An Admixture Scan in 1,484 African American Women with Breast Cancer

Introduction

Breast cancer incidence and mortality varies widely among women of different population groups in the United States. African American women have lower age-adjusted incidence of breast cancer compared with European Americans (1). However, breast cancer incidence is higher in African Americans who are 35 years of age or younger (2). African American women are also diagnosed, on average, with later stage of disease, larger tumors, and are more likely to present with lymph node metastases at the time of diagnosis (2, 3). Thus, despite the lower lifetime incidence of breast cancer among African American women compared with European American women, their breast cancer mortality rates are higher (4-6), particularly among younger women (7).

The expression of steroid hormone receptors (estrogen and progesterone receptors) in breast cancer tumors also varies substantially by population. African American women are diagnosed more frequently with estrogen receptor–negative (ER−) and progesterone receptor–negative (PR−) breast cancer compared with European American women (5, 7-9). In the Women's Health Initiative, 32% of breast cancers among postmenopausal African American women were ER− with poor/anaplastic grade in comparison to only 10% among European American women, a difference which remained after adjustment for multiple potentially confounding factors, including differential access to health care (10). Given the greater incidence of hormone receptor–negative, high-grade disease among African Americans, we hypothesized that there may be one or more genetic variants with increased frequency in populations of African origin, which predispose women to this more aggressive form of breast cancer.

Admixture mapping is a powerful approach for identifying genetic variants for common phenotypes that have large allele frequency differences between ancestral populations (11-14). Admixed populations are defined as populations in which two or more ancestral groups have been mixing over several generations. Recently admixed populations show extended linkage disequilibrium between markers that have a large difference in allele frequency between ancestral populations and are, therefore, informative about ancestry (ancestry-informative markers or “AIM”; refs. 13, 15). The principle of admixture mapping is to identify regions of the genome with greater estimated ancestry from one of the ancestral populations than the chromosomal average in individuals from an admixed group. These regions may highlight candidate risk loci that are associated with complex phenotypes. We have previously used this approach to identify risk variants for prostate cancer at 8q24 that are common in African American men and contribute to their increased disease incidence (16).

Here, we did an admixture-based genome-wide scan in 1,484 African American women with invasive breast cancer pooled from six population-based studies. Samples were typed at ∼1,500 AIMs to search for loci that might harbor predisposing variants for breast cancer, and more specifically, loci that may contribute to specific breast cancer phenotypes, such as ER− disease, a trait which is more common in African American women.

Results

Descriptive and tumor characteristics for cases in each of the six studies as well as for the combined sample of 1,484 women are summarized in Table 1. The mean age at diagnosis of all cases was 54 years (range, 22-83). The average percentage of European ancestry over all cases was 23% (range, 1-98) and was relatively homogeneous among studies. The Women's Circle of Health Study had the lowest average percentage of European ancestry (19%) and the Multiethnic Cohort had the highest (25%). We observed 31% of individuals with ER− tumors, 53% with ER+ tumors, and 16% with missing status. ER− tumors were overrepresented among younger cases as noted in the LIFE study (42%) and the Los Angeles component of the Women's CARE study (35%), which is consistent with previous reports (28-30). Regarding tumor stage, 53% of the individuals had localized tumors, 34% were non-localized and 13% had missing data. In the LIFE and CARE studies, which included higher proportions of younger cases, only ∼50% of the tumors were localized. For tumor grade, we observed a similar pattern, with a smaller proportion of lower grade tumors (grades 1 and 2) in the two studies that targeted younger women compared with the other studies. The percentage of European ancestry was significantly higher among individuals with hormone receptor–positive tumors compared with hormone receptor–negative tumors and women with localized disease compared with women with non-localized disease (Tables 1 and 2). We also observed a significantly higher percentage of European ancestry in women who were never pregnant compared with women who had one or more full-term pregnancies (Table 1).

Compared to women with ER− tumors, women with ER+ tumors had higher European ancestry [OR, 2.35; 95% confidence interval (CI), 1.06-5.20; Table 2]. This trend was observed both in cases with localized and non–localized tumors (localized: OR, 1.60; P = 0.39, n = 641; non-localized: OR, 2.08; P = 0.30, n = 429). For ER+PR+ (versus ER−PR−) tumors the association between European ancestry and positive receptor status became stronger (OR, 4.73; 95% CI, 1.56-14.33). We adjusted the models to include factors that have been found to correlate with hormone receptor status (i.e., number of full-term pregnancies, age at first full-term pregnancy, hormone replacement therapy, menopausal status, age at menarche, BMI, and family history of breast cancer). In the adjusted model, ER status alone was no longer significantly associated with ancestry (OR, 2.06; 95% CI, 0.90-4.71). The ER+PR+ versus ER−PR− analysis showed a significant ancestry effect. The OR of the unadjusted model was 4.73 (95% CI, 1.56-14.33; P < 0.01). After we adjusted for potential confounders, the effect of ancestry was reduced but remained statistically significant (OR, 2.84; 95% CI, 1.13-7.14; P = 0.026). Among the factors included in the adjusted model, the number of full-term pregnancies had the strongest effect, with nulliparous women being more likely to have ER+PR+ tumors compared with women who have one or more children (OR for being ER+PR+ if woman has one or more children: 0.40; 95% CI, 0.26-0.60; P < 0.01). We observed an association between European ancestry and disease stage (localized versus non-localized), with higher European ancestry among women with localized tumors (multivariate adjusted OR, 2.65; 95% CI, 1.11-6.35) compared with women with non-localized disease. We did not find a significant relationship between tumor grade and European ancestry (Table 2).

