Abstract
Background Activating mutations in the estrogen receptor 1 (ESR1) gene are recurrent mechanisms of acquired resistance to aromatase inhibitors (AI), and may be the target of other selective estrogen receptor down-regulators. To assess the clinical utility of monitoring ESR1 resistant mutations, a droplet digital PCR (ddPCR)-based assay compatible with body fluids is ideal due to its cost-effectiveness and quick turnaround.
Methods We designed a multiplex ddPCR, which combines a drop-off assay, targeting the clustered hotspot mutations found in exon 8, with another pair of probes interrogating the E380Q mutation in exon 5. We assessed its sensitivity in vitro using synthetic oligonucleotides, harboring E380Q, L536R, Y537C, Y537N, Y537S or D538G mutations. Validation of the assay was performed on plasma samples from a prospective study and compared to next generation sequencing (NGS) data.
Results The multiplex ESR1-ddPCR showed a high sensitivity with a limit of detection ranging from 0.07 to 0.19% in mutant allele frequency depending on the mutation tested. The screening of plasma samples from patients with AI-resistant metastatic breast cancer identified ESR1 mutations in 29% of them with perfect concordance (and higher sensitivity) to NGS data obtained in parallel. Additionally, this test identifies patients harboring polyclonal alterations. Furthermore, the monitoring of ctDNA using this technique during treatment follow-up predicts the radiological response to palbociclib-fulvestrant.
Conclusion The multiplex ESR1-ddPCR detects, in a single reaction, the most frequent ESR1 activating mutations and is compatible with plasma samples. This method is thus suitable for real-time ESR1 mutation monitoring in large cohorts of patients.
Statement of translational relevance Exons 5 and 8 mutations in ESR1 are recurrent mechanisms of resistance to aromatase inhibitors (AI) in estrogen receptor (ER)-positive metastatic breast cancer and may be targeted by selective ER down-regulators. We implemented a novel droplet digital PCR, which allows for the detection of the most frequent ESR1 mutations in circulating cell-free DNA. In prospectively collected plasma samples, ESR1 mutations were found in 29% of AI-resistant patients, with excellent concordance and higher sensitivity to next generation sequencing. Moreover, circulating ESR1 mutations appear to be reliable markers for ctDNA monitoring in order to predict treatment response. Ultimately, the short turnaround time, high sensitivity and limited cost of the ESR1-ddPCR are compatible with repeated samplings to detect the onset of resistance to AI before the radiological progression. This opens a window of opportunity to develop new clinical strategies for breast cancer hormone therapy, as tested in an ongoing phase 3 trial.
- AI
- Aromatase Inhibitor
- cfDNA
- Cell-free DNA
- ctDNA
- Circulating tumor DNA
- ddPCR
- Droplet digital PCR
- ER+ HER2-MBC
- ER+ HER2-negative Metastatic Breast Cancer
- ER
- Estrogen Receptor
- ER+
- Estrogen Receptor positive
- LOB
- Limit of blank
- LOD
- Limit of detection
- MAF
- Mutant Allele Frequency
- PBMC
- Peripheral blood mononuclear cells
- PD
- Progressive disease
- SD
- Standard deviation
- ToP
- Time of progression
- WT
- Wild type
ESR1: Estrogen Receptor 1
HER2: Human Epidermal Growth Factor Receptor 2
EGFR: Epithelial Growth Factor Receptor
KRAS: KRAS proto-oncogene, GTPase
BRAF: B-Raf Proto-Oncogene, Serine/Threonine kinase
Footnotes
↵* These authors share senior co-authorship
Financial support: SIRIC 2 Curie (grant INCa-DGOS-Inserm_12554) and the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115749 (project Cancer ID). The funding sources of the study had no role in the design of the study, collection, analysis or interpretation of the data or in the writing of this report.
Disclosure: C.P., E.J., M.H.S., and F.C.B. have ongoing patent applications relating to circulating tumor DNA analysis.