Sensitive detection of MYCN amplified neuroblastoma in blood samples with structural variants using standard whole genome sequencing

Background: MYCN amplification (MNA) is a defining biomarker of high‑risk neuroblastoma with a poor clinical outcome. While tumor‑based whole‑genome sequencing (WGS) coupled with copy‑number analysis (CNA) reliably identifies MNA, its detection in blood has remained uncertain due to low circulating tumor cell (CTC) burden. There is increasing interest in exploiting structural variants (SVs), which exhibit extremely low sequencing error rates, to enable ultra-sensitive tumor detection in liquid biopsies. Methods: We analyzed MNA neuroblastoma cases across three institutional cohorts with available paired tumor and peripheral blood (PB) WGS data. Mathematical modeling was performed to model limit of detection in using CNA versus SV‑based approaches under varying tumor purities and sequencing depths. CNA detection was performed using CNVkit, ichorCNA, and DELLY, whereas tumor‑specific SVs were genotyped in matching PB with our recently published error suppression method SVInDelGenotyper, and their error profiles are investigated using gnomAD and a healthy control cohort SJLIFE. Buccal samples and blood samples at remission were evaluated as alternative germline references. Results: CNA‑based detection identified MNA in 32% of PB samples (9/28), with method‑specific sensitivities ranging from 4-25%. Integration of tumor‑informed SV genotyping uncovered additional MYCN -linked breakpoints in CNA‑negative PB samples, increasing overall detection sensitivity to 64% (18/28). SVs remained detectable even when PB samples exhibited no visible copy‑number elevation (diploid‑level coverage), revealing tumor fractions as low as ~0.01%. Deeper WGS coverage and SV burden correlated with improved SV detection across cohorts. Our findings highlight the risk of missing detection of key mutations for childhood neuroblastoma by standard tumor vs normal comparison when diagnostic PB is used as normal control. Buccal samples and remission‑stage blood showed no tumor‑specific SVs, supporting their role as reliable germline controls. Conclusions: Standard‑depth WGS (30-100×), when combined with SV‑based liquid‑biopsy analysis, enables sensitive detection of MNA neuroblastoma from blood, outperforming CNA‑only approaches especially in low‑purity samples. These findings establish tumor‑specific SV genotyping as a powerful non‑invasive strategy for identifying high‑risk MNA neuroblastoma. Our data further suggests the future study design of childhood neuroblastoma liquid biopsy to employ buccal samples as normal controls.