Longitudinal plasma and CSF ctDNA profiling as used to define therapeutic landmarks and compartmentalized clonal evolution in rhabdomyosarcoma.

Abstract

11517 Background: Current risk stratification in rhabdomyosarcoma (RMS) relies on static pretreatment variables, which fail to capture dynamic tumor evolution and molecular residual disease under therapeutic pressure. Although liquid biopsy offers a non-invasive solution, clinical implementation has been constrained by a reliance on retrospective datasets and the lack of prospectively validated therapeutic landmarks or compartmentalized monitoring strategies. Methods: We conducted the largest multi-center, prospective liquid biopsy study to date, enrolling 143 treatment-naïve RMS patients (Aug 2020–Feb 2025; 91.6% intermediate/high-risk). We utilized ultra-deep (>5,000×) sequencing (506-gene panel) on matched tumor, longitudinal plasma (n=292), and cerebrospinal fluid (CSF) from patients with parameningeal or central nervous system (CNS) metastatic disease. Critically, paired tissue and plasma were collected at disease progression (PD) to map evolutionary trajectories. Genomic alterations were correlated with progression-free survival (PFS) and overall survival (OS) using univariate and multivariate Cox regression. Results: Baseline profiling identified MYOD1 mutation as the strongest independent predictor of inferior PFS (HR 6.52, P=0.003), while MYCN amplification defined a novel "ultra-high-risk" fusion-positive subset (OS, P<0.001). Baseline ctDNA positivity (66.7%) correlated with advanced disease (P<0.001). Longitudinally, persistent ctDNA at Cycle 5 Day 1 (C5D1) conferred a 12-fold increased progression risk (HR=12.2, P=0.004), establishing C5D1 as the definitive prognostic landmark. Patients with positive longitudinal ctDNA harbored a nearly 6-fold increased risk of progression (HR = 5.97, 95% CI 2.25–15.85, P < 0.001) and a 12-fold increased risk of death (HR = 12.4, 95% CI 1.42–108.37, P = 0.023) compared to those with sustained negativity. Analysis of paired samples at relapse revealed subclonal replacement driven by therapeutic pressure, enriching for acquired resistance alterations in TP53 and DNA damage response (DDR) pathways undetectable by static assays. Additionally, paired CSF-plasma profiling in parameningeal/CNS disease identified the CNS as an evolutionary "sanctuary site" harboring distinct, compartment-specific drivers (e.g., TP53, HRAS) divergent from plasma. Conclusions: This prospective study establishes ctDNA as a critical biomarker for RMS, defining MYOD1, MYCN, and TP53 as key genomic risk factors and validating C5D1 as the optimal therapeutic landmark. Deep longitudinal profiling uncovers subclonal replacement and compartmentalized CNS evolution as drivers of treatment failure, advocating for comprehensive, compartment-specific monitoring in next-generation risk-adapted trials. Clinical trial information: NCT05778955 .