determinant of cervical cancer risk after HPV-status, the associations of all variables in Table 2Go were recalculated after adjustment for previous screening. This demonstrated that the findings described above for indicators of sexual and reproductive behaviour and education did not materially change with the exception of parity, for which the association with cancer risk among HPV-positives was no longer statistically significant.

The risk associated with exposure to wood smoke among HPV-positives was not confounded by any of the variables in Table 2Go except previous screening. Adjustment for screening resulted in a non-significant association among HPV-negatives and a still borderline significant association among HPV-positives.

Contraceptives
The risk of cervical cancer associated with contraceptive practices is presented in Table 3Go. Of women in the control group, some 61% had ever used any kind of contraceptive method. Of controls, 44% had used oral contraceptives and 21% had ever used an intra-uterine device (IUD). Injections and condoms had not been used by most women. A diaphragm had been used by only one woman. Women in the case group had used every one of these methods less frequently than controls and the difference was statistically significant for the ones that were most frequently used. However, adjusting these associations for the number of previous cytological screens reduced the respective protective effects to insignificance.


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Table 3 Association of ever/never contraceptive use and invasive carcinoma of the cervix in Tegucigalpa, Honduras
 
In Table 4Go a re-analysis of Table 1Go is presented restricted to cases (45) and controls (58) who had at least one but not more than nine previous cytological screens. Women who had never been screened and women who had been screened 10 times or more were excluded from this analysis.


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Table 4 Re-analyses of risk factors from Table 1Goa
 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
This is the first case-control study of cervical cancer ever conducted in Honduras. It has for the first time generated data on the occurrence of HPV among women of low socioeconomic status in an urban and peri-urban area in this country. Our findings speak for the powerful role that both primary and secondary education plays in stimulating women to adopt a lifestyle that reduces their risk of invasive cervical cancer. The data suggest that important elements of such a lifestyle include later age at first sexual intercourse, a limited number of pregnancies, greater likelihood of undergoing cytological screening and reduced exposure to carcinogens in the household environment.

This suggestion is strengthened by the observation among controls of associations between education and other variables. Women with at least primary education were more likely to initiate sexual relationships at a later age (P = 0.0001) and to have fewer pregnancies (P = 0.0021); they were also exposed to wood smoke during fewer years (P = 0.0001). The positive correlation between education and previous screening was not statistically significant (P = 0.105). There was no relationship between education and number of sexual partners (P = 0.547).

It is conceivable that a number of false-negatives were included in the 20 cases of cervical cancer in whom no HPV-DNA was detected. This may be due in part to the difficulty of sampling cells from women who are in advanced stages of the disease. In the present study, all HPV-negative cases were in FIGO class II or higher. This would explain the association of the number of sexual partners with cancer risk among HPV-negatives, acting as a surrogate measure of HPV-infection.7

In the present study, later age at first sexual intercourse had a significant protective effect, particularly among HPV-positives, as was also observed by others.18,19 This effect was independent of previous cytological screening. It has been suggested that the epithelial cells of the cervix are more susceptible to HPV-infection at an early age.7,8 Bosch et al.10 point out that the effect of early pregnancy may be more important than the effect of early HPV-infection as such. Age at first intercourse among HPV-positives in the present study was more strongly associated with cancer risk than age at first pregnancy or parity.

We observed a strong association with parity in the present study, but the association was stronger among HPV-negatives than among HPV-positives, contrary to findings in Morocco8 and Brazil.20 A negative association of parity with number of previous Pap smears in the present study caused the association to disappear among HPV-positive women after adjustment for previous screening.