Admixture Mapping Does Not Show Significant or Suggestive Results Either for Breast Cancer Risk or for Tumor Characteristics

We next conducted a series of genome-wide admixture scans evaluating a number of breast cancer phenotypes (as described in Materials and Methods) among 1,484 African American women with breast cancer and 1,370 AIMs per subject, on average. The data were analyzed using an affected-only statistic, which calculates the likelihood of association based on an estimate of the ancestry at a particular location relative to the overall average ancestry of the individual's genome.

No genome-wide statistically significant association was observed between European or African ancestry and breast cancer at any specific locus (Table 3). The largest LOD score genome-wide was 2.9 (we set a threshold of >5 for significance; ref. 24) on chromosome X and 2.4 on chromosome 10 (in both cases, the African allele was associated with increased risk).

A series of analyses looking at hormone receptor status and at hormone receptor status and grade combined (Table 3) were not significant. Stratifying the analyses by age did not significantly alter the results. Case to case analyses were done comparing women with tumors that were hormone receptor–negative to those with hormone receptor–positive tumors as well as women with localized tumors versus non–localized tumors. The differences in locus-specific ancestry were not significant.

Analysis of Known Breast Cancer Risk Loci

We also searched for ancestry associations within regions that have previously been reported to be associated with breast cancer risk in other populations. Four genome-wide scans have been reported to date; all of them have been conducted in populations of European or Asian ancestry. The different regions that were found to be associated with risk were 4p14, 6q22, 7q22, 10q26, 5q11, 16q12, 11p15, 8q24, 2p24, 5p12, and 2q35 (31-36). Many of these regions have also been more strongly associated with ER+ status in Europeans (37). We found a weak deviation towards higher African ancestry within the 10q26 region compared with the rest of the chromosome; this region includes the FGFR2 gene. The FGFR2 gene has been repeatedly identified as a breast cancer susceptibility locus by genome-wide association studies (31, 33-35), and has also recently been fine-mapped to identify specific variants (38).

Exclusion Map

We prepared an exclusion map for the three case definitions with the largest sample sizes: all cases, ER+, and ER− cases. At least 98% of the genome can be excluded as having a European effect on risk of 1.4 or more, and at least 96% can be excluded as having an African effect on risk of 1.5 or more (Table 4). The power of the ER status analysis is less than that for all cases because of the smaller sample size. In the case of ER− disease, we can exclude 87% of the genome as having an increased risk of 1.8 or higher due to African ancestry and 92% as having an increased risk of 1.7 or higher due to European ancestry. In the case of ER+ disease, we can exclude 89% of the genome as having an increased risk of 1.6 or higher associated with African ancestry and 92% as having an increased risk of 1.5 or higher associated with European ancestry (Table 4).

Discussion

The present study represents the first genome-wide admixture scan conducted in African American women with breast cancer. In this study, we did not find an association between breast cancer risk and African or European ancestry at any specific loci among all cases or within subtypes of breast cancer, at genome-wide levels of significance. We detected European ancestry to be overrepresented among women with ER+ tumors. However, adjustment for known breast cancer risk factors could explain this association. A significant association remained for ER+PR+ tumors following adjustment, which could be due to misclassification of these risk factors, other risk factors which we did not consider (e.g., alcohol consumption), or that we do not know about that do correlate with ancestry and influence tumor characteristics. At the same time, it is possible that this association is due to genetic risk factors that correlate with ancestry. We observed that nulliparity was associated with both ER+PR+ disease as well as European ancestry. The association between number of full-term pregnancies and hormone receptors status has been reported previously in African Americans and white women (29), and our data replicates these results. The association between nulliparity and ER+PR+ disease could be the result of an underlying biological mechanism or could be due to the correlation between this risk factor and other known or unknown risk factors that we did not account for. The association between European ancestry and nulliparity was also significant (P = 0.01) but could not completely explain the association that we observed between ancestry and ER/PR status. We also detected European ancestry to be significantly overrepresented among women with localized tumors compared with women with non–localized tumors (OR, 2.65; 95% CI, 1.11-6.35; P = 0.029). This association could not be explained by the known breast cancer risk factors.

The exclusion map shows that for the analysis of the ER− cases, we had reasonable power to detect an increased risk due to an African allele of 1.8 and above and an increased risk due to a European allele of 1.6 and above. Therefore, the fact that our scan did not detect any significant signal does not discard the possibility that ancestry effects of 1.7 or lower are present. The observed association between ancestry and ER/PR status supports this possibility and suggests that further analyses are needed with adequate power to detect ancestry effects on risk of 1.7 or less.

