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Carcinogenesis Advance Access originally published online on September 6, 2006
Carcinogenesis 2007 28(2):427-434; doi:10.1093/carcin/bgl170
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Peroxisome profilerator-activated receptor{gamma}2 Pro12Ala, interaction with alcohol intake and NSAID use, in relation to risk of breast cancer in a prospective study of Danes

Ulla Vogel1,2,*, Jane Christensen3, Bjørn A Nexø4, Håkan Wallin1, Søren Friis3 and Anne Tjønneland3

1 National Institute of Occupational Health, Copenhagen Denmark
2 Institute of Science, Systems and Models, Roskilde University Roskilde
3 Institute of Cancer Epidemiology, Danish Cancer Society Copenhagen, Denmark
4 Institute of Human Genetics, University of Aarhus Aarhus, Denmark

*To whom correspondence should be addressed. Tel: +45 3916 5227; Fax: +45 3916 5201; Email: ubv{at}ami.dk


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Use of non-steroid anti-inflammatory drugs (NSAIDs) has been associated with decreased risk of breast cancer in epidemiological studies. Thus, a high-inflammatory response may be associated with increased breast cancer risk. It is thus possible that genetic variations leading to a modified inflammatory response will influence breast cancer risk. The purpose of this study was to determine if polymorphisms associated with an altered inflammatory response are associated with breast cancer risk, and to investigate the possible interaction with lifestyle factors such as alcohol use, smoking and NSAID use. We matched 361 female breast cancer cases with 361 controls, nested within the prospective ‘Diet, Cancer and Health’ study. Carriers of the variant Ala-allele of PPAR{gamma}2 Pro12Ala were at lower risk of breast cancer (IRR = 0.67, 95% CI = 0.46–0.97). This was primarily due to an interaction with alcohol consumption. Alcohol consumption was associated with a 1.21-fold increased risk of breast cancer per 10 g alcohol/day (95% CI = 1.06–1.35) among homozygous wild-type carriers, whereas alcohol was not associated with breast cancer risk among variant allele carriers (P for interaction = 0.005). Non-users of NSAID, who were carriers of the variant allele of PPAR{gamma}2 Pro12Ala, were at lower risk of breast cancer (IRR = 0.44; 95% CI = 0.26–0.73) compared with non-users carrying wild-type alleles (P for interaction = 0.03). No effects were found for IL6 G-174C, IL8 T-251A and COX2 T8473C. Our results suggest that PPAR{gamma}2 Pro12Ala is an important determinant in alcohol mediated breast carcinogenesis. We also observe interaction between NSAID, alcohol consumption and PPAR{gamma}2 Pro12Ala genotype in relation to breast cancer risk.

Abbreviations: BMI, body mass index; COX, cyclooxygenase; HRT, hormone replacement therapy; IRR, incidence rate ratios; NSAIDs, non-steroid anti-inflammatory drugs; PPAR{gamma}, peroxisome profilerator-activated receptor {gamma}


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The incidence of breast cancer is high in the Western world. In Denmark, 1 in 10 women will develop breast cancer before the age of 75. The established risk factors for breast cancer are mainly endocrine and reproductive, but lifestyle factors, such as alcohol consumption and NSAID use, also modulate breast cancer risk. Oxidative stress is another suggested risk factor for breast cancer (1–6). Inflammation is one of the sources of oxidative stress. Several functional polymorphisms have been identified in genes involved in the inflammatory response. It is thus possible that genetic variations, which lead to a modified inflammatory response, will modify breast cancer risk. Moreover, there may be gene–environment interactions between polymorphisms on onehand and lifestyle factors on the other hand, which both modulate the inflammatory response. Such interactions would provide evidence that inflammation and oxidative stress contribute to breast cancer risk and give more insight into the mechanism behind breast cancer carcinogenesis by identifying genes and lifestyle factors that are involved in the process.