The control group consisted of women who had come to a clinic for cytological screening. In the absence of a national screening programme, physicians will often refer women for screening in the context of monitoring the use of contraceptives, as has been observed in other Latin American countries.21 As a consequence, a high proportion of control women had ever used some form of contraception and had been screened several times before entering the study. This was not true of most cases, most of whom were symptomatic as is strongly suggested by the fact that all but five out of 99 cases were in FIGO class II or higher at the time of diagnosis. To eliminate this bias, in Table 4Go the analysis was repeated for only those women having one to nine previous cytological screens. This excluded women who had never been screened (49% cases, 8% controls) and women who had been screened 10 times or more (5% cases, 28% controls) i.e. the categories where the imbalance between cases and controls was most marked. Under these conditions, presence of HPV was again the predominant risk factor. Education continued to confer a protective effect and the number of sex partners remained significantly associated with cervical cancer risk. The OR for successive levels of exposure to wood smoke were in the same direction as before, though no longer statistically significant. Women with more than four pregnancies were again at a significantly higher risk of cervical cancer, but the dose-response relationship was no longer evident. Associations with age at first intercourse and age at first pregnancy were no longer observable.

Thus, most of the associations were in the same direction as before although sometimes no longer significant due to the sharp drop in statistical power after exclusion of 54% of the cases. After adjustment for the presence or absence of HPV-infection, education and high parity (5+ versus 1–4 pregnancies) continued to be independent determinants of risk, with education showing the stronger association (data not shown). Restricting the dataset to HPV-positive women with 1–9 previous screens, the OR for exposure to wood smoke increased with increasing exposure from 2.4 to 4.9 and the likelihood ratio test was 2.89 (P = 0.089).

Indoor air pollution, in particular through the combustion of fossil fuels such as coal, wood and straw, has been related to lung cancer22,23 and to cancers of the head and neck.24,25 During the combustion of wood, carbon monoxide, particulates, sulphur and nitrogen oxides, and mixtures of polycyclic aromatic hydrocarbons (PAH) are released; most PAH have been classified by the World Health Organization as possibly carcinogenic to humans while three have been classified as probably carcinogenic to humans—mainly on the basis of experimental data.26

To our knowledge, the use of wood for cooking fuel has not been linked to cervical cancer before. In the present study, the OR was 3.67 among women with 25–34 years of exposure to wood smoke and 6.35 among women who were exposed for >=35 years. The association was even stronger among HPV-infected women and remained significant even after adjustment for education. This fact strongly suggests that exposure to wood smoke is not just an indicator of socioeconomic status, but inclines us to believe that there could be a biological effect. Given the differential probability of cases and controls to have undergone previous cytological screening—as explained above—it is not surprising that adjustment for cytological screening reduced this association to borderline significance in this relatively small study (P = 0.089). The present data suggest that the use of wood for cooking is a strong risk factor, particularly among women with HPV-infection. The latter suggests that its effect is dependent on HPV, perhaps favouring the development of a tumour once HPV has created the necessary conditions for a carcinogenic process to occur.

It is plausible that inhalation of wood smoke over many years could have an effect on the progression of malignant tumours of the cervical epithelium, similar to what has been observed for the inhalation of cigarette smoke.27,28 However, the possibility that the association of wood smoke and cervical cancer is in fact due to other factors cannot be excluded. The use of wood in the kitchen brings other possibly relevant exposures such as to ashes, charred wood and the soot which abundantly covers the walls of many kitchens in which wood is used. Unfortunately, we were unable to investigate the possible confounding effect of dietary factors which likely play a role in the aetiology of cervical cancer,29–31 but we know that such adjustment did not explain the association between the use of woodstoves and cancer of the head and neck observed in Brazil. Although not conclusive, the present evidence, coupled with the high prevalence of the use of wood for cooking all over Latin America, is important enough to warrant further investigation.

In conclusion, the present study confirms the known association of HPV with invasive cervical cancer in Honduras and demonstrates the importance of education as a means of fostering a healthy lifestyle in this population of low socioeconomic status. Regular cytological screening, later age at first sexual intercourse, a limited number of pregnancies and reduction in exposure to carcinogens in the household environment are relevant elements of such a lifestyle identified in the present study.


    Acknowledgments
 
This study was funded by Grant CI1*-CT92-0003 from the Commission of the European Community. JPV received support from the ‘Vereniging Trust Fonds Erasmus Universiteit Rotterdam’ in the Netherlands. The authors wish to thank Mrs L Diaz and Mrs D Maradiaga for their vital role in the collection of the data and Dr FX Bosch for helpful comments during various stages of the work.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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