Risk Factors for Triple-Negative Breast Cancer in Women Under the Age of 45 Years

Breast cancer is a strikingly heterogeneous disease with variable clinical, pathologic, and molecular features. Microarray expression patterns and immunohistochemical (IHC) signatures can distinguish breast cancer subtypes and likely reflect important differences in pathogenesis and etiology (1-4). Current breast cancer treatment strategies rely on the characterization of estrogen and progesterone hormone receptor [estrogen receptor (ER)/progesterone receptor (PR)] protein expression status and more recently on human epidermal growth factor (HER2) protein expression or gene amplification. Breast tumors that fail to express ER/PR and HER2 (triple-negative breast cancer) account for 10% to 17% of all breast cancers (5-12).

Recently, five distinct gene expression profile-based “intrinsic” subtypes were identified by cDNA microarray analysis, two derived from ER+ (estrogen receptor positive) subtypes (luminal A and B) and three from ER- (estrogen receptor negative) subtypes (HER2+; basal-like and normal-like; refs. 1, 2, 13). More than 90% of triple-negative breast cancer tumors fall within the basal-like subgroup, so called for its gene expression profile that mimics basal epithelial cells in other parts of the body (usually identified by IHC staining for the expression of cytokeratin 5/6, reduced ER/PR, and HER2 expression) and a characteristic morphology that includes high proliferative rate, central necrosis, and a pushing border (14, 15). Basal-like breast cancer is associated with aggressive histology, unresponsiveness to typical endocrine therapies, poor prognosis, and BRCA1-related breast cancer (1-3, 16).

Triple-negative breast cancer constitutes a clinically challenging type of breast cancer that occurs more frequently in younger women (<50 years; refs. 6, 7, 9, 10) and African-American women (10-12) and is associated with significant aggressiveness as compared with other subtypes (5-7, 9-11). Although triple-negative breast cancer is of growing interest in the clinical and research community, its etiology remains understudied. We undertook this study to evaluate the contribution of known and suspected breast cancer risk factors to triple-negative breast cancer in a large population-based study.

The cases included in this study were originally ascertained for two previous studies through the population-based Seattle–Puget Sound Surveillance, Epidemiology, and End Results cancer registry. Eligible cases from the first study population included all primary invasive breast cancers within the three-county Seattle metropolitan area, diagnosed between January 1, 1983, and April 30, 1990 (ages, 21-45 y). The methods for this study have been described elsewhere (17, 18). The study was confined to Caucasians because of the small representation of minorities in the region. Of 898 eligible invasive cases, 744 (83%) were interviewed. Nine hundred sixty-one controls were interviewed, representing a 76% overall response rate (97% of dialed known residential households successfully screened; 78% interviewed). For both studies, controls were identified by random digit dialing and frequency matched to cases by 5-y age groups.

The second population included the Seattle site participants of the multicenter Women's Interview Study of Health, the methods for which have been described (19). Eligible cases included women in the Seattle area diagnosed with invasive breast cancer between May 1, 1990, and December 31, 1992 (ages 21-44 y). In-person interviews were completed on 542 women (86% of eligible Seattle cases with invasive disease). Six hundred eight Seattle controls were interviewed, representing a 71% overall response rate (90% of dialed known residential households successfully screened; 78% interviewed). Reference dates were assigned to all participants: age at diagnosis for cases and an assigned age for each control to result in an approximately similar age distribution for cases and controls. Because the present study focuses on invasive triple-negative breast cancer, in situ cases were excluded. The appropriate institutional review boards approved all protocols.

In-person interviews of comparable format, covering a broad range of risk factors that included lifestyle/demographic factors, reproductive history, and oral contraceptive use, were administered to participants in both studies. Tumor specimens were obtained for 1,019 of the 1,286 cases with invasive breast cancer who were accrued in the two previous studies. Tissue collection, pathology review, and testing for prognostic markers have been discussed previously (20). Briefly, tumor tissue was sufficient for immunoperoxidase (IHC) assay on 907 (89.0%) of the tumors. Antibody staining for ER, PR, and HER2 was assessed as negative, 1+ (low positive), 2+ (intermediate positive) or 3+ (high positive). Scores above negative were considered positive for ER and PR. A distinct membranous staining pattern above 1+ was considered positive for HER2. The current study is restricted to cases for whom complete ER, PR, and HER2 results were obtained (n = 897).

