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. 2018 Apr 1;29(4):872-880.
doi: 10.1093/annonc/mdy025.

Recurrent Hyperactive ESR1 Fusion Proteins in Endocrine Therapy-Resistant Breast Cancer

Free PMC article

Recurrent Hyperactive ESR1 Fusion Proteins in Endocrine Therapy-Resistant Breast Cancer

R J Hartmaier et al. Ann Oncol. .
Free PMC article


Background: Estrogen receptor-positive (ER-positive) metastatic breast cancer is often intractable due to endocrine therapy resistance. Although ESR1 promoter switching events have been associated with endocrine-therapy resistance, recurrent ESR1 fusion proteins have yet to be identified in advanced breast cancer.

Patients and methods: To identify genomic structural rearrangements (REs) including gene fusions in acquired resistance, we undertook a multimodal sequencing effort in three breast cancer patient cohorts: (i) mate-pair and/or RNAseq in 6 patient-matched primary-metastatic tumors and 51 metastases, (ii) high coverage (>500×) comprehensive genomic profiling of 287-395 cancer-related genes across 9542 solid tumors (5216 from metastatic disease), and (iii) ultra-high coverage (>5000×) genomic profiling of 62 cancer-related genes in 254 ctDNA samples. In addition to traditional gene fusion detection methods (i.e. discordant reads, split reads), ESR1 REs were detected from targeted sequencing data by applying a novel algorithm (copyshift) that identifies major copy number shifts at rearrangement hotspots.

Results: We identify 88 ESR1 REs across 83 unique patients with direct confirmation of 9 ESR1 fusion proteins (including 2 via immunoblot). ESR1 REs are highly enriched in ER-positive, metastatic disease and co-occur with known ESR1 missense alterations, suggestive of polyclonal resistance. Importantly, all fusions result from a breakpoint in or near ESR1 intron 6 and therefore lack an intact ligand binding domain (LBD). In vitro characterization of three fusions reveals ligand-independence and hyperactivity dependent upon the 3' partner gene. Our lower-bound estimate of ESR1 fusions is at least 1% of metastatic solid breast cancers, the prevalence in ctDNA is at least 10× enriched. We postulate this enrichment may represent secondary resistance to more aggressive endocrine therapies applied to patients with ESR1 LBD missense alterations.

Conclusions: Collectively, these data indicate that N-terminal ESR1 fusions involving exons 6-7 are a recurrent driver of endocrine therapy resistance and are impervious to ER-targeted therapies.


Figure 1.
Figure 1.
Rearrangements (REs) in metastatic breast cancer and the identification of ESR1 fusion proteins. (A) Tileplots show the distribution of REs across tumors from Pitt patients 1–6. REs with breakpoints in two genes and supporting RNAseq gene fusion evidence are indicated in green. (inset) Circos plot of copy number variation data for primary (outer ring) and nodal recurrence (inner ring) and structural variants specific to the nodal recurrence (arcs in center). (B, E) RNAseq split read evidence, (C, F) RT-PCR validation, and (D, G) immunoblot of ESR1 fusions in Pitt-1 and Pitt-7, respectively. (D) Overexpression in HEK293 cells was used for positive controls.
Figure 2.
Figure 2.
ESR1 fusion proteins identified via direct discordant read evidence. Gene fusions have clustered junctions in ESR1 between exons 6 and 7. The ESR1 reference transcript is shown at the top with functional domains overlaid. AF1, ligand-independent activation domain; DBD, DNA-binding domain; H, hinge region; AF2, ligand-dependent activation domain. *FMI-78 was called initially as copyshift-positive only, with subsequent RNAseq identified the ESR1-TFG fusion. (B–D) Clinical history summaries for patients with ESR1 fusion positive tumors. Timelines are drawn to scale. Green vertical lines represent the primary tumor surgery while red lines represent major metastatic events (see supplementary Table S9, available at Annals of Oncology online, for more details).
Figure 3.
Figure 3.
ESR1 fusion proteins are constitutively hyperactive. (A) ESR1 fusion activity determined via ERE-Tk-Luc assay in HEK293 cells. Data points represent mean firefly luciferase values normalized to renilla (transfection control) from three independent transfections in one experiment. Similar results were repeated in three independent experiments. Drug concentrations are as follows: E2 (estradiol: 0.1 nM), 4OHT (4-hydroxy-tamoxifen: 100 nM), ICI (ICI-182, 780: 100 nM). Error bars represent standard deviation. (B) HEK293 cells were transfected with ESR1 and/or ESR1-DAB2. Immunoblot analysis was performed for both the WT and fusion forms of ESR1 and for pS118. Untransfected MCF-7 cells in the absence (i.e. charcoal stripped) and presence of signaling (FBS) were included as a positive control of pS118. (C) Heatmap showing expression of classic estrogen responsive genes in metastatic tumor samples (normalized to paired primary tumor) and MCF-7 (–/+ estrogen as a positive control of E2 signaling) by NanoString analysis.
Figure 4.
Figure 4.
Recurrent ESR1 copyshift at exon 6 is enriched in ER-positive metastatic breast cancer. (A) Samples containing ESR1 fusion proteins are often positive for copyshift. Adapting the method to mate pair sequencing data also showed ESR1-DAB2 is copyshift-positive (dashed line). (B) Heatmap showing the shift in copy number across ESR1. Of the 9542 breast cancer genomic profiles examined, the 83 copyshift positive are shown. FMI-52 is highlighted with the red arrowhead. (C) Tileplot illustrates the genomic context of ESR1 fusions and copyshift positivity. ESR1 alteration containing samples (point mutations, fusions, copyshift) are found predominantly in metastatic disease.

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