Epidemiological studies have consistently shown that alcohol consumption is associated with an increased risk of breast cancer (7–10). One alcoholic drink a day is associated with a 1.1-fold increased risk of breast cancer (95% CI = 1.1–1.2) (10). Alcohol intake has been shown to alter the cytokine level in several tissues as well as in vitro (11,12).

Epidemiological evidence have suggested that use of non-steroid anti-inflammatory drugs (NSAIDs) is associated with a decreased risk of breast cancer (13,14), indicating that a high- inflammatory response may increase breast cancer risk. Both animal and human in vitro studies indicate that Cyclooxygenase-2 (COX-2) inhibitors and other NSAIDs have chemopreventive effects against breast cancer (15), but the epidemiological evidence is now conflicting (15,16). The majority of case–control studies have found NSAID use to be associated with a lower risk of breast cancer (13,14,16), but results from cohort studies, including four recent large population-based studies, have yielded largely null results (17–20). In one cohort study, use of non-aspirin NSAID (ibuprofen) was associated with an increased risk of breast cancer (20).

Smoking has not been previously been regarded a risk factor for breast cancer, but recent epidemiological studies suggest that smoking may indeed be a risk factor for breast cancer (21,22). Smoking leads to increased IL-8 production in stimulated blood cells, indicating that smoking has systemic effects on an inflammatory response (23).

A number of polymorphisms modulate an inflammatory response. The IL6 G-174C promoter polymorphism affects the transcription of the gene (24,25) with the G-allele (reading in the sense direction) having a higher transcriptional level than the C-allele (25). Landi et al. (26) found that carriers of the variant C-allele were at 1.53-fold (95% CI = 1.12–2.09) higher risk of colorectal cancer than homozygous carriers of the G-allele (26), whereas no association with risk of lung cancer was found in a study by Campa et al. (27).

The IL-8 T-251A promoter polymorphism has been shown to modify IL8 production in vitro (28), with the variant A-allele associated with increased IL8 response. In the study by Landi et al. (26) carriers of the variant A-allele were at lower risk of colorectal cancer than homozygous carriers of the wild-type T-allele, whereas no association was found with risk of lung cancer in Campa et al.'s study (27).

The gene PPAR{gamma} encodes the peroxisome profilerator-activated receptor {gamma}, which is a transcription factor and member of the nuclear hormone receptor superfamily. A polymorphism in the PPAR{gamma}2 isoform results in a Pro to Ala amino acid substitution at codon 12 which is present in the PPAR{gamma}2 isoform only, because this transcript is the only one to contain exon B (29). PPAR{gamma}2 is primarily expressed in adipose tissue. PPAR{gamma} regulates the expression of many adipose-specific genes via binding of a heterodimer of PPAR{gamma} and RXR to regulatory response elements (PPRE) in target gene promoters (29). In vitro studies have shown that the PPAR{gamma}2 12Ala gives less transcriptional activation of target genes (30). In human studies, the variant Ala allele of PPAR{gamma}2 Pro12Ala polymorphism has rather consistently been associated with a lowered risk of developing metabolic syndrome and improved insulin sensitivity (29). Carriers of the variant allele were found to have half the risk of colorectal cancer than homozygous carriers of the wild-type allele (26).

The COX2 T8473C polymorphism in the 3'-UTR region of the transcript has been associated with lung cancer risk. However, while carriers of the variant allele were found to be at higher risk of lung cancer in one study (27), variant carriers were found to be at decreased risk in another study (31).

In the present study we wanted to study the association of polymorphisms in genes involved in the inflammatory response and risk of breast cancer and how these polymorphisms interact with life style factors which modulate inflammation. The chosen lifestyle factors were alcohol, smoking and use of NSAIDs. We performed a case–control study nested in the prospective ‘Diet, Cancer and Health cohort’.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Subjects
The case–control study of breast cancer has been described previously (32–34). The subjects were selected from the Danish ‘Diet, Cancer and Health’ study, an ongoing prospective cohort study (35). Between December 1993 and May 1997, 79 729 women aged 50 to 64 years, born in Denmark, living in the Copenhagen and Aarhus areas and having no previous cancers at the time of invitation, were invited to participate in the study. A total of 29 875 women accepted the invitation.

