Purpose Given the clinical relevance of ESR1 mutations as potential drivers of resistance to endocrine therapy, this study used sensitive detection methods to determine the frequency of ESR1 mutations in primary and metastatic breast cancer, and in cell free DNA (cfDNA). Patients and Methods Six ESR1 mutations (K303R, S463P, Y537C, Y537N, Y537S, D538G) were assessed by digital droplet PCR (ddPCR), with lower limits of detection of 0.05% to 0.16%, in primary tumors (n=43), bone (n=12) and brain metastases (n=38), and cfDNA (n=29). Correlations between ESR1 mutations in metastatic lesions and single (1 patient) or serial blood draws (4 patients) were assessed. Results ESR1 mutations were detected for D538G (n=13), Y537S (n=3) and Y537C (n=1), and not for K303R, S463P or Y537N. Mutation rates were 7.0% (3/43 primary tumors), 9.1% (1/11 bone metastases), 12.5% (3/24 brain metastases), and 24.1% (7/29 cfDNA). Two patients showed polyclonal disease with more than one ESR1 mutation. Mutation allele frequencies were 0.07% to 0.2% in primary tumors, 1.4% in bone metastases, 34.3 to 44.9% in brain metastases, and 0.2% to 13.7% in cfDNA. In cases with both cfDNA and metastatic samples (n=5), mutations were detected in both (n=3) or in cfDNA only (n=2). Treatment was associated with changes in ESR1 mutation detection and allele frequency. Conclusions ESR1 mutations were detected at very low allele frequencies in some primary breast cancers, and at high allele frequency in metastases, suggesting that in some tumors rare ESR1 mutant clones are enriched by endocrine therapy. Further studies should address if sensitive detection of ESR1 mutations in primary breast cancer and in serial blood draws may be predictive for development of resistant disease.

RNA-seq profiling of longitudinally collected specimens uncovered recurrent gene expression acquisitions in metastatic tumors, distinct from matched primary tumors. Critically, we identify aberrations in key oncogenic pathways and provide functional evidence for their suitability as therapeutic targets. Altogether, this study establishes recurrent, acquired vulnerabilities in BrM that warrant immediate clinical investigation and suggests paired specimen expression profiling as a compelling and underutilized strategy to identify targetable dependencies in advanced cancers.

BackgroundEstrogen 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 methodsTo 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.ResultsWe 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.ConclusionsCollectively, 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.

Purpose: We examined the role of ERBB2 activating mutations in endocrine therapy resistance in estrogen receptor positive (ER+) breast cancer. Design: ERBB2 mutation frequency was determined from large genomic databases. Isogenic knock-in ERBB2 mutations in ER+ MCF7 cells and xenografts were used to investigate estrogen-independent growth. Structural analysis was used to determine the molecular interaction of HERL755S with HER3. Small molecules and siRNAs were used to inhibit PI3Kα, TORC1 and HER3. Results: Genomic data revealed a higher rate of ERBB2 mutations in metastatic vs. primary ER+ tumors. MCF7 cells with isogenically incorporated ERBB2 kinase domain mutations exhibited resistance to estrogen deprivation and to fulvestrant both in vitro and in vivo, despite maintaining inhibition of ERα transcriptional activity. Addition of the irreversible HER2 tyrosine kinase inhibitor neratinib restored sensitivity to fulvestrant. HER2-mutant MCF7 cells expressed higher levels of p-HER3, p-AKT and p-S6 than cells with wild type HER2. Structural analysis of the HER2L755S variant implicated a more flexible active state, potentially allowing for enhanced dimerization with HER3. Treatment with a PI3Kα inhibitor, a TORC1 inhibitor or HER3 siRNA, but not a MEK inhibitor, restored sensitivity to fulvestrant and to estrogen deprivation. Inhibition of mutant HER2 or TORC1, when combined with fulvestrant, equipotently inhibited growth of MCF7/ERBB2V777L xenografts, suggesting a role for TORC1 in antiestrogen resistance induced by ERBB2 mutations. Conclusions: ERBB2 mutations hyperactivate the HER3/PI3K/AKT/mTOR axis, leading to antiestrogen resistance in ER+ breast cancer. Dual blockade of the HER2 and ER pathways is required for the treatment of ER+/HER2 mutant breast cancers. Translational Significance ERBB2 activating mutations occur with increased frequency in ER+ breast cancers after progression on antiestrogen therapy. Inhibition of mutant HER2 function with neratinib restores the efficacy of antiestrogen therapy. Thus, we propose dual blockade of the HER2 and ER pathways is required for the treatment of ER+/HER2 mutant breast cancers.