Emergence of constitutively active estrogen receptor-α mutations in pretreated advanced estrogen receptor-positive breast cancer

Abstract

Purpose: We undertook this study to determine the prevalence of estrogen receptor (ER) α (ESR1) mutations throughout the natural history of hormone-dependent breast cancer and to delineate the functional roles of the most commonly detected alterations.

Experimental design: We studied a total of 249 tumor specimens from 208 patients. The specimens include 134 ER-positive (ER(+)/HER2(-)) and, as controls, 115 ER-negative (ER(-)) tumors. The ER(+) samples consist of 58 primary breast cancers and 76 metastatic samples. All tumors were sequenced to high unique coverage using next-generation sequencing targeting the coding sequence of the estrogen receptor and an additional 182 cancer-related genes.

Results: Recurring somatic mutations in codons 537 and 538 within the ligand-binding domain of ER were detected in ER(+) metastatic disease. Overall, the frequency of these mutations was 12% [9/76; 95% confidence interval (CI), 6%-21%] in metastatic tumors and in a subgroup of patients who received an average of 7 lines of treatment the frequency was 20% (5/25; 95% CI, 7%-41%). These mutations were not detected in primary or treatment-naïve ER(+) cancer or in any stage of ER(-) disease. Functional studies in cell line models demonstrate that these mutations render estrogen receptor constitutive activity and confer partial resistance to currently available endocrine treatments.

Conclusions: In this study, we show evidence for the temporal selection of functional ESR1 mutations as potential drivers of endocrine resistance during the progression of ER(+) breast cancer.

Fig. 1. Location of the ER mutations and frequencies per cohort

(A) Diagram of ER with detected predicted somatic point mutations designated with a circle at the representative protein position. * Denotes a mutation found in a primary ER negative breast cancer. (B) Frequency of the 538/537 substitution mutations and ER amplifications per cohort in ER positive tumors showing an increase in frequency with the progression and increase in number of treatment lines for ER positive breast cancer. Each bar represents the percentage of patients with a mutation and the different colors within the bars represent the frequency of the indicated alteration. In the EM+ cohort one patient had both an ER amplification and an Y537S mutation.

Fig. 2. Genetic alteration in primary versus metastatic breast cancer

The frequencies of the common genomic alterations found in our cohorts comparing primary tumors versus metastatic samples shows a significant increase in the ESR1 alterations in metastatic samples. P-value calculated using the Fisher's exact test.

Fig. 3. Analysis of the transcriptional activity of the recurring mutant ER and dose response to tamoxifen and fulvestrant

(A) Transient transfection of 293T cells with wild-type (WT) or mutant ER expression vectors in addition to ERE-TK-Luc reporter vector. ER expression levels were determined to be equivalent by western-blot. β-actin expression level is shown as an internal loading control (left panel). Luciferase activity comparison of transfected 293 cells then treated for 24 hours with 10nM estrogen (E₂) or vehicle (ethanol) for 24hours. Experiments were done in triplicates and repeated three times. Data represent mean +/−SD and are normalized by Beta-galactosidase internal control activity (left panel). (B) Luciferase activity comparison of 293T cells transfected with WT or mutant ER expression vectors in addition to ERE-TK-Luc reporter vector, then treated with vehicle (ethanol) or indicated dose of E2 and increasing doses of 4-hydroxytamoxifen (4-OHT) or fulvestrant (Fulv.) for 24hours. Response to 4-OHT and fulvestrant is normalized to the response to E₂ stimulation. Data represent mean +/−SD and are normalized by Beta-galactosidase internal control activity. Unpaired two-tail t-test was used to examine the statistical differences between the WT and Y537N mutant and p-values are shown. (C) Luciferase activity levels of 293T cells transfected with WT or mutant ER expression vectors in addition to ERE-TK-Luc reporter vector after treatment with a wide range of E2 doses. Activity levels were not altered by high doses of E2.

Fig.4. Transcriptional activity and growth of mutant ER in a breast cancer cell line

(A) Transient transfection of MCF7 cells with empty vector (E.V.), wild-type (wt) or mutant Y537N ER expression vectors, then grown in complete medium (grey bars), hormone depleted medium (white bars) or hormone depleted medium with 10nM E2 (black bars). Relative mRNA levels of ER target genes GREB1 and PR (left panel) and pS2/TFF1 and CA12 (right panel) were determined by real-time PCR. Bars represent fold change +/− SD of two independent experiments. (B) MCF7 cells were transfected with WT or Y537N ER. Cells were grown in E₂ deprived conditions alone, with 4-OHT or fulvestrant and monitored for 5 days. Histograms depict cell count at day 5. Unpaired two-tail t-test was used to examine the statistical difference and p-value is shown. (C) Western blot of ER levels in MCF7 cells after transfection of either WT-ER or Y537N mutant ER showing similar expression levels.