Of the initial 29 875 women, a total of 326 women were excluded from the study because they were diagnosed with a cancer prior to enrolment. Further 4844 women who were not post-menopausal at study entry and 8 women who did not fill in the lifestyle questionnaire were excluded. A total of 24 697 post-menopausal women were eligible for analysis.

Cohort members were followed up for diagnosis of breast cancer from date of entry until either the date of diagnosis of any cancer (except for non-melanoma skin cancer) using record linkage to the Danish Cancer Registry, date of death, date of emigration or December 31, 2000, whichever came first.

A total of 434 women were diagnosed with breast cancer during the follow-up period. For each case with diagnosed breast cancer, one matched control was selected. The control was cancer-free at the exact age at diagnosis of the case and was further matched on age at inclusion into the cohort (half-year intervals), on use of hormone replacement therapy (HRT) (current/former/never) and on certainty of post-menopausal status (known/probably post-menopausal) at inclusion into the cohort. Known post-menopausal women were either (i) non-hysterectomized and reporting no menstruation during 12 months before inclusion, (ii) reporting bilateral oophorectomy, or (iii) reporting age at last menstruation lower than age at hysterectomy. Probably post-menopausal women were either (i) reporting menstruation during the 12 months prior to inclusion and current use of HRT (we assumed the bleeding was caused by HRT), (ii) reporting hysterectomy with a unilateral oophorectomy or an oophorectomy of unknown laterality, or (iii) reporting last menstruation at the same age as age of hysterectomy. In this nested case–control study, 13 pairs were excluded due to lack of blood samples or failure, 33 pairs due to missing information on confounders and 27 pairs due to one or more missing genotypes, leaving 361 pairs for study.

‘Diet, Cancer and Health’ and the present sub-study were approved by the regional Ethical Committees on Human Studies in Copenhagen and Aarhus [jr.nr.(KF)11-037/01] and [jr.nr.(KF)01-045/93], and by the Danish Data Protection Agency.

Blood sampling and storage
From non-fasting participants a total of 30 ml blood was collected in citrated (2 x 10 ml) and plain (1 x 10 ml) venojects from each participant. Plasma, serum, lymphocytes and erythrocytes were isolated and frozen at –20°C within 2 h. At the end of the day of collection, all samples were stored in liquid nitrogen, at –150°C.

Genotyping
DNA was isolated from frozen lymphocytes as described by Miller et al. (36). Generally, 100 µg DNA was obtained from 107 lymphocytes and 20 ng of DNA was genotyped in 5 µl (microliter) containing 1x Mastermix (Applied Biosystems, Nærum, Denmark), 100 nM probes, and 900 nM primers. Controls were included in each run, and repeated genotyping of a random 10% subset yielded 100% identical genotypes.

IL8 T-251A (rs4073) and COX2 C8473T (rs5275) were genotyped as described previously (27).

For IL6 G-174C (rs1800795) primers and probes were: primers: 5'-AGC TGC ACT TTT CCC CCT AGT -3' and 5'-TTG GAA ACC TTA TTA AGA TTG TGC AA-3'. Probes were: G-allele: 5'-FAM- TGT CTT GCG ATG CTA AAG GAC GTC-BHQ-1.3', C-allele: 5'-Yakima Yellow-TGT CTT GCC ATG CTA AAG GAC GTC-BHQ-1-3'.

For PPAR{gamma}2 Pro12Ala (rs1801282) primers and probes were: primers: 5'-GTT ATG GGT GAA ACT CTG GGA GAT -3', 5'-TGT TTG CAG ACA GTG TAT CAG TGA A-3', probes: G-allele: 5'-FAM- CTC CTA TTG ACG CAG AAA GCG ATT C-BHQ-1.3', C-allele: 5'-Yakima Yellow-TCC TAT TGA CCC AGA AAG CGA TTC C-BHQ-1-3'.

