HSD17B1 Genetic Variants and Hormone Receptor–Defined Breast Cancer

Genetic variation in the estrogen synthesis pathway may affect breast cancer risk by increasing lifetime exposure to the cell proliferation and genotoxic effects of estrogen (1). 17β-hydroxysteroid dehydrogenase 1 (HSD17B1) has been suggested to be an important candidate gene in breast carcinogenesis because its coding enzyme, 17HSD1, catalyzes the conversion of estrone to estradiol, the final step of estrogen synthesis (2, 3). In conjunction with 17HSD1, 17HSD2, which catalyzes the reverse estradiol to estrone, predominates the control of available active estrogen (4). However, 17HSD1 is overexpressed in malignant breast tissue of pre- and postmenopausal women (5) and in breast tumors with lower estrogen receptor (ER) expression (6).

A nonsynonymous G-to-A (rs605059) single-nucleotide polymorphism in HSD17B1 has been examined in relationship to breast cancer risk in eight studies with inconclusive results (7-11). In a recent report of five U.S. and European cohorts (5,370 cases, 7,480 controls; ref. 12), additional genetic variation in the gene and its flanking regions, as designated by four haplotype-tagging single-nucleotide polymorphisms (rs676387, rs605059, rs598126, and rs2010750), was not found to be associated with breast cancer risk. However, among a subset of 2,091 Caucasian cases with ER data and 2,982 controls, risk for ER-negative (ER-) tumors (n = 354) was lower among women with at least 1 copy of the variant A allele of rs676387 and higher among those with minor alleles of 3 strongly-correlated haplotype-tagging single-nucleotide polymorphisms, rs605059, rs598126, and rs2010750. We followed up these findings in two studies, in which we genotyped the same four haplotype-tagging single-nucleotide polymorphisms in the Polish Breast Cancer Study (PBCS; 1,995 cases; 2,296 controls) and rs676387 and rs605059 in the Studies of Epidemiology and Risk Factors in Cancer Heredity (SEARCH; 4,470 cases; 4,560 controls).

The present analysis is based on data from two case-control studies conducted in Poland (13) and England (14) that have been previously described in detail. Both studies received approval from their respective institutional review committees, and all study respondents provided informed consent.

The PBCS study was conducted between 2000 and 2003 among women residing in Warsaw and Lodz, Poland (13). Eligible cases were women with the age of 20 to 74 years, who were newly diagnosed with either histologically or cytologically confirmed in situ or invasive breast cancer. Study personnel identified cases through a rapid identification system and cancer registries to ensure complete case ascertainment. Controls with no history of breast cancer were randomly selected through a database of all Polish residents. Controls were frequency matched to cases by city and age in 5-year categories. A total of 1,995 cases (65% of eligible cases identified) and 2,296 controls (63% of eligible controls identified) provided a personal interview on known and suspected risk factors and donated a venous blood sample. Medical records of cases were obtained and extracted for tumor characteristics and initial breast cancer treatment regimens. Hormone receptor status was available for 76% of cases and determined by immunohistochemical assays (91% of cases) and biochemical methods (9%).

In SEARCH, cases were drawn from the East Anglian Cancer Registry. All patients diagnosed with invasive breast cancer before the age of 55 years since 1991 and still alive in 1996 (prevalent cases; median age, 48 years), together with all those diagnosed at <70 years between 1996 and the present (incident cases; median age, 54 years), were eligible to take part. Of the eligible breast cancer patients, 67% returned a questionnaire and 64% provided a blood sample for DNA analysis. Female controls were randomly selected from the Norfolk component of the European Prospective Investigation of Cancer (EPIC) in approximate order of recruitment through general practice age-sex registers. EPIC is a prospective study of diet and cancer being carried out in nine European countries. The EPIC-Norfolk cohort comprises 25,000 residents of Norfolk, East Anglia, the same region from which the cases were recruited. Controls were not matched to cases but were broadly similar with respect to age (range, 42-81 years). This analysis is based on a subset of 4,470 cases and 4,560 controls all of whom completed an epidemiologic questionnaire and provided a blood sample for DNA analysis. Tumor characteristics, including ER status, were extracted from tumor registry data. ER data were available for 52% of cases in SEARCH.

