Clinical Impact of Integrating RNA-Based Next-Generation Sequencing Into the Diagnostic Evaluation of Soft Tissue Sarcomas: Insights From a Single-Center Multidisciplinary Workflow

PURPOSE Sarcomas exhibit clinicopathologic heterogeneity, leading to diagnostic and therapeutic uncertainty. This study assesses the clinical value of integrating targeted RNA-based next-generation sequencing (NGS) into a multidisciplinary sarcoma workflow to improve diagnosis, enable comprehensive molecular characterization, and inform personalized treatment strategies. METHODS In this prospective single-center study, 68 patients with suspected or confirmed soft tissue sarcoma underwent RNA-based NGS using a 138-gene fusion panel, integrated within a multidisciplinary sarcoma framework. Genomic findings integrated with histopathology and immunohistochemistry for diagnostic reclassification and treatment changes. RESULTS Of 70 patients advised RNA-based NGS, 68 (97.1%) were evaluable (median age, 38.5 years). Soft tissue sarcomas predominated (77.1%), with 67.1% presenting with advanced disease. Initial histology included 17 round cell sarcomas (RC), 19 undifferentiated sarcomas (US), and 34 other subtypes. Among evaluable RC cases (n = 16), NGS identified diagnostic fusions in 87.5%, enabling reclassification in 68.7% and treatment change in 18.7%. In US, fusions were detected in 21.1%, all reclassified, with treatment altered in 75%. In other subtypes, fusions were found in 48.4%, enabling reclassification in 39.3% and treatment impact in 21.2%. Nondiagnostic mutations (eg, TP53 , SLX4 , NF1 ) were observed in 47.4% of US and 18.2% of other subtypes. Potentially actionable variants ( KIT , PIK3CA ) were seen in 5.8%. Overall, RNA-NGS was clinically informative in 50%, refined diagnosis in 41.1%, and influenced treatment in 26.4% of patients. CONCLUSION To our knowledge, this first-of-its-kind study highlights the clinical utility of integrating targeted RNA-based NGS into multidisciplinary sarcoma care, enabling diagnostic reclassification in 41.1% of cases most notably in round cell sarcomas with atypical morphology. Both canonical and rare fusions informed individualized therapy, reinforcing RNA-NGS as a pivotal tool for diagnostic refinement and precision oncology in sarcoma management.