Data on alcohol and NSAID
Data on alcohol has been described earlier (8,34).

In the food-frequency questionnaire, alcohol intake was recorded as the average frequency of intake of six types of alcoholic beverage over the preceding year: the frequency of consumption of three types of beer was recorded in bottles (330 ml), wine in glasses (125 ml), fortified wine in drinks (60 ml) and spirits in drinks (30 ml). The predefined responses were in 12 categories, ranging from ‘never’ to ‘≥8 times a day’. The alcohol content was calculated as follows: one bottle of light beer, 8.9 g ethanol; one bottle of regular beer, 12.2 g ethanol; one bottle of strong beer, 17.5 g ethanol; one glass of wine, 12.2 g ethanol; one drink of fortified wine, 9.3 g ethanol; and one drink of spirits, 9.9 g ethanol. We did not differentiate between red and white wine.

Information on drinking patterns was obtained from the lifestyle questionnaire, on which subjects were asked about the frequency of alcohol drinking occasions in the following categories: never, less than once per month, 1–3 times per month, once a week, 2–4 times per week, 5–6 times per week, and daily. Abstainers were defined as those who reported no intake of alcohol on the food-frequency questionnaire and no drinking occasions on the lifestyle questionnaire.

The lifestyle questionnaire included this question regarding use of NSAID: Have you taken more than one pain relieving pill per month during the last year? If the answer was yes, the participant was asked to record how frequent they took each of the following medications: ‘aspirin’, ‘paracetamol’, ‘ibuprofen’, or ‘other pain relievers’. The latter category included NSAID preparations other than aspirin and ibuprofen. Based on all records, we classified study subjects according to use of ‘any NSAID’ (≥2 pills per month during one year) at baseline.

Statistical methods
The analyses of the association between the exposure variables and breast cancer incidence rate ratios (IRR) were based on a conditional logistic regression analysis corresponding to a Cox Proportional Hazard model in view of the study design used (37). Age was used as the time axis, because of the perfect matching on age at cancer diagnosis and ensured that the estimation procedure was based on comparison of individuals of the same age to prevent confounding by age.

All models were adjusted for baseline values of established risk factors for breast cancer such as parity (entered as two variables; parous/nulliparous and number of births), age at first birth, length of school education (low, medium and high), the duration of HRT, body mass index (BMI) and alcohol intake.

All quantitative variables were entered linearly in the model. This is biologically more reasonable than the step functions corresponding to categorization and furthermore increases the power of the analyses (38). The linearity of the associations was evaluated graphically by linear splines with three boundaries placed at the quartiles (25th, 50th and 75th) of intake among cases (39). We found no significant departures from linearity.

For the different genes we investigated different interactions with alcohol intake and NSAID use, using the likelihood ratio test.

Two-sided 95% confidence intervals (CI) for the IRRs were calculated on the basis of a Wald's test of the Cox regression parameter, that is, on the log rate ratio scale. The procedure PHREG in SAS (release 8.2; SAS Institute Inc., Cary, NC, USA) was used for the statistical analyses.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Baseline characteristics of women diagnosed with breast cancer and their matched controls are presented in Table I as medians and 5 and 95% percentiles. Findings regarding all the included risk factors except for the effect of NSAID use have been reported previously both for the whole Diet Cancer and Health cohort (8,40–42) as well as for the presently used nested case–control study (34). Ever-use of NSAID was associated with an increased risk of breast cancer that was not statistically significant.


