Detection of activating estrogen receptor gene (ESR1) mutations in single circulating tumor cells

Purpose: Early detection is essential for treatment plans before onset of metastatic disease. Our purpose was to demonstrate feasibility to detect and monitor estrogen receptor 1 (ESR1) gene mutations at the single circulating tumor cell (CTC) level in metastatic breast cancer (MBC). Experimental Design: We used a CTC molecular characterization approach to investigate heterogeneity of 14 hotspot mutations in ESR1 and their correlation with endocrine resistance. Combining the CellSearch and DEPArray technologies allowed recovery of 71 single CTCs and 12 WBC from 3 ER-positive MBC patients. Forty CTCs and 12 WBC were subjected to whole genome amplification by MALBAC and Sanger sequencing. Results: Among 3 selected patients, 2 had an ESR1 mutation (Y537). One showed two different ESR1 variants in a single CTC and another showed loss of heterozygosity. All mutations were detected in matched cell-free DNA (cfDNA). Furthermore, one had 2 serial blood samples analyzed and showed changes in both cfDNA and CTCs with emergence of mutations in ESR1 (Y537S and T570I), which has not been reported previously. Conclusions: CTCs are easily accessible biomarkers to monitor and better personalize management of patients with previously demonstrated ER-MBC who are progressing on endocrine therapy. We showed that single CTC analysis can yield important information on clonal heterogeneity and can be a source of discovery of novel and potential driver mutations. Finally, we also validate a workflow for liquid biopsy that will facilitate early detection of ESR1 mutations, the emergence of endocrine resistance and the choice of further target therapy. ©2017 AACR

Genomic modelling of the ESR1 Y537S mutation for evaluating function and new therapeutic approaches for metastatic breast cancer

Drugs that inhibit estrogen receptor-α (ER) activity have been highly successful in treating and reducing breast cancer progression in ER-positive disease. However, resistance to these therapies presents a major clinical problem. Recent genetic studies have shown that mutations in the ER gene are found in >20% of tumours that progress on endocrine therapies. Remarkably, the great majority of these mutations localize to just a few amino acids within or near the critical helix 12 region of the ER hormone binding domain, where they are likely to be single allele mutations. Understanding how these mutations impact on ER function is a prerequisite for identifying methods to treat breast cancer patients featuring such mutations. Towards this end, we used CRISPR-Cas9 genome editing to make a single allele knock-in of the most commonly mutated amino acid residue, tyrosine 537, in the estrogen-responsive MCF7 breast cancer cell line. Genomic analyses using RNA-seq and ER ChIP-seq demonstrated that the Y537S mutation promotes constitutive ER activity globally, resulting in estrogen-independent growth. MCF7-Y537S cells were resistant to the anti-estrogen tamoxifen and fulvestrant. Further, we show that the basal transcription factor TFIIH is constitutively recruited by ER-Y537S, resulting in ligand-independent phosphorylation of Serine 118 (Ser118) by the TFIIH kinase, cyclin-dependent kinase (CDK)7. The CDK7 inhibitor, THZ1 prevented Ser118 phosphorylation and inhibited growth of MCF7-Y537S cells. These studies confirm the functional importance of ER mutations in endocrine resistance, demonstrate the utility of knock-in mutational models for investigating alternative therapeutic approaches and highlight CDK7 inhibition as a potential therapy for endocrine-resistant breast cancer mediated by ER mutations

Targeted Mutation Detection in Advanced Breast Cancer Using MammaSeq Identifies RET as a Potential Contributor to Breast Cancer Metastasis

