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. Here, ESR1 is linked to neoplasm.