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  		Table I Baseline characteristics of study participants selected from the Danish ‘Diet, Cancer and Health’ prospective cohort study

 
The genotype distribution of all the polymorphisms was in Hardy–Weinberg equilibrium among the controls. No association between genotype and risk of breast cancer was found for IL6 G-174C and IL8 T-251A (Table II). Adjustment for parity, number of births, age at first birth, education, the duration of HRT, BMI and alcohol consumption had only minor effect on the risk estimates.


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  		Table II IRR for breast cancer in relation to the studied genotypes

 
Both heterozygous carriers and homozygous carriers of the variant Ala-allele of PPAR{gamma}2 Pro12Ala were at lower risk of breast cancer (0.66, 95% CI = 0.45–0.96 and 0.81, 95% CI = 0.29–2.19, respectively). Carriers of the variant allele of PPAR{gamma}2 Pro12Ala were at lower risk of breast cancer than homozygous carriers of the wild-type allele (0.67, 95% CI = 0.46–0.97). The polymorphism COX2 T8473C was not associated with breast cancer risk.

Alcohol intake (per 10 g/day) was associated with 1.20-fold (95% CI = 1.04–1.40) increased risk of breast cancer among heterozygous carriers of IL6 G-174C, but not in the two other genotype groups (Table III). There was no statistically significant interaction between alcohol intake and genotype. The effect of alcohol intake was not influenced by IL8 T-251A or COX2 T8473C.


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  		Table III IRR for breast cancer per 10 g alcohol/day for different genotypes

 
Among homozygous carriers of the wild-type Pro-allele of PPAR{gamma}2 Pro12Ala, alcohol intake was associated with 1.20-fold (95% CI = 1.06–1.35) increased risk of breast cancer, whereas alcohol intake was not associated with breast cancer risk among carriers of the variant allele (Table III). The interaction was statistically significant, both before (P = 0.003) and after adjustment for parity, number of births, age at first birth, education, duration of HRT and BMI (P = 0.005).

We estimated the effect of PPAR{gamma}2 Pro12Ala genotypes on breast cancer without alcohol consumption (Table IV). Because only 3% of the present study group were non-drinkers, low alcohol consumption was defined as <3 g/day (~2 drinks/week). This subgroup comprised 25% of the study group. Among women with low alcohol consumption, carriers of the variant allele were at 1.32-fold higher risk of breast cancer (95% CI = 0.61–2.88) compared to homozygous wild-type carriers. Thus, among non-drinkers, there was no effect of the PPAR{gamma}2 Pro12Ala genotype. Homozygous wild-type allele carriers with moderate to high alcohol consumption (>3 g/day) had 74% higher risk of breast cancer (95% CI = 1.14–2.65) than those with a low alcohol consumption. Conversely, carriers of the variant allele with moderate to high alcohol consumption were not at higher breast cancer risk than women with low alcohol consumption (IRR = 1.02, 95% CI = 0.59–1.76).


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  		Table IV Rate ratios for breast cancer in relation to PPAR{gamma}2 Pro12Ala genotype and alcohol intake among post-menopausal women in the ‘Diet, Cancer and Health’ study

 
The effect of ever-use of NSAIDs including aspirin was investigated for the different genotypes. Use of NSAIDs did not change the risk estimates for breast cancer in relation to IL6 G-174C, IL8 T-251A or COX2 T8473C (Table V). Carriers of the variant allele of PPAR{gamma}2 Pro12Ala were only at lower risk of breast cancer among non-users of NSAID. Variant allele carriers who were non-users had an RR = 0.44 (95% CI = 0.26–0.73). There was a statistically significant interaction between genotype and NSAID when heterozygous and homozygous carriers of the variant allele were grouped together (P = 0.03).


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  		Table V Rate ratios for breast cancer in relation to genotypes and NSAID use among post-menopausal women in the ‘Diet, Cancer and Health’ study

 
Smoking status at baseline did not change the risk estimates for breast cancer in relation to IL6 G-174C, IL8 T-251A, PPAR{gamma}2 Pro12Ala or COX2 T8473C (Table VI).