The lack of any reported breast cancer specific diagnostic NGS tests inspired the development of MammaSeq, an amplicon based NGS panel built specifically for use in advanced breast cancer. In a pilot study to define the clinical utility of the panel, 46 solid tumor samples, plus an additional 14 samples of circulating-free DNA (cfDNA) from patients with advanced breast cancer were sequenced and analyzed using the OncoKB precision oncology database. We identified 26 clinically actionable variants (levels 1-3) annotated by the OncoKB precision oncology database, distributed across 20 out of 46 solid tumor cases (40%), and 4 clinically actionable mutations distributed across 4 samples in the 14 cfDNA sample cohort (29%). The mutation allele (MAF) frequencies of ESR1-D538G and FOXA1-Y175C mutations correlated with CA.27.29 levels in patient-matched blood, indicating that MAF may be a reliable marker for disease burden. Interestingly, 4 of the mutations found in metastatic samples occurred in the gene RET, an oncogenic receptor tyrosine kinase. In an orthogonal study, the lab has recently identified RET as one of the most recurrently upregulated genes in breast cancer brain metastases. Interestingly, the ligand for RET is the family of glial-cell derived neurotrophic factors (GDNF), a growth factor secreted by glial cells of the central nervous system. This lead to the hypothesis that RET overexpression facilitates breast cancer brain metastasis in response to the high levels of GDNF, while RET activating point mutations increase metastatic capacity without specific organ tropism. While the effect of GDNF treatment on proliferation in 2D was limited, in ultra-low attachment (ULA) plates we saw a significant increase in anchorage independent growth of MCF-7 cells. To determine if GDNF acts as a chemoattractant for RET positive BrCa cells, we utilized a transwell migration assay, with GDNF as the sole chemoattractant. When RET was overexpressed, there was a visual increase in cell migration. Together, these studies demonstrate the clinical feasibility of using MammaSeq to detect clinically actionable mutations in breast cancer patients, and provides provisional data supporting the investigation of RET signaling as a potentially targetable mediator of breast cancer brain metastasis

Estrogen receptor mutations and splice variants determined in liquid biopsies from metastatic breast cancer patients

Mutations and splice variants in the estrogen receptor (ER) gene, ESR1, may yield endocrine resistance in metastatic breast cancer (MBC) patients. These putative endocrine resistance markers are likely to emerge during treatment, and therefore, its detection in liquid biopsies, such as circulating tumor cells (CTCs) and cell-free DNA (cfDNA), is of great interest. This research aimed to determine whether ESR1 mutations and splice variants occur more frequently in CTCs of MBC patients progressing on endocrine treatment. In addition, the presence of ESR1 mutations was evaluated in matched cfDNA and compared to CTCs. CellSearch-enriched CTC fractions (≥5/7.5 mL) of two MBC cohorts were evaluated, namely (a) patients starting first-line endocrine therapy (n = 43, baseline cohort) and (b) patients progressing on any line of endocrine therapy (n = 40, progressing cohort). ESR1 hotspot mutations (D538G and Y537S/N/C) were evaluated in CTC-enriched DNA using digital PCR and compared with matched cfDNA (n = 18 baseline cohort; n = 26 progressing cohort). Expression of ESR1 full-length and 4 of its splice variants ((increment)5, (increment)7, 36 kDa, and 46 kDa) was evaluated in CTC-enriched mRNA. It was observed that in the CTCs, the ESR1 mutations were not enriched in the progressing cohort (8%), when compared with the baseline cohort (5%) (P = 0.66). In the cfDNA, however, ESR1 mutations were more prevalent in the progressing cohort (42%) than in the baseline cohort (11%) (P = 0.04). Three of the same mutations were observed in both CTCs and cfDNA, 1 mutation in CTCs only, and 11 in cfDNA only. Only the (increment)5 ESR1 splice variant was CTC-specific expressed, but was not enriched in the progressing cohort. In conclusion, sensitivity for detecting ESR1 mutations in CTC-enriched fractions was lower than for cfDNA. ESR1 mutations detected in cfDNA, rarely present at the start of first-line endocrine therapy, were enriched at progression, strongly suggesting a role in conferring endocrine resistance in MBC

Endocrine resistance in hormone receptor positive breast cancer–from mechanism to therapy

