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Optimizing Liquid Biopsy: Spike-In Panel Approach for ctDNA Copy Number Variation Analysis
Hyunji Kim1,2, Byoung Mok Kim2, Jinho Kim1,2,3
1Department of Laboratory Medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Republic of Korea.
Cancer Genomics & Proteomics
|January 2, 2026
Summary
A custom spike-in panel improved circulating tumor DNA (ctDNA) next-generation sequencing (NGS) assays for detecting MYC and MYCN amplifications. This enhancement meets clinical requirements for copy-number variation (CNV) detection in precision oncology.
Area of Science:
- Oncology
- Genetics
- Molecular Diagnostics
Background:
- Circulating tumor DNA (ctDNA) testing is crucial for precision oncology and treatment monitoring.
- Commercial ctDNA NGS assays often lack coverage for key oncogenes like MYC and MYCN, hindering copy-number variation (CNV) assessment.
- MYC and MYCN amplifications are significant drivers of tumor progression and therapeutic resistance, with detection mandated by Korean National Health Insurance.
Purpose of the Study:
- To evaluate the efficacy of a custom spike-in panel added to the Avenio ctDNA Expanded Kit.
- To determine if the enhanced assay improves CNV detection for MYC and MYCN.
- To assess if the improved detection meets clinical and regulatory requirements for reimbursement.
Main Methods:
- A custom spike-in panel was designed and integrated into the Avenio ctDNA Expanded Kit.
- Reference materials with known MYCN and MYC copy numbers were analyzed.
- Plasma samples from cancer-free individuals established a baseline for CNV analysis.
- Sequencing was performed on a NextSeq 550Dx, and CNV analysis utilized CNVkit with adjusted parameters.
Main Results:
- Mean exon coverage for MYCN and MYC was high (698.5 and 740.3, respectively).
- The assay reliably detected high-level MYCN amplifications (mean fold change 4.2, inferred CNV ≈8.2).
- Detection of MYC amplification was less sensitive, with an estimated limit of detection around 3 copies.
Conclusions:
- Integrating a spike-in panel enhances the Avenio ctDNA assay for detecting high-level MYC/MYCN amplifications.
- The modified assay meets practical requirements for local reimbursement in South Korea.
- Further validation with clinical specimens is recommended to refine sensitivity and interlaboratory robustness.