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  		Table VI Rate ratios for breast cancer in relation to genotypes and smoking status at baseline among post-menopausal women in the ‘Diet, Cancer and Health’ study

 
Since PPAR{gamma}2 Pro12Ala interacts with both alcohol consumption and NSAID use in relation to breast cancer risk, we investigated the effect of NSAID use on alcohol associated breast cancer risk among homozygote PPAR{gamma}2 Pro12 carriers and among carriers of the variant allele (Table VII). Among non-users of NSAID, wild-type carriers had a 1.13-fold increased risk of breast cancer per 10 g alcohol/day (95% CI = 0.97–1.32), but among variant allele carriers, alcohol consumption was associated with a decreased breast cancer risk (IRR = 0.61, 95% CI = 0.44–0.86). Among NSAID users, wild-type carriers had 1.22-fold increased risk per 10 g alcohol/day (95% CI = 1.04–1.43), whereas alcohol was not associated with breast cancer risk among variant allele carriers (IRR = 1.09, 95% CI = 0.82–1.44). The interaction was statistically significant (P = 0.0019).


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  		Table VII IRR for breast cancer per 10 g alcohol/day for different genotypes in combination with NSAID use

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
We have found that the Ala-allele of PPAR{gamma}2 Pro12Ala is associated with lower risk of breast cancer than the Pro-allele. However, this was due to a high alcohol consumption and interaction between alcohol and PPAR{gamma}2 Pro12Ala. Among women with low alcohol consumption, the Ala-allele of PPAR{gamma}2 Pro12Ala was not associated with decreased risk of breast cancer. Alcohol intake is associated with 21% increased risk of breast cancer among homozygous carriers of the wild-type Pro-allele.

We observed a weak interaction between NSAID use and PPAR{gamma}2 Pro12Ala in relation to breast cancer risk. There was a clear tendency that the protective effect of PPAR{gamma}2 12Ala was absent among NSAID users. A number of commonly used NSAIDs, including ibuprofen, activate PPAR{gamma} (43). Thus, low PPAR-{gamma} activity is associated with lowered breast cancer risk, and use of PPAR{gamma} activating NSAIDs may be associated with slightly higher breast cancer risk. We also observed interaction between NSAID use and genotype in relation to alcohol associated breast cancer risk. Caution must be taken in the interpretation of the effect of NSAID use, since we have used a very broad definition of NSAID use by including anyone who used a least 2 pills/month in the last year.

The combined effects of alcohol and NSAIDs in relation to breast cancer risk may be an explanation why diverging results regarding NSAID use and breast cancer risk has been obtained in different studies of different populations. Alcohol has been shown to suppress an inflammatory response both in vitro and in vivo in lung and liver (11,12). Thus, assuming that alcohol intake has the same effect in breast tissue, alcohol and NSAID could both be anti-inflammatory. The observed interaction between NSAID use and alcohol in relation to PPAR-{gamma}2 Pro12Ala and risk of breast cancer could indicate that both NSAID and alcohol modify breast cancer risk through a mechanism that involves a specific activation of PPAR{gamma}2 that differs between the two polymorphic forms of PPAR-{gamma}2.

There is ample evidence that the PPAR{gamma}2 12Ala is a less efficient transcription activator than the wild-type PPAR-{gamma}2 12Pro at least in reporter assays and that carriers of the PPAR{gamma}2 12Ala are at lower risk of developing metabolic syndrome and have improved insulin sensitivity compared to homozygous PPAR{gamma}2 12Pro carriers (29). Interaction between the PPAR-{gamma}2 Pro12Ala polymorphism and alcohol intake has been reported once before, where it was shown that carriers of PPAR{gamma}2 12Ala who drank alcohol had a higher level of total serum and HDL cholesterol than PPAR{gamma}2 12Pro carries, whereas the opposite relationship was found for non-drinkers (44). While this observation does not provide any indication of a possible mechanism, it indicates that it is possible that alcohol intake has opposite effects in PPAR{gamma}2 12Pro and PPAR{gamma}2 12Ala carriers. Recently, animal studies have shown that alcohol intake increases the oxidative stress in mammary tissue, leading to significantly increased levels of lipid hydroperoxides (45). In agreement with this observation, alcohol intake was shown to increase the levels of markers of oxidative stress and lipid peroxidation in blood samples in a controlled dietary study of 53 women (46).

