A single droplet digital PCR for ESR1 activating mutations detection in plasma
Authors: Jeannot Emmanuelle , Darrigues Lauren , Michel Marc , Stern Marc-Henri , Pierga Jean-Yves , Rampanou Aurore , Melaabi Samia , Benoist Camille , Bièche Ivan , Vincent-Salomon Anne , Ayachy Radouane El , Noret Aurélien , Epaillard Nicolas , Cabel Luc , Bidard François-Clément , Proudhon Charlotte 
Published: January 2019

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.

List of abbreviations

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

Human genes

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