ABSTRACT
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.
Subject(s)
Antineoplastic Agents, Hormonal/therapeutic use , Breast Neoplasms/drug therapy , Drug Resistance, Neoplasm/genetics , Estrogen Receptor alpha/metabolism , Recombinant Fusion Proteins/metabolism , Breast Neoplasms/pathology , Estrogen Receptor alpha/genetics , Female , High-Throughput Nucleotide Sequencing , Humans , Mutation , Neoplasm Metastasis , Recombinant Fusion Proteins/geneticsABSTRACT
Time-kill curve experiments were performed with linezolid, doripenem, tigecycline, moxifloxacin, and daptomycin against Staphylococcus aureus and with colistin, moxifloxacin, and doripenem against Pseudomonas aeruginosa to evaluate the effect of porcine pulmonary surfactant on antimicrobial activity. Pulmonary surfactant significantly impaired the activities of moxifloxacin and colistin. When antibiotics are being developed for respiratory tract infections, the method described here might be used to preliminarily quantify the effect of pulmonary surfactant on antimicrobial activity.
