Lessons learned from a candidate gene study investigating aromatase inhibitor treatment outcome in breast cancer

Abstract

The role of germline genetics in adjuvant aromatase inhibitor (AI) treatment efficacy in ER-positive breast cancer is poorly understood. We employed a two-stage candidate gene approach to examine associations between survival endpoints and common germline variants in 753 endocrine resistance-related genes. For a discovery cohort, we screened the Breast Cancer Association Consortium database (n ≥ 90,000 cases) and retrieved 2789 AI-treated patients. Cox model-based analysis revealed 125 variants associated with overall, distant relapse-free, and relapse-free survival (p-value ≤ 1E-04). In validation analysis using five independent cohorts (n = 8857), none of the six selected candidates representing major linkage blocks at CELA2B/CASP9, NR1I2/GSK3B, LRP1B, and MIR143HG (CARMN) were validated. We discuss potential reasons for the failed validation and replication of published findings, including study/treatment heterogeneity and other limitations inherent to genomic treatment outcome studies. For the future, we envision prospective longitudinal studies with sufficiently long follow-up and endpoints that reflect the dynamic nature of endocrine resistance.

Fig. 1. Flow diagram of BCAC patients with primary invasive ER-positive breast cancer, early-stage disease characteristics and AI therapy.

The BCAC database (version 12) comprised 92,933 patients with a confirmed diagnosis of invasive ER-positive breast cancer who are of European descent (based on genotype data) and older than or equal to 18 years of age. Of these, 27,887 patients had early-stage disease with documented adjuvant endocrine therapy (AI, Tamoxifen, switch therapy, or unspecified) and complete follow-up data. The subgroup of AI-treated patients analysed in this study comprised 2789 women of which 1335 had received anastrozole, 689 letrozole, and 114 exemestane. For 570 patients, AI treatment was not further specified and 81 patients had received an additional endocrine treatment. AI Aromatase Inhibitor, OS Overall survival, DRFS Distant relapse free survival, RFS Relapse free survival.

Fig. 2. Candidate gene association study of 15-year overall, distant relapse-free, and relapse-free survival in AI-treated BCAC patients (discovery cohort).

The Manhattan plots display the results of unadjusted analyses (stratified by country and corrected for genetic principal components). The y-axis shows the –log10 meta-analysis p-values and the x-axis the chromosomal positions of the variants. Variants are located within 753 selected candidate genes or surrounding gene region (defined as +/- 10 kb from gene start/end). For variants with association p-value ≤ 1E-04, the corresponding gene symbols are displayed on top (“intergenic” in case no gene was annotated). Genes marked in bold were part of the 753 candidate genes. Analysis of (a) patients treated with any AI (n = 2789) and approximately 170,000 variants with MAF ≥ 0.05, (b) patients treated with anastrozole (n = 1335) and approximately 138,000 variants with MAF ≥ 0.1, and (c) patients treated with letrozole (n = 689) and approximately 138,000 variants with MAF ≥ 0.1. Survival endpoints include OS (overall survival), DRFS (distant relapse-free survival), and RFS (relapse-free survival).

Fig. 3. Results of meta-analyses for selected candidate variants in validation cohorts and BCAC patients (discovery cohort).

Forest plots depicting per-allele hazard ratios (x-axis) and corresponding confidence intervals for (a) rs3107669, (b) rs353298, and (c) rs353296 in eligible validation cohorts. The size of the squares reflects the study size. The KARMA/pKARMA studies were omitted in DRFS and RFS analyses due to delayed study entry of nearly all 643 patients ( > 0.5 years after diagnosis). Diamonds represent results of random effects (RE) meta-analyses in validation cohorts and of the discovery analyses in BCAC patients, respectively. Endpoints are OS (overall survival), DRFS (distant relapse-free survival), and RFS (relapse-free survival).