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Updated: Jun 27, 2026
Detection of Rare Mutations in CtDNA Using Next Generation Sequencing
Published on: August 24, 2017
Development of a Detection System for ESR1 Mutations in Circulating Tumour DNA Using PNA-LNA-Mediated PCR Clamping
Yuki Kojima1,2, Emi Noguchi1, Tomomi Yoshino3
1Department of Medical Oncology, National Cancer Center Hospital, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, Japan.
Abstract:
Although circulating tumour DNA (ctDNA)-based next-generation sequencing (NGS) is a less invasive method for assessing ESR1 mutations that are essential mechanisms of endocrine therapy resistance in patients with oestrogen receptor-positive breast cancer, adequate amounts of DNA are required to assess polyclonal ESR1 mutations. By combining a peptide nucleic acid and locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamping assay, we have developed a novel detection system to screen for polyclonal ESR1 mutations in ctDNA. A validation assay was prospectively performed on clinical samples and compared with the NGS results. The PNA-LNA PCR clamp assay was validated using six and four blood samples in which ESR1 mutations were detected by NGS and no mutations were detected, respectively. The PNA-LNA assay results 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 out of 18 samples, including those in which mutations were not detected by NGS due to small amounts of ctDNA. The PNA-LNA PCR clamping method is a highly sensitive and minimally invasive assay for polyclonal ESR1 mutation detection in the ctDNA of patients with breast cancer.
Insights
A new PNA-LNA PCR clamping assay offers a sensitive method for detecting ESR1 mutations in circulating tumor DNA (ctDNA) from breast cancer patients, overcoming limitations of next-generation sequencing.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Estrogen receptor-positive (ER+) breast cancer often develops resistance to endocrine therapy due to ESR1 mutations.
- Circulating tumor DNA (ctDNA) analysis offers a less invasive approach for detecting these mutations.
- Next-generation sequencing (NGS) requires substantial DNA amounts, potentially missing low-level or polyclonal ESR1 mutations.
Purpose of the Study:
- To develop and validate a novel, highly sensitive assay for detecting polyclonal ESR1 mutations in ctDNA.
- To compare the performance of the new assay against established NGS methods.
Main Methods:
- Development of a peptide nucleic acid and locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamping assay.
- Prospective validation using clinical blood samples from breast cancer patients.
- Comparison of PNA-LNA PCR assay results with concurrent NGS analysis.
Main Results:
- The PNA-LNA PCR clamp assay demonstrated comparable results to NGS in initial validation.
- Prospective assessment showed high concordance between the PNA-LNA PCR method and NGS.
- The PNA-LNA PCR assay successfully detected ESR1 mutations in 5 out of 18 samples, including cases missed by NGS due to insufficient ctDNA.
Conclusions:
- The PNA-LNA PCR clamping method is a sensitive and minimally invasive assay for detecting polyclonal ESR1 mutations in ctDNA.
- This assay improves the detection of ESR1 mutations in breast cancer patients, especially when ctDNA amounts are limited.
