Optimizing Liquid Biopsy: Spike-In Panel Approach for ctDNA Copy Number Variation Analysis

Abstract Background/Aim: Circulating tumor DNA (ctDNA) testing has emerged as a minimally invasive tool for precision oncology, enabling dynamic monitoring of tumor burden and treatment response. However, commercial ctDNA NGS assays often omit clinically important oncogenes, limiting accurate assessment of copy-number variation (CNV). Amplifications of MYC and MYCN are key drivers of tumor progression and therapeutic resistance, and their detection is required under the Korean National Health Insurance coverage criteria. We evaluated whether a custom spike-in panel added to the Avenio ctDNA Expanded Kit improves CNV detection for MYC and MYCN to meet these clinical and regulatory requirements. Materials and Methods: Spike-in targets were designed with KAPA Target Enrichment Custom Designs and integrated into the Avenio panel. Reference materials (Horizon Structural Multiplex cfDNA Standard, 5% (MYCN ≈9.5 copies); Seraseq ctDNA Complete, 1% (MYC≈3.07 copies)) were measured in triplicate; Seraseq was additionally diluted 1:2 and 1:10. Eight cancer-free plasma samples established the baseline. Libraries were sequenced on a NextSeq 550Dx (high-output). CNV analysis used CNVkit v0.9.9 with custom parameters (reference spread threshold increased 1.0→1.5; GC upper limit relaxed 0.7→0.8, lower limit retained at 0.3). Log2 fold-change versus healthy controls assessed CNV signals. Results: Mean exon coverage was 698.5 for MYCN (range=325.4-1081.2) and 740.3 for MYC (range=438.8-1221.7). In the Horizon material, all MYCN exons showed ≥3.6-fold change (mean 4.2; inferred CNV ≈8.2), concordant with expected amplification. Seraseq showed a mean MYC fold change of 1.46 (inferred CNV ≈2.94); diluted samples yielded CNV estimates of 2.79 (1:2) and 2.67 (1:10), indicating limited sensitivity below ~3 copies. One MYC exon reproducibly underperformed despite adequate coverage. Conclusion: Incorporation of a spike-in panel into the Avenio ctDNA assay enabled reliable detection of high-level MYC/MYCN amplifications and fulfilled practical requirements for local reimbursement. The estimated CNV limit of detection in this setting is ≈3 copies. Further replicate testing and validation with clinical specimens are warranted to refine sensitivity and interlaboratory robustness.