Breast cancer risk factors were evaluated according to ER, PR, and HER2 status. Classification by these three markers results in eight different subtype combinations; however, our analyses focus primarily on comparisons between triple-negative breast cancer (n = 187, 20.8%) and non-triple-negative breast cancer tumors partly because of the small number of observations with dissimilar ER/PR status in our study population (e.g., ER+/PR-/HER2-, n = 57, 6.4%; ER-/PR+/HER2-, n = 65, 7.2%; ER+/PR-/HER2+, n = 23, 2.6%; ER-/PR+/HER2+, n = 26, 2.9%).

Secondary analyses focus on oral contraceptive variables and breast cancer defined separately and jointly by ER and HER2 status (collapsed across PR status; ER/PR r = 0.60) and also stratified by age (≤40 and 41-44), allowing us to determine whether one or two marker classification methods produced associations similar to that of triple-negative breast cancer and compare our results with ER and HER2 findings from previous studies. Furthermore, analyses were repeated stratified by source study and also restricted to participants with reference dates after 1985 (the latter because of an ascertainment delay for women with a reference date before the start of the study in 1986).

Unordered polytomous logistic regression (STATA mlogit; Stata Statistical Software: Release 9, StataCorp) was used to determine odds ratios (as an approximation of the relative risk) and 95% confidence interval (95% CI) for the risk for triple-negative breast cancer and non-triple-negative breast cancer, as well as for ER- and HER2-defined breast cancer. The following known and suspected breast cancer risk factors were examined separately as potential confounders for the main effects of all other risk factors in age-adjusted models: age (at reference), race, education, annual income, family history of breast cancer, body mass index (BMI; kilogram per square meter) 1 y before reference, smoking history, alcohol consumption, age at menarche, number of live births, age at first birth (still or live), lactation history (among parous women), abortion history (among gravid women), and oral contraceptive use (never/<1 y versus ≥1 y, oral contraceptive duration, age at first use, years since first use, and years since last use). Those variables that produced ≥10% change in the odds ratio for any triple-negative breast cancer risk factor were considered as adjustment factors in the final model. All final risk estimates are adjusted for age, family history, lactation history, and oral contraceptive duration (that is, multivariate adjusted). Trend tests for ordered categorical exposure variables were done by including a single grouped linear variable in the polytomous logistic regression model. We excluded nulliparous women from the trend test for age at first birth to evaluate whether an association with breast cancer risk existed beyond the effect of parity alone. To explore whether characteristics of oral contraceptive use were associated with breast cancer risk beyond any effect of never/<1 y versus ≥1 y use, we tested the trend of oral contraceptive duration, age at first use, years since first use, and years since last use among the oral contraceptive users (≥1 y) only.

Odds ratio heterogeneity between tumor subtypes was evaluated by logistic regression restricted to cases. For ordered categorical exposure variables, the P_{heterogeneity} value was based on the significance of a linear trend variable; for age at first birth and the characteristics of oral contraceptive use, P_{heterogeneity} was limited to parous women and oral contraceptive users ≥1 y, respectively. For dichotomous and nominal exposure variables, P_{heterogeneity} was derived from the significance of removing the variable from models based on log-likelihood ratio tests.

In analyses of all 897 breast cancer cases (subtypes combined), the multivariate-adjusted odds ratios for examined risk factors were consistent with the effects observed in previous studies on younger women (Table 1). Specifically, older age, family history of breast cancer, earlier menarche age, induced abortion, and oral contraceptive use were associated with an increased risk for breast cancer. Risk was decreased in relation to greater number of births and younger age at first birth. Oral contraceptive use ≥1 year was associated with a modest increased risk for breast cancer, and among oral contraceptive users only, earlier age at first use further elevated the risk.

