| Summary: | A considerable number of estrogen receptor-positive breast cancer (ER<sup>+ </sup>BrCa) patients develop resistance to endocrine treatment. One of the most important resistance mechanisms is the presence of <i>ESR1</i> mutations. We developed and analytically validated a highly sensitive and specific NaME-PrO-assisted ARMS (NAPA) assay for the detection of four <i>ESR1 </i>mutations (Y537S, Y537C, Y537N and D538G) in circulating tumour cells (CTCs) and paired plasma circulating tumour DNA (ctDNA) in patients with ER<sup>+ </sup>BrCa. The analytical specificity, analytical sensitivity and reproducibility of the assay were validated using synthetic oligos standards. We further applied the developed <i>ESR1</i> NAPA assay in 13 ER<sup>+ </sup>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 <i>ESR1</i> 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 <i>ESR1</i> profiling. The clinical performance of the <i>ESR1</i> NAPA assay will be prospectively evaluated in a large number of well-characterized patient cohorts.
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