Abstract S1-03: Identification of base pair mutations and structural rearrangements acquired in breast cancer metastases including a novel hyperactive ESR1-DAB2 fusion gene specifically in hormone-resistant recurrence
Authors: Hartmaier Ryan J , Puhalla Shannon L , Oesterreich Steffi , Bahreini Amir , Davidson Nancy E , Brufsky Adam M , Lee Adrian V 
Published: May 2015

Abstract
DNA structural variations (SVs) are a major source of genetic instability in cancer, but they remain understudied. Large-insert mate-pair sequencing (MPS) is a powerful method designed to detect SVs, even in highly repetitive regions. Using MPS and other methods, we performed a comprehensive analysis of genomic alterations in breast cancer progression.
Matched primary/recurrent frozen tumor samples from 6 patients, including two patients from our rapid autopsy program with multiple metastatic tissues (20 total samples; average 5.5 years to recurrence) were examined by multiple large-insert library (3-5, 5-8, 8-12kb) MPS to identify metastatic acquired SVs. This was supplemented with RNAseq (n=15), whole exome sequencing (n=18; ∼75x), whole genome sequencing (n=3; 40-65x), and SNP arrays.
A relatively small fraction (∼10%) of somatic single nucleotide variants (SNVs) in the primary tumor were identified in matched metastatic samples, and the majority of metastatic SNVs were not found in the matched primary tumor. This indicates that a rare sub-clone colonizes the metastatic site and evolves extensively before becoming clinically evident. For example, in one patient with an ER+ tumor who initially declined anti-estrogen therapy, the recently described ESR1 Y537S mutation was not present in the primary tumor or in metastatic disease 5 years later. However, after extensive anti-estrogen treatment for metastatic disease, the mutation was identified at rapid autopsy, indicating that this mutation can be acquired even after initial metastatic spread.
We observed extensive patient-to-patient variability in the number and types of SVs. In general, the overall pattern of SVs was remarkably similar between matched primary and metastatic samples, however, we identified a number of metastatic specific SVs that likely contribute to disease progression. Specifically, in one patient with an ER+ primary tumor treated with adjuvant Tamoxifen, we identified a novel fusion gene between ESR1 (estrogen receptor-α, ERα) and DAB2 (disabled-2) only in a lymph node recurrence. RT-PCR and western blot analysis confirmed that the fusion RNA/protein was expressed/translated only in the recurrent disease. The fusion retains the DNA-binding domain (DBD) and hinge region of ERα while the ligand-binding domain (LBD) is replaced with the majority of DAB2. We hypothesized that this is a functional genetic alteration conferring ligand-independent ERα-mediated signaling and growth. Confirming this, in vitro ERE-Tk-luc reporter assays showed that the ESR1-DAB2 fusion has ligand-independent activity that is 13-290x higher than wild-type ERα. Chromatin immunoprecipitation assays in metastatic tissue from tumors with mutant ERα show strong enrichment for ERα at classical ERα target genes. We are currently assessing the genome-wide binding of ESR1-DAB2 and the functional contribution of DAB2 to the fusion protein.
This study represents the most comprehensive analysis to date of genomic changes in breast cancer progression and indicates extensive changes occur during metastatic spread. A number of acquired changes likely represent therapeutically targetable metastatic dependencies.
Citation Format: Ryan J Hartmaier, Shannon L Puhalla, Steffi Oesterreich, Amir Bahreini, Nancy E Davidson, Adam M Brufsky, Adrian V Lee. Identification of base pair mutations and structural rearrangements acquired in breast cancer metastases including a novel hyperactive ESR1-DAB2 fusion gene specifically in hormone-resistant recurrence [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S1-03.