Haplotype-tagging single-nucleotide polymorphisms were selected based on the efforts of the Breast and Prostate Cohort Consortium (15), as previously described (12, 16). Given the close proximity of the adjacent genes, including N-acetylglucosaminidase-α (NAGLU) gene and a pseudogene for HSD17B1 (HSD17BP1) in the 5′ direction and CoA synthase (COASY) and transcription factor-like 4 (TCFL4) in the 3′ direction, the location of haplotype-tagging single-nucleotide polymorphisms for HSD17B1 extended into these neighboring genes. Four haplotype-tagging single-nucleotide polymorphisms (rs676387, rs605059, rs598126, and rs2010750) were selected that capture most common haplotype diversity among European-Americans (R_H² = 0.82).

Methods for genotype assays for rs676387, rs605059, rs598126, and rs2010750 performed on PBCS samples are described online: http://snp500cancer.nci.nih.gov (17). A total of 100 duplicate DNA pairs were interspersed throughout the PBCS DNA samples. All single-nucleotide polymorphisms were >97% concordant for duplicate pairs and >99% complete for all samples. Genotype frequencies were in Hardy-Weinberg equilibrium among controls (P > 0.44).

In the SEARCH DNA samples, only rs605059 and rs676387 were genotyped because there was a strong correlation between rs605059, rs598126, and rs2010750 in the PBCS and in previous studies (12). Genotyping was performed using a fluorescent 5′ exonuclease assay (Taqman) and the ABI PRISM 7900 Sequence Detection Sequence (PE Biosystems). Cases and controls were arrayed together in twelve 384-well plates and a 13th plate contained 8 duplicate samples from each of the 12 plates. All single-nucleotide polymorphisms were 100% concordant for duplicate pairs and >99% complete for all samples. Genotype frequencies were in Hardy-Weinberg equilibrium among controls (P > 0.33).

Unconditional logistic regression models were used to estimate odds ratios and 95% confidence intervals (95% CI), adjusted for study site (Warsaw, Lodz, or Norfolk), for the association between individual single-nucleotide polymorphisms and breast cancer, using STATA (version 10.0). Genotypes were evaluated using indicator variables. We assumed an additive mode of inheritance to calculate the P for trend. We evaluated effect modification by age at baseline (defined as age at diagnosis for cases and age at interview for controls; age categories, <45, 45-55, >55) and menopausal status (premenopausal and postmenopausal for PBCS only) comparing the -2 log likelihood of models with and without interaction terms. Breast cancer cases were further defined by ER [ER positive (ER+), ER-] and progesterone receptor [PR positive (PR+), PR negative (PR−)] status of the breast tumor, excluding cases with unknown hormone receptor status. Polytomous logistic regression models were used to estimate odds ratios and 95% CIs for 2 different case groups and controls. To test for differences in genotype associations between case groups, we evaluated case-only logistic regression models, adjusted for age and study, with ER status (ER+, ER-) or PR status (PR+, PR−) as the outcome variable and genotype as the explanatory variable.

Study-specific odds ratios (95% CI) were extracted for the risk for invasive breast cancer overall and by ER status among the Caucasian participants of the five cohorts previously involved in a pooling project (that is, American Cancer Society Cancer Prevention Study II (9), EPIC cohort, Nurses' Health Study (8), Hawaii–Los Angeles Multiethnic Cohort, and Women's Health Study; study-specific odds ratios and 95% CIs provided in Supplemental Table 2 in ref. 12), as well as those for PBCS and SEARCH. Additional published studies were found in PubMed searches conducted using the key words “HSD17B1” or “17β-hydroxysteroid dehydrogenase 1” and “breast cancer risk.” Four studies (7, 10, 11, 18) were identified that published data on rs605059 and breast cancer risk prior to June 2008; however, we excluded two studies conducted among Asian women (7, 18). Studies, conducted in Germany (Gene Environment Interaction and Breast Cancer in Germany; ref. 19) and Finland (10) were included in our meta-analysis. Odds ratios (95% CI) of breast cancer risk overall (risk estimates by ER status were not available) were extracted from these publications. Summary odds ratios (95% CIs) were calculated using fixed-effect and random-effect models, with each study result weighted by the within-study and between-study variance (20). A 2-sided P < 0.05 was deemed statistically significant. The presence of between-study heterogeneity was assessed by the Q test (20). Given the small number of studies included in our meta-analysis, as well as the low power of the aforementioned test (21-23), a 2-sided P < 0.10 was considered statistically significant and that of <0.20, borderline significant. Statistical analyses were performed in STATA10 (StataCorp).

