Estrogen receptor {alpha} gene polymorphisms and endometrial cancer risk

Elisabete Weiderpass3, Ingemar Persson, Håkan Melhus1, Sara Wedrén, Andreas Kindmark1 and John A. Baron2

Department of Medical Epidemiology, Karolinska Institutet, PO Box 281, 171 77 Stockholm, Sweden,
1 Department of Internal Medicine, Akademiska Sjukhuset, Uppsala, Sweden and
2 Department of Medicine and Department of Community and Family Medicine, Dartmouth Medical School, Hanover, NH, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since the estrogen receptor {alpha} (ER) is an important mediator of hormonal responses such as proliferation in estrogen-sensitive tissues, we hypothesized that polymorphisms in the ER gene could be functional and associated with endometrial cancer risk. We performed a population-based case–control study in Sweden, focusing on restriction fragment length polymorphisms for XbaI and PvuII and an upstream TA repeat polymorphism. In the main analysis, 154 cases and 205 controls who never used hormone replacement therapy took part and we calculated age-adjusted and multivariate odds ratios (OR) and 95% confidence intervals (CI) using unconditional logistic regression. The XbaI X allele appeared to confer a reduced risk for endometrial cancer. The multivariate OR for the XX genotype was 0.52 (95% CI 0.21–1.29) compared to the xx genotype and there were suggestions of decreasing risk with increasing number of X alleles (P for trend = 0.07). The PvuII PP genotype was also associated with a non-significantly decreased risk for endometrial cancer (multivariate OR 0.70, 95% CI 0.34–1.44) compared with the pp genotype (P for trend = 0.43). The multivariate OR for two short TA (<19 repeats) alleles versus two long alleles was 1.54 (95% CI 0.73–3.27) and there were suggestions of increasing risk with increasing number of short alleles (P for trend = 0.26). We observed the same pattern of results in an expanded group of subjects, which included women who had used hormone replacement (in total 288 cases and 392 controls). Our data suggest that variants of the ER gene may be associated with an altered risk of endometrial cancer.

Abbreviations: CI, confidence interval; ER, estrogen receptor {alpha}; OR, odds ratio; RFLPs, restriction fragment length polymorphisms.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The estrogen receptor {alpha} (ER) is an important mediator of the hormonal response in estrogen-sensitive tissues such as the endometrium, breast and bone. Since ER functioning is reflected in the proliferation of these tissues, it is plausible that variation in the function of the receptor could have clinically significant effects. Polymorphisms in the ER gene, hypothetically related to biological function, have inconsistently been associated with bone density (17) but seem not to be related to breast cancer risk (812).

Endometrial cancer is the most `estrogen sensitive malignancy' in women (13). Therefore, we hypothesized that functionally significant polymorphisms in the ER gene might be associated with the risk of endometrial cancer, through regulation of estrogenic effects at the cellular level. Because alterations in estrogen signaling may be most apparent in women who have not taken menopausal estrogens, we were especially interested in associations among women who had never taken these drugs. To investigate these possibilities, we conducted a population-based case–control study in Sweden to assess if polymorphisms in the ER gene, in particular restriction fragment length polymorphisms (RFLPs) for XbaI and PvuII (2,9,10,14) and an upstream microsatellite polymorphism, a TA repeat (1,15), were associated with susceptibility to endometrial cancer.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study is based on an investigation of organochlorine serum levels and endometrial cancer, which was conducted among women who had never used hormone replacement therapy. To increase sample size for the molecular analysis presented here, the study population was enlarged to include women who had used hormone replacement.

Study population
Our study included women aged 50–74 years, resident between February 1996 and November 1997 in 12 Swedish counties on the coasts of the Gulf of Bothnia, the Baltic Sea or the largest Swedish lakes. These counties were chosen because we hypothesized that women living there would have a higher consumption of food contamined with organochlorines. Women were eligible if they were born in Sweden and had no prior hysterectomy.

Cases
We attempted to collect and store blood samples before treatment from all women with an incident endometrial cancer diagnosed between February 1996 and Novenber 1997 in the study area. These case patients were identified through a network of personnel at the 26 departments of gynecology/gynecology–oncology in the study area. (One of the departments did not collaborate.) A total of 396 cases were reported, ~95% of the number expected (16). Of these, 288 (73%) were contacted before surgery, volunteered to donate blood samples and subsequently completed the study questionnaire a few months after surgery. Forty-one patient cases refused to participate and 67 patient cases were not approached (due to failure of the medical staff to collect a blood sample before surgery). Of the enrolled cases, 154 (53.5%) had never used hormone replacement and were included in the organochlorine (and ER polymorphisms) analysis. A further 134 cases with blood samples reported using hormone replacement and were also included in the analysis of ER polymorphism.