Our results indicate that alcohol intake is only associated with breast cancer risk among homozygote carriers of PPAR{gamma}2 12Pro. This implies that there would be a large fraction of women for whom alcohol consumption was not associated with breast cancer risk, and conversely, a large fraction of women who are homozygous wild-type carriers (70% in Denmark) for whom even moderate alcohol consumption would be associated with an increased breast cancer risk. It was recently shown that alcohol associated breast cancer risk was modified by ADH3 genotypes, indicating that women who metabolized ethanol to acetaldehyde very efficiently were at greater breast cancer risk (47).

The association between PPAR{gamma}2 Pro12Ala polymorphism and breast cancer risk has only been investigated in one previous study of 725 incident breast cancer cases and 953 matched controls nested within the prospective Nurses' Health Study, in which no association was found (48). The discrepancy between the results of this study and ours may be explained by the relatively high alcohol consumption among Danish women. If only women with low alcohol consumption were analysed, then the variant Ala-allele is not associated with lower risk of breast cancer. Interaction with alcohol consumption was not examined in the above mentioned study (48), but in another study nested in the Nurses Health cohort, alcohol intake was reported to be 5.8 g/day in contrast to 10 g/day in the present study, indicating that alcohol intake probably was considerably lower in that cohort than in the Diet, Cancer and Health cohort (49).

In a recent published case–control study of IL6 G-174C among 269 breast cancer cases and 227 controls, homozygous and heterozygous carriers of the variant C-allele were found to be at 1.5-fold (CI = 1.04–2.3) and 2.0-fold (CI = 1.1–3.6) higher risk of breast cancer than homozygous G-allele carriers (50). We were unable to reproduce these findings. Assuming Hardy–Weinberg equilibrium and the allele frequency of the control group, we had 95% chance of detecting the reported OR at a 5% significance level. The IL6 G-174C promoter polymorphism affects the transcription of the gene although two studies have reported conflicting results as to which allele has the highest response (24,25). Fishman et al. (25) reported that the G-allele (reading in the sense direction) had a higher transcriptional level than the C-allele. In the study by Terry et al. (24), the nomenclature of the primers indicated that the single nucleotide polymorphism (SNP) had been read on the opposite strand, resulting in naming the usual G-allele as the C-allele and vice versa. Thus, there may not be conflicting results, and the wild-type G-allele would then have the highest inflammatory response.

Our cases and controls were selected from the same cohort, which together with complete follow-up of the participants minimized the risk for selection bias. For all participants, information lifestyle factors were collected at enrolment, which minimized the risk for differential misclassification between the cases and controls.

In conclusion, we found interaction between the polymorphism PPAR{gamma}2 Pro12Ala and alcohol consumption and NSAID use. Our results indicate that PPAR{gamma}2 Pro12Ala is an important effect modifier of the association between alcohol use and breast cancer risk. We also observe interaction between NSAID, alcohol consumption and PPAR{gamma}2 Pro12Ala genotype in relation to breast cancer risk. The findings should be replicated in other breast cancer study groups.


   Acknowledgments
 
We thank Yonit Bertelsen, Anne-Karin Jensen, Lourdes Pedersen and Katja Boll for excellent technical support. This work was supported by the Danish Cancer Society, grant DP00027, the grant ENGAGE from the Novo Nordisk Foundation and a grant from the Danish Ministry of Health, Research Centre for Environmental Health's Fund, ENGAGE.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 18, 2006; revised August 29, 2006; accepted September 2, 2006.


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