The average age of the PBCS controls under study was 55.7 (±10.0) years, and all were Caucasian. The SEARCH controls were slightly older (mean age, 57.8 ± 10.7), and >98% were self-reported Caucasian. The SEARCH cases tended to be diagnosed with ER+ tumors (79.1%), whereas slightly less PBCS cases were diagnosed with ER+ tumors (65.1%). The Polish cases were on average older (55.8 ± 10.0 years) than the SEARCH cases (51.7 ± 8.7 years).

Although rs598126 and rs2010750 were also genotyped in PBCS, only results for rs605059 are presented as results were similar for these three haplotype-tagging single-nucleotide polymorphisms given the high correlation between them in Polish controls [rs605059-rs598126 (r² = 0.98), rs605059-rs2010750 (r² = 0.80), and rs2010750-rs598126 (r² = 0.82)]. The minor allele frequencies in controls were 22% in PBCS and 27% in SEARCH for rs676387 and 48% and 44%, respectively, for rs605059.

Compared with the AA genotype, the GA and GG genotypes of rs605059 were associated with weak decreases in breast cancer risk in SEARCH [odds ratio (95% CI), 0.88 (0.79-0.99) and 0.85 (0.73-0.98), respectively]. However, neither genotype was associated with risk in PBCS [odds ratio (95% CI), 0.94 (0.82-1.08) and 1.00 (0.85-1.18), respectively; Table 1]. The summary odds ratios (95% CIs), based on 11,762 cases and 14,329 controls from 10 studies, were 0.93 (0.87-0.99) for the GA genotype and 0.95 (0.85-1.08) for the GG genotype (Fig. 1 and Supplemental Table 1). Significant between-study heterogeneity was detected for the summary odds ratios for the GG genotype (P value for between-study heterogeneity = 0.04) but not for the AG genotype (P value for between-study heterogeneity = 0.51). In models restricted to the 6 studies (that is, Hawaii–Los Angeles Multiethnic Cohort, American Cancer Society Cancer Prevention Study II, Women's Health Study, Nurses' Health Study, PBCS, and SEARCH) included in the ER-specific breast cancer risk analyses below (that is, 5,982 invasive Caucasian cases and 9,912 Caucasian controls), the summary odds ratio (95% CI) for rs605059 GG genotype was 0.98 (0.86-1.11), with evidence of significant between-study heterogeneity (P = 0.04), similar to the findings based on all studies (Fig. 1 and Supplemental Table 1).

Overall, breast cancer risk for rs676387 was slightly elevated for AA genotype compared with the CC genotype in PBCS (odds ratio, 1.22; 95% CI, 0.94-1.57) and SEARCH (odds ratio, 1.16; 95% CI, 0.96-1.40), although neither odds ratio was statistically significant. The summary odds ratio (95% CI), based on 11,074 cases and 13,605 controls in 7 studies, for the AA genotypes compared with the CC genotypes was 1.12 (95% CI, 0.99-1.27; P for between-study heterogeneity = 0.30; Supplemental Table 1; Fig. 1).

Although there was no significant difference in odds ratio estimates for rs605059 in the PBCS between ER+ and ER- breast tumors (P for case heterogeneity = 0.75 for AG versus AA, and 0.18 for homozygote GG versus AA; Table 1), SEARCH results suggested a stronger decreased risk of the GG genotype for ER- (odds ratio, 0.58; 95% CI, 0.41-0.83) than ER+ (odds ratio, 0.85; 95% CI, 0.71-1.01; P for case heterogeneity = 0.01; Table 1) tumors. For our meta-analysis, ER data were available for cases from 6 studies (that is, Hawaii–Los Angeles Multiethnic Cohort, American Cancer Society Cancer Prevention Study II, Women's Health Study, Nurses' Health Study, PBCS, and SEARCH), including 4,635 ER+ cases and 1,347 ER- cases. No significant associations were found between rs605059 and ER+ breast cancers (Fig. 1 and Supplemental Table 2); summary odds ratios (95% CIs) were 0.94 (0.86-1.03) for the GA genotype and 0.97 (0.84-1.11) for the AA genotype, with no evidence of between-study heterogeneity (P = 0.48 and 0.16, respectively). There was also no significant association between the GG genotype of rs605059 and risk for ER- tumors (odds ratio, 1.18; 95% CI, 0.78-1.80), but these odds ratios were not consistent across studies (P for between-study heterogeneity = 0.001). To determine whether an individual study was the source of between-study heterogeneity, we systematically dropped each study and recalculated the summary odds ratio for the GG genotype of rs605059. Exclusion of each study with the exception of SEARCH did not affect the test of between-study heterogeneity nor the random effect estimates. When we excluded SEARCH, the summary odds ratio (95% CI) for the GG genotype of rs605059 was 1.39 (1.04-1.87) and P for the test of between-study heterogeneity was 0.20.