Controls
Population controls, randomly selected from a continuously up-dated population registry, were frequency matched to cases by 5 year age groups. The period of control recruitment coincided with that of the cases, since we sampled and enrolled controls in four phases: spring 1996; fall 1996; spring 1997; fall 1997. In contrast to cases, controls were approached first for questionnaire information and subsequently asked to donate a blood sample. We did not request samples from some in the first group of control women (n = 54) because they had used hormone replacement and so were not eligible for the organochlorine analysis (see above). Subsequently we attempted to recruit all control women, whether or not they had used hormone replacement therapy, to correspond to the expected recruitment of the cases. Of 688 control women selected, 505 (73.4%) responded to the questionnaire and 438 (63.7%) also agreed to donate blood samples. After excluding 46 women because of prior hysterectomy, 392 control women were included in the study; of these, 205 (52.3%) had never used hormone replacement therapy.

The self-administered study questionnaire requested information on weight, height, reproductive history, hormone use, smoking history and medical history, among others. Missing information was supplemented by a telephone interview in ~50% of cases and controls.

The study was originally approved by The Ethics Committee, Uppsala University, and by The Ethics Committee at Karolinska Institutet, Stockholm.

Blood sampling
Blood samples from fasting case women were drawn at the hospital departments before surgery or any cancer treatment and from controls at a primary health care unit or at home. Leukocyte genomic DNA was extracted from whole blood (EDTA) according to standard procedures.

Molecular analyses
The PvuII and XbaI RFLPs are located in intron 1, only 50 bp apart, 400 bp upstream of exon 2 of the ER gene (2,9,10,14). They were analyzed as described (2) with the following exceptions: 50 pmol of each oligonucleotide primer was used in the PCR which was performed for 30 cycles at 94°C for 30 s, 62°C for 20 s and 72°C for 90 s, with a final extension for 5 min at 72°C using a GeneAmp PCR 9600 (Perkin Elmer, Norwalk, CT).

The TA repeat at –1174 bp upstream of exon 1 of the human ER gene was analyzed by PCR amplification using the oligonuclotide primer sequences described (1), but with a forward primer that was labeled with a fluorescent dye, TET (Perkin Elmer) (17). Genomic DNA (100 ng) was amplified with 10 pmol of each primer, in a total volume 50 µl containing 10x PCR buffer, 2.5 U Taq DNA polymerase, 2.5 mM MgCl2 and 0.2 mM dNTPs. There was an initial denaturation at 94°C followed by 25 cycles of 30 s at 94°C, 30 s at 55°C and 30 s at 72°C. Fluorescence-labeled PCR products were separated on a 6% polyacrylamide gel using an ABI 373 A automatic DNA sequenator (Perkin Elmer). A fluorescence-labeled size marker, GS-350 TAMRA (Perkin Elmer), was used as an internal lane standard. A GENESCAN 672 kit (Perkin Elmer) was used to quantitate fluorescence-labeled PCR products in base pairs and by amount of fluorescence. DNA fragment sizes were confirmed by automated DNA sequencing using a DNA sequencing kit (ABI Prism dye terminator cycle sequencing ready reaction kit, with Amplitaq DNA polymerase FS; Perkin Elmer) exactly according to the manufacturer's instructions.