We detected a nonsignificant deviation towards higher African ancestry on chromosome 10q26 compared with the chromosomal average. This region includes the FGFR2 gene and a common variant that is associated with increased risk of breast cancer in Asian and European populations (33, 34, 38). A recently published study investigated FGFR2 variants in African Americans, Asians, and Europeans to search for causative variants and to evaluate if the same variants were associated with risk of breast cancer in the different racial/ethnic groups (38). Based on association results, and an analysis of DNase I hypersensitive sites looking at chromatin accessibility, the conclusion was reached that two variants, rs2981578 and rs10736303, are the most likely to be causal variants. The frequency of these two variants is different in African populations compared with Europeans or Asians. The frequency of the risk allele for the variant rs2981578 is 0.93 in the HapMap African sample and 0.46 in the HapMap European samples. A similar difference was observed for rs10736303, with the risk allele having a frequency of 0.92 in Africans and 0.60 in Europeans.¹⁶ The increase in African ancestry that we observed in the admixture mapping analysis within the 10q26 region could potentially be explained by the higher frequency of causal risk alleles in this region, which are likely to be more common in African than European populations.

There was no apparent deviation from the average chromosomal ancestry for any other region of the genome previously reported to have a risk variant. Different studies have reported associations between variants in the FGFR2 gene and breast cancer risk, with per allele ORs that varied between 1.20 and 1.30 (33, 34, 38-40). The reported ORs for the FGFR2 gene are among the higher reported ORs compared with those of other risk variants discovered through whole genome association studies (∼1.25 compared with <1.20; ref. 39). Adding to this, the candidate variants within the FGFR2 gene show a large allele frequency difference between Europeans and Africans. Therefore, it is likely that we did not observe any other ancestry deviations because of lack of power (we had power >80% to detect risk variants with an allele effect of 1.5 or larger; if the allele effect was ∼1.2, then the allele frequency difference between the ancestral populations needed to be larger than 0.7 to achieve a power above 40%).

One limitation of this study is the sample size. Although the study included >1,400 women, ER−PR− cases are still a minority of cases, even among African Americans, and thus, we had limited power to assess associations for the different breast cancer phenotypes.

Her2 status was not available for the majority of cases because most of the cases in the different studies were recruited at a time when Her2 status was not routinely assayed for clinical testing. Therefore, we were unable to analyze ER−PR−Her2-negative breast cancer cases (i.e., “triple negatives”), an aggressive subset of tumors that has been estimated to be more common in African Americans than in European Americans (28-30, 41). Much larger studies in African populations, with available tumor specimen resources for tumor phenotyping, will be needed to evaluate the genetic contribution to the various breast cancer subtypes.

Information about ER and PR status, grade, and stage, comes from pathology reports or from the cancer registry, depending on the study. Therefore, it is likely that there were differences in how the tumors were classified. This potential misclassification could have contributed to the negative results observed. However, the frequency of the different tumor characteristics in the six studies are similar and when they differ, they do it in the expected direction given the age distribution of the women in the studies. This suggests that misclassification might not be a serious problem for these data, although caution must be taken in the interpretation of the results. Future studies involving centralized tumor marker data collection will be necessary to avoid the potential effect of misclassification in genetic epidemiology studies with multiple data sources.

The AIMs selected to infer genetic ancestry are assumed to have homogenous frequency within the African continent. Given that African Americans are likely to have a mixed ancestry from different regions of Western Africa (42), which might not share the same allele frequencies for the markers used in the present study, results must be interpreted with caution.

The clinical implications of the differences in tumor presentation of African American women with breast cancer compared with European American patients are substantial. Although the overall incidence of breast cancer is lower in African American women, the mortality rate is higher in African American women than in European American women (43). This may be in part be due to higher rates of ER− disease because hormonal treatment, either with selective estrogen receptor modifiers (tamoxifen or raloxifene) or with aromatase inhibitors, is highly effective for ER+ disease only (44). Furthermore, ER− disease often occurs in younger women who have never had screening because they are younger than the standard screening age and because screening with mammography is less sensitive among younger women (45). The high rates of ER− disease among African Americans may also have implications for breast cancer prevention. Tamoxifen and raloxifene have been shown to prevent ER+ breast cancer in primary prevention studies, and some have advocated that the medications be used in women at high risk (44, 46). In addition, aromatase inhibitors may also be useful in the prevention of breast cancer (47). However, there is no clear preventive strategy for ER− breast cancers. Identifying the causal factors that explain the difference in incidence of hormone receptor–negative tumors between European American and African American women should be a high priority.

The present admixture mapping scan in 1,484 African American women with breast cancer suggests that the difference in breast cancer risk between Europeans and African Americans is unlikely to be due to an effect of a European or African allele on risk larger than 1.7. It also excludes an effect on risk for ER+ status larger than 1.9 and for ER− status larger than 2.4. Global ancestry association results, however, show a positive association of European ancestry with stage of disease, and with ER+PR+ disease. These associations could result from population differences in nongenetic risk factors or from the effect of multiple genetic variants each with a relatively moderate contribution to the ancestry-related risk difference.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.