In PBCS and SEARCH, no significant difference between risk for ER+ breast cancer and risk for ER- breast cancer was found for the AA genotype of rs676387 (P = 0.56 and 0.16, respectively). However, risk for ER- tumors were elevated for rs676387 AA genotype in PBCS (odds ratio, 1.34; 95% CI, 0.92-1.93) and SEARCH (odds ratio, 1.53; 95% CI, 1.03-2.27; Table 1). These findings were not consistent with summary odds ratio (95% CI), based on 1,347 ER- cases and 9,912 controls, which suggested no association (odds ratio, 1.02; 95% CI, 0.70-1.50; Supplemental Table 2; Figure 2) between the AA genotype of rs676387 and risk for ER- tumors, although there was weak evidence of between-study heterogeneity in this estimate (P for between-study heterogeneity = 0.11). None of the individual studies found an association between rs676387 AA genotype and ER+ breast cancer risk (P for between-study heterogeneity = 0.95); the summary odds ratio (95% CI) was 1.05 (0.91-1.22; Supplemental Table 2; Figure 2).

Menopausal status did not modify any of the genotype associations in the PBCS (P for interactions > 0.42; no additional data shown). Data on menopausal status was not available for SEARCH; therefore, we used age at baseline as a proxy of menopausal status. We also did not observe modification by age of the association between breast cancer risk and rs676387 or rs605059 (Supplemental Table 3).

Data from two independent studies in Poland and the UK totaling 6,465 cases and 6,856 controls showed weak to null associations with overall breast cancer risk for two single-nucleotide polymorphisms (rs605059 or rs676387) that captured most of the known common variation in HSB17B1. Meta-analysis results including data from these two studies and previously published data (up to 11,762 cases and 14,329 controls in total) were consistent with these findings.

Previous analyses of 2,091 cases and 2,982 controls in 5 prospective cohort studies found ER- tumors (n = 354) were negatively associated with the rs676387 AA genotype and positively associated with the rs605059 GG genotype (12). However, data from the Polish (n = 586 ER- cases) and British (n = 407 ER- cases) studies did not support these findings, and meta-analyses including previously published data demonstrated significant evidence for heterogeneous relative risk estimates for ER- disease across studies. Factors that could explain the observed heterogeneity include unidentified biases, a true effect modified by a factor with varying prevalences across study populations, or chance. All of the study participants were Caucasian and had similar minor allele frequencies; therefore, ethnic differences are unlikely to explain the observed heterogeneity. Furthermore, the differences in main effect estimates across studies were minimal, and confounding by population stratification is unlikely to more strongly affect subgroup analyses, such as those for ER- tumors.

Studies differed in the distribution of age and menopausal status; study subjects in the previous report (12) were all postmenopausal women, whereas PBCS and SEARCH included both premenopausal and postmenopausal women. Differences in study population characteristics may explain between-study heterogeneity. However, we did not observe evidence of modification of genotype associations by menopausal status in PBCS or by age at baseline in SEARCH or PBCS. Thus, differences in age distributions between studies are unlikely to appreciably contribute to disparate results across studies.

Although it is unlikely that genotype is related to selection or participation in case-control or cohort studies, other study-specific biases may influence observed relative risks. Bias may have been introduced by the restriction of cases to those with information on hormone receptor status. In PBCS and SEARCH, genotype frequencies were similar for cases with and without ER data. In the previous report (12), one of the largest studies (1,610 cases; 2,884 controls) included in analyses of the association between genotypes and overall breast cancer risk was not included in the analyses by hormone receptor data. Interestingly, estimates from this study showed an overall 33% risk increase for rs676387 (12), which is inconsistent with the lower risk for ER- tumors and lack of association with ER+ tumors observed in the other cohorts (12). If individual studies had specific biases, it is unlikely that they would be in the same direction for all studies; this may explain some of the heterogeneity across studies.

In conclusion, it does not appear that common genetic variation in HSD17B1 is associated with a moderate modulation in breast cancer risk overall; however, we cannot exclude the possibility of a very weak effect. Additional data for HSD17B1 polymorphisms and breast cancer risk from studies with well characterized tumors is needed to clarify the findings for ER- tumors.

No potential conflicts of interest were disclosed.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Witold Zatonski and Neonila Szeszenia-Dabrowska for their contributions to the PBCS, Shahana Ahmedand, and the entire SEARCH breast study team.