Statistical analysis
Using standard {chi}2 statistics, we tested if the allele frequencies deviated from the Hardy–Weinberg equilibrium. Considering the polymorphic genotypes as `exposures', we calculated odds ratios (ORs) and 95% confidence intervals (CIs) using unconditional logistic regression models, fitted by maximum likelihood (18). Genotype frequencies for the PvuII and XbaI polymorphisms were compared using subjects homozygous for the most common allele as the reference. For the TA repeats in cases and controls, the distribution of allele sizes from 6 to 30 was bimodal. Therefore, we categorized genotypes to avoid making strong assumptions about the functional form of the relationship between allele size and disease status. Categories were chosen to correspond to the naturally occurring short (<19 TA repeats) and long (>=19 TA repeats) alleles as occurring in our study population (in both cases and controls). We computed ORs using subjects homozygous for long TA alleles as the reference. We estimated unadjusted (univariate) ORs for various genotypes and subsequently included in the logistic regression models variables known to be associated with endometrial cancer risk. These variables were age (<55, 55–59, 60–64, 65–69, >=70 years), menopausal status (pre- or post-menopausal), age at menopause (<50, 50–52, >=53 years), age at last birth (<30, >=30 years), nulliparity, number of births (1, 2, 3 or more), body mass index (i.e. weight in kg/(height in m)2, as a continuous variable), use of oral contraceptives (ever or never), smoking (ever or never smoked regularly), clinical history of diabetes mellitus and hypertension (self-reported) and, in the expanded subject group only, use of different hormone replacement therapy regimens (classified according to ever or never exposed to the following compounds: estrogens without progestins, estrogens with cyclic addition of progestins, estrogens with continuous addition of progestins, progestins without estrogens, oral estriol and vaginal use of estriol, dienoestrol or estradiol).

Adjustment for age did not substantially affect the crude risk estimates. However, inclusion of other covariates in the models (namely diabetes mellitus, hypertension, body mass index, menopausal status and age at menopause) did change risk estimates modestly, and both age-adjusted and multivariate models are presented. We computed tests for trend by the introduction of ordinal variables obtained by assigning consecutive integers to levels of the categorized genotype variables.

We separately present results for women who never used hormone replacement, a focus of our analysis, and results for all women (i.e. users and non-users of hormone replacement). This analytical strategy responds to our concern regarding potential selection bias introduced by the under-representation of control subjects who used hormone replacement and also to our interest in studying separately women who were unexposed to hormone replacement.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Compared to controls, cases were slightly older, had a higher age at menopause, a lower parity and a greater body mass index. Proportionally more cases than controls reported being nulliparous, having never smoked or used oral contraceptives and having a history of diabetes mellitus or hypertension. Characteristics of subjects who took hormone replacement therapy were similar to all women (data not shown). Thus, these case–control differences largely reflect known epidemiological associations (Table IGo).


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Table I. Selected characteristics of endometrial cancer patients and control women
 
From the 288 enrolled cases, 261 were genotyped for PvuII and XbaI polymorphisms and TA repeats. Of the 392 controls enrolled, 380 were successfully genotyped for PvuII and XbaI and 372 for TA repeats. None of the genotype frequency distributions deviated significantly from the Hardy–Weinberg equilibrium among cases or among controls.

PvuII polymorphisms
The age-adjusted relative risks for the genotypes Pp and PP were not substantially different from that of the reference group pp. However, after multiple adjustment, the PP genotype was associated with a non-significantly decreased risk: OR 0.70 (95% CI 0.34–1.44) (Table IIGo). A similar pattern of ORs was found in the expanded subject group. A trend of decreasing risk with increasing number of P alleles was present, but it was not statistically significant (multivariate P = 0.43 in women unexposed to hormone replacement, multivariate P = 0.25 in the expanded study group).


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Table II. Genotype distribution among endometrial cancer cases and controls of PvuII, XbaI and TA repeat polymorphisms of the ER
 
The XbaI polymorphisms
The X allele appeared to confer a reduced risk of endometrial cancer, the ORs for genotypes XX and Xx being <1.0, and risk decreased with increasing numbers of X alleles (P for trend for the multivariate ORs = 0.07). For the XX genotype the multivariate OR was 0.52 (95% CI 0.21–1.29). With the expanded dataset, this was 0.60 (95% CI 0.33–1.10, P for trend = 0.05) (Table IIGo).

Dinucleotide repeats
Among women who had never used hormone replacement, there was a tendency toward a possible allele dose effect. The multivariate OR for heterozygous long/short was 1.30 (95% CI 0.66–2.58) and for short/short 1.54 (95% CI 0.73–3.27) when compared with the long/long genotype (P for trend = 0.26). A similar pattern of results was observed in the analysis including all women (Table IIGo, P for trend = 0.09).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We found suggestive evidence of an association between ER gene polymorphisms and endometrial cancer risk. The P, X and long alleles seemed to confer decreased relative risks, especially when trends over the number of alleles were considered. Effects were similar among women who had never used hormone replacement therapy and in the broader subject population.

Our study has several strengths, including its population-based design, the relative ethnic homogeneity of the Swedish population and the availability of detailed questionnaire information which allowed us to consider potential confounding factors.

