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Updated: Mar 10, 2026
Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization
Published on: May 15, 2018
Next Generation Sequencing of Circulating Cell-Free DNA for Evaluating Mutations and Gene Amplification in Metastatic
Karen Page1, David S Guttery1, Daniel Fernandez-Garcia1
1Department of Cancer Studies and Cancer Research UK Leicester Centre, University of Leicester, Leicester, UK.
Background:
Breast cancer tissues are heterogeneous and show diverse somatic mutations and somatic copy number alterations (CNAs). We used a novel targeted next generation sequencing (NGS) panel to examine cell-free DNA (cfDNA) to detect somatic mutations and gene amplification in women with metastatic breast cancer (MBC).
Methods:
cfDNA from pretreated patients (n = 42) and 9 healthy controls were compared with matched lymphocyte DNA by NGS, using a custom 158 amplicon panel covering hot-spot mutations and CNAs in 16 genes, with further validation of results by droplet digital PCR.
Results:
No mutations were identified in cfDNA of healthy controls, whereas exactly half the patients with metastatic breast cancer had at least one mutation or amplification in cfDNA (mean 2, range 1-6) across a total of 13 genes. Longitudinal follow up showed dynamic changes to mutations and gene amplification in cfDNA indicating clonal and subclonal response to treatment that was more dynamic than cancer antigen 15-3 (CA15-3). Interestingly, at the time of blood sampling disease progression was occurring in 7 patients with erb-b2 receptor tyrosine kinase 2 (ERBB2) gene amplification in their cfDNA and 3 of these patients were human epidermal growth factor receptor 2 (HER2) negative at diagnosis, suggesting clonal evolution to a more aggressive phenotype. Lastly, 6 patients harbored estrogen receptor 1 (ESR1) mutations in cfDNA, suggesting resistance to endocrine therapy. Overall 9 of 42 patients (21%) had alterations in cfDNA that could herald a change in treatment.
Conclusions:
Targeted NGS of cfDNA has potential for monitoring response to targeted therapies through both mutations and gene amplification, for analysis of dynamic tumor heterogeneity and stratification to targeted therapy.
Insights
Targeted next-generation sequencing of cell-free DNA (cfDNA) detects mutations and gene amplifications in metastatic breast cancer (MBC). This approach reveals dynamic tumor heterogeneity and aids in treatment stratification.
Area of Science:
- Oncology
- Genomics
- Molecular Diagnostics
Background:
- Breast cancer exhibits significant heterogeneity with diverse somatic mutations and copy number alterations (CNAs).
- Metastatic breast cancer (MBC) presents challenges in monitoring tumor evolution and treatment response.
Purpose of the Study:
- To evaluate the utility of targeted next-generation sequencing (NGS) of cell-free DNA (cfDNA) for detecting somatic mutations and gene amplifications in MBC.
- To assess the potential of cfDNA analysis for monitoring treatment response and tumor heterogeneity.
Main Methods:
- A custom 158-amplicon NGS panel was used to analyze cfDNA from 42 pretreated MBC patients and 9 healthy controls.
- The panel covered hot-spot mutations and CNAs in 16 genes, with results validated by droplet digital PCR.
Main Results:
- Half of MBC patients (21/42) had cfDNA mutations or amplifications across 13 genes; healthy controls had none.
- Longitudinal cfDNA analysis showed dynamic changes in mutations and amplifications, reflecting treatment response.
- ERBB2 amplifications were observed in progressing patients, some initially HER2-negative, indicating clonal evolution.
- ESR1 mutations suggested resistance to endocrine therapy, and 21% of patients had cfDNA alterations indicating potential treatment changes.
Conclusions:
- Targeted NGS of cfDNA is a promising tool for monitoring response to targeted therapies in MBC.
- cfDNA analysis facilitates the study of dynamic tumor heterogeneity and patient stratification for personalized treatment.
