肌肉横纹肉瘤（Rhabdomyosarcoma，RMS）从初发到复发的基因组谱系尚未完全理解。在这项试点研究中，我们探索了各种靶向和全基因组测序平台的敏感性，以评估在未来前瞻性临床试验中使用液体活检的最佳基因组方法。此外，我们通过靶向DNA或全外显子测序（WES）对两个机构共计的35例配对原发/复发RMS进行了研究，其中18例融合阳性（FP-RMS）和17例融合阴性RMS（FN-RMS）。在10例病例中，通过临床照护和进展的多个时间点分析了循环肿瘤DNA（ctDNA），以评估液体活检在监测治疗反应/复发方面的可行性。采用一个针对36个基因的定制RMS靶向面板进行了ctDNA变异的评估，其中包括高覆盖度的单核苷酸变异和融合检测，以及用于拷贝数变异的浅表全基因组测序。FP-RMS在复发时具有稳定的基因组，在诊断时存在常见的二次改变CDKN2A/B，MYCN和CDK4，对存活率产生影响。在基线时缺少主要的二次事件的FP-RMS会获得反复出现的MYCN和AKT1变异。FN-RMS获得了更多的新变异，最常见的是SMARCA2错义突变。在我们的病例集中，不论变异类型如何，ctDNA分析能够在所有RMS患者的诊断时检测到病理学变异，并在86%的FP-RMS和100%的FN-RMS的复发时进行确认。此外，在病情加重时以及患者复发时，融合序列的数量增加。这些结果强调了肿瘤进展的模式，并为使用液体活检监测治疗反应提供了理论基础。© 2023. Nature Publishing Group UK.

The genomic spectrum of rhabdomyosarcoma (RMS) progression from primary to relapse is not fully understood. In this pilot study, we explore the sensitivity of various targeted and whole-genome NGS platforms in order to assess the best genomic approach of using liquid biopsy in future prospective clinical trials. Moreover, we investigate 35 paired primary/relapsed RMS from two contributing institutions, 18 fusion-positive (FP-RMS) and 17 fusion-negative RMS (FN-RMS) by either targeted DNA or whole exome sequencing (WES). In 10 cases, circulating tumor DNA (ctDNA) from multiple timepoints through clinical care and progression was analyzed for feasibility of liquid biopsy in monitoring treatment response/relapse. ctDNA alterations were evaluated using a targeted 36-gene custom RMS panel at high coverage for single-nucleotide variation and fusion detection, and a shallow whole-genome sequencing for copy number variation. FP-RMS have a stable genome with relapse, with common secondary alterations CDKN2A/B, MYCN, and CDK4 present at diagnosis and impacting survival. FP-RMS lacking major secondary events at baseline acquire recurrent MYCN and AKT1 alterations. FN-RMS acquire a higher number of new alterations, most commonly SMARCA2 missense mutations. ctDNA analyses detect pathognomonic variants in all RMS patients within our collection at diagnosis, regardless of type of alterations, and confirmed at relapse in 86% of FP-RMS and 100% FN-RMS. Moreover, a higher number of fusion reads is detected with increased disease burden and at relapse in patients following a fatal outcome. These results underscore patterns of tumor progression and provide rationale for using liquid biopsy to monitor treatment response.© 2023. Nature Publishing Group UK.