ESR1 NAPA Assay: Development and Analytical Validation of a Highly Sensitive and Specific Blood-Based Assay for the Detection of ESR1 Mutations in Liquid Biopsies

Dimitra Stergiopoulou; Athina Markou; Eleni Tzanikou; Ioannis Ladas; G Mike Makrigiorgos; Vassilis Georgoulias; Evi Lianidou

doi:10.3390/cancers13030556

ESR1 NAPA Assay: Development and Analytical Validation of a Highly Sensitive and Specific Blood-Based Assay for the Detection of ESR1 Mutations in Liquid Biopsies

Cancers (Basel). 2021 Feb 1;13(3):556. doi: 10.3390/cancers13030556.

Authors

Dimitra Stergiopoulou¹, Athina Markou¹, Eleni Tzanikou¹, Ioannis Ladas², G Mike Makrigiorgos², Vassilis Georgoulias^{3

4}, Evi Lianidou¹

Affiliations

¹ Analysis of Circulating Tumour Cells, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, 15771 Athens, Greece.
² Department of Radiation Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02215-5450, USA.
³ First Department of Medical Oncology, Metropolitan General Hospital, 155 62 Athens, Greece.
⁴ Department of Medical Oncology, School of Medicine, University of Crete, 71500 Crete, Greece.

Abstract

A considerable number of estrogen receptor-positive breast cancer (ER⁺ BrCa) patients develop resistance to endocrine treatment. One of the most important resistance mechanisms is the presence of ESR1 mutations. We developed and analytically validated a highly sensitive and specific NaME-PrO-assisted ARMS (NAPA) assay for the detection of four ESR1 mutations (Y537S, Y537C, Y537N and D538G) in circulating tumour cells (CTCs) and paired plasma circulating tumour DNA (ctDNA) in patients with ER⁺ BrCa. The analytical specificity, analytical sensitivity and reproducibility of the assay were validated using synthetic oligos standards. We further applied the developed ESR1 NAPA assay in 13 ER⁺ BrCa primary tumour tissues, 13 non-cancerous breast tissues (mammoplasties) and 64 liquid biopsy samples: 32 EpCAM-positive cell fractions and 32 paired plasma ctDNA samples obtained at different time points from 8 ER+ metastatic breast cancer patients, during a 5-year follow-up period. Peripheral blood from 11 healthy donors (HD) was used as a control. The developed assay is highly sensitive (a detection of mutation-allelic-frequency (MAF) of 0.5% for D538G and 0.1% for Y537S, Y537C, Y537N), and highly specific (0/13 mammoplasties and 0/11 HD for all mutations). In the plasma ctDNA, ESR1 mutations were not identified at the baseline, whereas the D538G mutation was detected in five sequential ctDNA samples during the follow-up period in the same patient. In the EpCAM-isolated cell fractions, only the Y537C mutation was detected in one patient sample at the baseline. A direct comparison of the ESR1 NAPA assay with the drop-off ddPCR using 32 identical plasma ctDNA samples gave a concordance of 90.6%. We present a low cost, highly specific, sensitive and robust assay for blood-based ESR1 profiling. The clinical performance of the ESR1 NAPA assay will be prospectively evaluated in a large number of well-characterized patient cohorts.

Keywords: ARMS-PCR; CTCs; ER+ breast cancer; ESR1; NAME-Pro; cell free DNA; circulating tumour DNA; liquid biopsy; mutation analysis.

Abstract

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