PO.MCB05.02 · 分子与细胞生物学
从视网膜母细胞瘤房水cfDNA解码致癌基因扩增结构，作为染色体碎裂和ecDNA生物学的模型
Decoding oncogene amplification structures from retinoblastoma aqueous humor cfDNA as a model for chromothripsis and ecDNA biology
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
目的：复杂的致癌基因扩增——包括MDM4 1q获得和MYCN高水平扩增——常通过染色体碎裂（chromothripsis）、串联重复循环和染色体外DNA（ecDNA）等机制产生。这些结构驱动因素塑造了各种癌症的肿瘤侵袭性和治疗抵抗性，然而由于肿瘤分数低和片段限制，它们在血浆ctDNA中仍大多难以获取。视网膜母细胞瘤房水（AH）中主要含有肿瘤来源的cfDNA，且具有基因组学上简单的背景，提供了一个独特而强大的人类模型系统，用于对基于cfDNA的扩增分析的技术可行性、数据特征和分析要求进行基准评估。
方法：对四只具有MDM4局灶获得或MYCN高水平扩增的RB眼球的AH cfDNA进行深度全基因组测序（>20×）。为经典亚型细胞系生成配对的长读长纳米孔WGS：WERI-Rb1（MDM4）和Y79（MYCN），作为高置信度参考扩增图谱。我们评估了cfDNA相对于长读长肿瘤DNA的拷贝数分辨率、断裂点特征和跨接合片段的呈现。
结果：所有AH cfDNA样本在Illumina PE150 NGS设置下均达到>20×全基因组覆盖度，推断肿瘤分数>90%，从而实现高分辨率的局灶扩增评估。MDM4阳性AH样本显示出多片段局灶1q获得，伴中度拷贝升高（较基线约3-8×）和清晰的区域边界，而MYCN扩增样本表现出超高水平扩增（>50×拷贝），伴密集、局部化的断裂点聚集。长读长肿瘤DNA（>1.5×全基因组覆盖度）提供了足以确认扩增拓扑结构和断裂点位置的结构参考。跨接合的cfDNA片段与预测边界比对一致，支持从液体活检直接恢复局灶扩增。
结论：房水cfDNA为表征局灶扩增特征以及定义与液体活检检测开发相关的技术性能阈值提供了一个高性能试验平台。这些结果确立了分析参数——最小测序深度、拷贝数分辨率极限和接合片段可检测性——可为未来针对肿瘤分数较低的生物体液（如血浆和脑脊液）的平台优化提供参考。
查看英文原文 English abstract
Purpose: Complex oncogene amplifications-including MDM4 1q gains and MYCN high-level amplification-frequently arise through mechanisms such as chromothripsis, tandem-duplication cycles, and extrachromosomal DNA (ecDNA). These structural drivers shape tumor aggression and therapy resistance across cancers, yet they remain largely inaccessible in plasma ctDNA due to low tumor fraction and fragment constraints. Retinoblastoma aqueous humor (AH) contains predominantly tumor-derived cfDNA with a genomically simple background, providing a uniquely powerful human model system to benchmark the technical feasibility, data characteristics, and analytical requirements for cfDNA-based amplification profiling.
Methods: Deep whole-genome sequencing (>20×) was performed on AH cfDNA from four RB eyes having MDM4 focal gain or MYCN high-level amplification. Matched long-read nanopore WGS was generated for canonical subtype cell lines: WERI-Rb1 (MDM4) and Y79 (MYCN) to serve as high-confidence reference amplification profiles. We evaluated copy-number resolution, breakpoint features, and junction-spanning fragment representation in cfDNA relative to long-read tumor DNA.
Results: All AH cfDNA samples achieved >20× genome-wide coverage with >90% inferred tumor fraction from Illumina PE150 NGS setting, enabling high-resolution focal amplification assessment. MDM4-positive AH samples demonstrated multi-segment focal 1q gain with moderate copy elevation (~3-8× over baseline) and sharp regional boundaries, while MYCN-amplified samples exhibited ultra-high amplification (>50× copies) with dense, localized breakpoint clustering. Long-read tumor DNA (>1.5× whole-genome coverage) provided a structural reference sufficient to confirm amplification topology and breakpoint positions. Junction-spanning cfDNA fragments aligned to predicted boundaries, supporting direct focal amplification recovery from liquid biopsy.
Conclusions: Aqueous humor cfDNA provides a high-performance testbed for characterizing focal amplification signatures and defining technical performance thresholds relevant to liquid biopsy assay development. These results establish analytical parameters-minimum sequencing depth, copy-number resolution limits, and junction-fragment detectability-that may inform future platform optimization for lower-tumor-fraction biofluids such as plasma and CSF.
利益披露 Disclosure
Y. Chen, None..
L. Xu, None.