One concern is that selection bias could have occurred if non-participation was related differently to genotype among eligible cases and controls. Among cases, the principal reason for non-participation was the failure of the hospital staff to collect blood samples, a reflection of the characteristics of the medical personnel, not of the patients. However, in our expanded study population, our control recruitment procedure led to a higher participation rate among women who had never used hormone replacement than among women who used such hormones. This could have influenced our findings if ER polymorphisms were associated with use of hormone replacement therapy. However, we observed similar patterns of results whether or not we included women who had used hormone replacement therapy and conclude that substantial distortion from selection bias is unlikely in the analysis of all subjects.

Misclassification of cancer cases may have occurred. In another recently completed study in Sweden we found that ~10% of cases reported as endometrial cancer to the cancer register were reclassified as severe atypical hyperplasia after blind histopathological review. Atypical hyperplasia is considered a pre-malignant lesion and its association with hormonal exposure is stronger than the association observed for invasive endometrial lesions (19). Therefore, the possible inclusion of atypical hyperplasias in our study could have biased results towards stronger associations, if the polymorphisms are linked to increased sensitivity to estrogens. Our focus on women who had never used hormone replacement therapy, and therefore were less likely to be erroneously classified as endometrial cancers, reduces this potential problem. Misclassification of genotypes is unlikely, since we obtained allele frequencies among controls which were similar to those reported by others (15).

The confidence limits for the ORs in most analyses of our data included unity and so the findings are consistent with chance. However, two of the trends in risk over alleles were statistically significant or nearly so. Clearly our suggestive finding of weak associations needs confirmation by a study with greater statistical power to detect weak associations.

The associations between endometrial cancer risk and ER polymorphisms were nearly null before the multivariate adjustment and became more pronounced after multiple adjustment. This occurred because both ER polymorphisms and case/control status were associated with several known risk factors for endometrial cancer, i.e. diabetes mellitus, hypertension, body mass index, menopausal status and age at menopause. Our study was too small to allow separate analyses within categories of these covariates and the associations between the polymorphisms and these characteristics need to be assessed in a larger study.

Estrogen mediates cellular growth and differentiation in tissues such as the endometrium, mammary gland, bone, cardiovascular system, brain and urogenital tract in men and women (2023), with the intracellular ER functioning as a hormone-dependent transcriptional regulator (24). Polymorphisms in the ER have been studied mostly in relation to bone mass and mammary cancer. To our knowledge, no studies have previously been published on the relationship between ER PvuII, XbaI or TA repeat polymorphisms and endometrial cancer.

Reports on associations between the studied polymorphisms and bone mineral density are inconsistent. While in some studies no association between PvuII or XbaI ER genotypes and bone mineral density were reported (4,7), other studies suggest an association between the xx (3) and PP genotypes (5) and low bone mineral density. Since estrogen levels and bone mineral density are related and a high bone mineral density is associated with an increased risk of breast cancer (25,26), we believe that any estrogenic effect of ER genotypes would also be detectable on the endometrium, i.e. through changes in the risk of endometrial cancer.

The XbaI X allele has been associated with an increased risk of breast cancer in a study from Norway (11). However, the PvuII restriction site polymorphism is apparently not associated with expression of ER in breast cancer (810) and was not related to breast cancer risk in two analyses that did not consider covariates such as hormone replacement therapy and reproductive history (9,11). The study by Parl et al. (8) found the pp genotype to be related to a younger age at breast cancer diagnosis.

The ER gene loci that we are studying are not in the coding domains of the gene. However, these polymorphisms may be markers of altered cellular function in several other ways. Receptor function could be affected through differential splicing of mRNA (27,28) or alteration of transcriptional elements within introns (12). Further, it is possible that some of these polymorphisms serve as markers by being in linkage disequilibrium with other, as yet undetected, sequence alterations of functional significance for the gene (20).

In conclusion, our data provided suggestions that some endometrial cancer patients carry variants of the ER gene that may be associated with increased susceptibility to the disease.


    Acknowledgments
 
We are indebted to Prof. Hans-Olov Adami, Karolinska Institutet, for valuable help in early phases of this study and for critical review of the manuscript. We thank B.Wilén, who provided expert technical assistance. This work was supported by grants from the Swedish Cancer Society, American Cancer Society and The Chlorine Chemistry Council, USA.


    Notes
 
3 To whom correspondence should be addressed Email: elisabete.weiderpass{at}mep.ki.se Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 27, 1999; revised November 29, 1999; accepted December 6, 1999.