Upon examination of the same risk factors in cases with (n = 187) and without (n = 710) triple-negative breast cancer (Table 1), we found that oral contraceptive use ≥1 year (P_{heterogeneity} = 0.008), oral contraceptive duration (P_{heterogeneity} = 0.02), and years since last oral contraceptive use (P_{heterogeneity} = 0.01) conferred significantly different risk estimates by case group, and BMI ≥30 k/m² was associated with a borderline significant increased risk for triple-negative breast cancer (odds ratio, 1.3; 95% CI, 0.8-2.2) and a nonsignificant decreased risk for non-triple-negative breast cancer (odds ratio, 0.8; 95% CI, 0.6-1.2) in women of all ages. Upon restriction to women ages 41 to 45 years, the risk for triple-negative breast cancer in relation to BMI ≥30 k/m² was further elevated (odds ratio, 2.2; 95% CI, 0.9-5.24) whereas that of non-triple-negative breast cancer did not change substantively (odds ratio, 0.9; 95% CI, 0.5-1.6; results not presented). Oral contraceptive use ≥1 year was associated with a 2.5-fold increased risk for triple-negative breast cancer (95% CI, 1.4-4.3) and no significantly increased risk for non-triple-negative breast cancer. Among oral contraceptive users, risk for triple-negative breast cancer increased with longer duration of oral contraceptive use (P_trend = 0.05) and fewer years since last oral contraceptive use (P_trend = 0.04), relationships that were absent for non-triple-negative breast cancer. We attempted to disentangle the effect of oral contraceptive duration versus recency via stratified and adjusted polytomous logistic regression analyses and found that neither risk factor was a more important determinant of risk.

We also examined the effect of oral contraceptive variables across HER2- and ER-defined breast cancer risk to evaluate the influence of each marker separately (Table 2). We found a 2-fold increased risk for ER- breast cancer conferred by oral contraceptive use ≥1 year (odds ratio, 2.0; 95% CI, 1.3-2.9), which differed significantly from the absence of an association with ER+ breast cancer (odds ratio, 1.1; 95% CI, 0.8-1.4; P_{heterogeneity} = 0.005), as did the risk conferred by oral contraceptive duration (P_{heterogeneity} = 0.004) and years since last use (P_{heterogeneity} < 0.001). The risk for ER- breast cancer increased substantially with longer oral contraceptive duration (P_trend = 0.05) and fewer years since use (P_trend = 0.02).

Ever use of oral contraceptives was associated with a modest increased risk for HER2- disease (odds ratio, 1.4; 95% CI, 1.1-1.9) and a lower nonstatistically significant risk for HER2+ disease (odds ratio, 1.2; 95% CI, 0.8-1.7). No significant trends across oral contraceptive use features were observed in relation to the risk for HER2- breast cancer, but risk for HER2+ disease did seem to increase with younger age at first use (P_trend = 0.05). Heterogeneity between HER2 subtypes was not statistically significant for any oral contraceptive use variable. For all aspects of oral contraceptive use, risk estimates were greater for ER- breast cancer than for HER2- breast cancer.

Upon further cross-classification by both ER and HER2 status (Table 3), we observed significantly elevated risk for breast cancer across all oral contraceptive variables consistently and almost exclusively in the ER-/HER2- subset; odds ratios were comparable, only slightly less than those seen in relation to the risk for triple-negative breast cancer. The risk for ER-/HER2- breast cancer increased with longer oral contraceptive duration (P_trend = 0.03) and fewer years since last use (P_trend = 0.04). We observed a large degree of heterogeneity between HER2- ER subtypes according to oral contraceptive use ≥ 1 year (P_{heterogeneity} = 0.01), as well as oral contraceptive duration (P_{heterogeneity} < 0.001) and years since last use (P_{heterogeneity} < 0.001).

Finally, we examined the effect of oral contraceptive use according to triple-negative breast cancer status stratified by age at breast cancer diagnosis ≤40 and 41 to 45 years (Table 4). Among women 41 to 45 years of age, there was no significantly increased risk for breast cancer for any aspect of oral contraceptive use, overall and within triple-negative breast cancer–defined subgroups; however, we did find significant heterogeneity between the risk for triple-negative breast cancer and non-triple-negative breast cancer according to years since last use (P_{heterogeneity} = 0.01). Among triple-negative breast cancer cases ≤40 years of age, all risk estimates for oral contraceptive use variables were approximately two times greater than those in the combined triple-negative breast cancer age group estimates. In women ≤40 years of age, oral contraceptive use ≥1 year was associated with a >4-fold increased risk for triple-negative breast cancer (odds ratio, 4.2; 95% CI, 1.9-9.3) and no increased risk for non-triple-negative breast cancer (odds ratio, 1.2; 95% CI, 0.9-1.7; P_{heterogeneity} < 0.001). Also, among women ≤40 years of age, we found that the risk for breast cancer overall and of non-triple-negative breast cancer increased with younger age at first use (P_trend = 0.02 and 0.04, respectively).