The importance and role of the estrogen receptor (ER) pathway has been well-documented in both breast cancer (BC) development and progression. The treatment of choice in women with metastatic breast cancer (MBC) is classically divided into a variety of endocrine therapies, 3 of the most common being: selective estrogen receptor modulators (SERM), aromatase inhibitors (AI) and selective estrogen receptor down-regulators (SERD). In a proportion of patients, resistance develops to endocrine therapy due to a sophisticated and at times redundant interference, at the molecular level between the ER and growth factor. The progression to endocrine resistance is considered to be a gradual, step-wise process. Several mechanisms have been proposed but thus far none of them can be defined as the complete explanation behind the phenomenon of endocrine resistance. Although multiple cellular, molecular and immune mechanisms have been and are being extensively studied, their individual roles are often poorly understood. In this review, we summarize current progress in our understanding of ER biology and the molecular mechanisms that predispose and determine endocrine resistance in breast cancer patients

What to expect from high throughput genomics in metastatic breast cancers?

peer reviewe

Positron emission tomography of tumour [18F]fluoroestradiol uptake in patients with acquired hormone-resistant metastatic breast cancer prior to oestradiol therapy

Purpose Whereas anti-oestrogen therapy is widely applied to treat oestrogen receptor (ER) positive breast cancer, paradoxically, oestrogens can also induce tumour regression. Upregulation of ER expression is a marker for oestrogen hypersensitivity. We, therefore, performed an exploratory study to evaluate positron emission tomography (PET) with the tracer 16 alpha-[F-18] fluoro-17 beta-oestradiol (F-18-FES) as potential marker to select breast cancer patients for oestradiol therapy. Methods Eligible patients had acquired endocrine-resistant metastatic breast cancer that progressed after >= 2 lines of endocrine therapy. All patients had prior ER-positive histology. Treatment consisted of oestradiol 2 mg, three times daily, orally. Patients underwent F-18-FES-PET/CT imaging at baseline. Tumour F-18-FES-uptake was quantified for a maximum of 20 lesions and expressed as maximum standardised uptake value (SUVmax). CT-scan was repeated every 3 months to evaluate treatment response. Clinical benefit was defined as time to radiologic or clinical progression >= 24 weeks. Results F-18-FES uptake, quantified for 255 lesions in 19 patients, varied greatly between lesions (median 2.8; range 0.6-24.3) and between patients (median 2.5; range 1.1-15.5). Seven (37 %) patients experienced clinical benefit of oestrogen therapy, eight progressed (PD), and four were non-evaluable due to side effects. The positive and negative predictive value PPV/NPV) of F-18-FES-PET for response to treatment were 60 % (95 % CI: 31-83 %) and 80 % (95 % CI: 38-96 %), respectively, using SUVmax >1.5. Conclusion F-18-FES-PET may aid identification of patients with acquired antihormone resistant breast cancer that are unlikely to benefit from oestradiol therapy

Cell-free DNA mutations as biomarkers in breast cancer patients receiving tamoxifen

The aim was to identify mutations in serum cell-free DNA (cfDNA) associated with disease progression on tamoxifen treatment in metastatic breast cancer (MBC). Sera available at start of therapy, during therapy and at disease progression were selected from 10 estrogen receptor (ER)-positive breast cancer patients. DNA from primary tumor and normal tissue and cfDNA from minute amounts of sera were analyzed by targeted next generation sequencing (NGS) of 45 genes (1,242 exons). At disease progression, stop-gain single nucleotide variants (SNVs) for CREBBP (1 patient) and SMAD4 (1 patient) and non-synonymous SNVs for AKAP9 (1 patient), PIK3CA (2 patients) and TP53 (2 patients) were found. Mutations in CREBBP and SMAD4 have only been occasionally reported in breast cancer. All mutations, except for AKAP9, were also present in the primary tumor but not detected in all blood specimens preceding progression. More sensitive detection by deeper re-sequencing and digital PCR confirmed the occurrence of circulating tumor DNA (ctDNA) and these biomarkers in blood specimens