Background: ESR1 gene mutations are an essential mechanism of resistance to endocrine therapy in patients with oestrogen receptor-positive breast cancer. ESR1 mutations can exist at multiple sites and are typically detected polyclonally after endocrine therapy. The use of circulating tumour DNA (ctDNA) is a less invasive and less expensive method for assessing ESR1 mutations. Adequate amounts of DNA are required to assess polyclonal ESR1 mutations using next-generation sequencing (NGS). The aim of this study was to develop a method to assess polyclonal ESR1 mutations in blood samples.
Methods: We developed a novel detection system to screen for ESR1-activated mutations in ctDNA by combining a peptide nucleic acid and locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamping assay for detection of the most common mutations: E380Q, Y537S, and D538G. Other mutations in codons 536-538 were detected by direct sequencing of the amplified product following clamp PCR. A validation assay was prospectively performed on clinical samples and compared with the NGS results.
Results: The PNA-LNA PCR clamp assay was validated using six blood samples in which ESR1 mutations were detected by NGS and four samples in which no mutations were detected. Results of the PNA-LNA assay were comparable with those of NGS. We prospectively assessed the concordance between the PNA-LNA PCR clamp method and NGS. Using the PNA-LNA PCR clamp method, ESR1 mutations were detected in 5 of 18 samples, including those in which mutations were not detected by NGS due to the small amount of ctDNA.
Conclusions: The PNA-LNA PCR clamping method is a highly sensitive and minimally invasive assay for the detection of polyclonal ESR1 mutations in the ctDNA of patients with breast cancer.