LBPO.CL01 · 临床研究 · Late-Breaking
利用标准全基因组测序通过结构变异在血液样本中灵敏检测MYCN扩增型神经母细胞瘤
Sensitive detection of MYCN amplified neuroblastoma in blood samples with structural variants using standard whole genome sequencing
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：MYCN扩增（MNA）是高危神经母细胞瘤的一个决定性生物标志物，预后不良。虽然基于肿瘤的全基因组测序（WGS）结合拷贝数分析（CNA）可可靠地识别MNA，但由于循环肿瘤细胞（CTC）负荷低，其在血液中的检测一直不确定。人们对利用具有极低测序错误率的结构变异（SV）以实现液体活检中超灵敏肿瘤检测的兴趣日益增加。
方法：我们分析了三个机构队列中具有配对肿瘤和外周血（PB）WGS数据的MNA神经母细胞瘤病例。进行了数学建模，以模拟在不同肿瘤纯度和测序深度下使用CNA与基于SV的方法的检测限。CNA检测使用CNVkit、ichorCNA和DELLY进行，而肿瘤特异性SV则使用我们近期发表的错误抑制方法SVInDelGenotyper在匹配的PB中进行基因分型，并使用gnomAD和健康对照队列SJLIFE研究其错误谱。评估了颊拭子样本和缓解期血液样本作为替代的种系参照。
结果：基于CNA的检测在32%的PB样本中识别出MNA（9/28），方法特异性灵敏度为4-25%。整合肿瘤知情的SV基因分型在CNA阴性的PB样本中发现了额外的MYCN相关断点，将总体检测灵敏度提高至64%（18/28）。即使在PB样本未显示可见的拷贝数升高（二倍体水平覆盖度）时，SV仍可检测到，揭示了低至约0.01%的肿瘤分数。更深的WGS覆盖度和SV负荷与各队列中改善的SV检测相关。我们的发现凸显了当使用诊断性PB作为正常对照时，标准肿瘤对正常比较可能漏检儿童神经母细胞瘤关键突变的风险。颊拭子样本和缓解期血液未显示肿瘤特异性SV，支持其作为可靠种系对照的作用。
结论：标准深度WGS（30-100×）与基于SV的液体活检分析相结合，可从血液中灵敏检测MNA神经母细胞瘤，尤其在低纯度样本中优于仅基于CNA的方法。这些发现确立了肿瘤特异性SV基因分型作为识别高危MNA神经母细胞瘤的一种强大的非侵入性策略。我们的数据进一步提示，未来儿童神经母细胞瘤液体活检的研究设计应采用颊拭子样本作为正常对照。
查看英文原文 English abstract
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.
利益披露 Disclosure
P. Kolekar, None..
R. S. Kaufman, None..
H. Li, None..
Y. Liu, None..
Y. Feng, None..
B. Wang, None..
X. Wang, None..
L. Fan, None..
L. Wang, None..
J. Zhang, None..
J. M. Maris, None..
S. J. Diskin, None..
X. Ma, None.