COMPREHENSIVE FUSION PROfiLING OF PILOCYTIC ASTROCYTOMAS: EVALUATING NANOSTRING NCOUNTER® FOR MOLECULAR DIAGNOSTICS

Abstract AIMS Pilocytic astrocytomas (PAs) constitute ~15% of pediatric brain tumors, with surgery as the primary treatment, but incomplete resection leads to recurrence in 20–50% of cases. The pathogenesis of PAs is linked to MAPK pathway alterations, with KIAA1549-BRAF being the most frequent fusion event. However, additional fusions are involved in tumor biology, which is critical for accurate diagnosis and therapeutic decision-making. The study aimed to comprehensively profile fusions in PAs and assess their correlation with clinical, pathological, and radiological features to enhance molecular diagnostics in CNS tumors. METHODS 200 PA cases were screened for KIAA1549-BRAF fusions (15-9, 16-9, and 16-11) using FISH and qRT-PCR, iden- tifying 52 fusion-negative cases. To further explore the fusion landscape, 60 cases were analyzed using the NanoString nCounter® platform with a customized pan-CNS fusion panel comprising 106 fusion events. This cohort included 30 KIAA1549-BRAF fusion-positive cases to assess NanoString’s accuracy in detecting known KIAA1549-BRAF fusions and 30 fusion-negative cases to uncover rare fusions in PAs. RESULTS Of 60 cases, 55 (91.6%) harbored fusions. Among 30 KIAA1549-BRAF fusion-positive cases, 29 were con- firmed by NanoString, accurately identifying subtypes: e16-e9 (56.7%), e15-e9 (33.3%), and e16-e11 (6.7%). Among 30 fusion-negative cases, 26 harbored other fusions: FGFR1-FGFR1b (50%), KIAA1549-BRAF_e18-e10 (23.3%), RNF130-BRAF_e3-e9 (6.7%), and YAP1-FAM118B_e7-e3 (6.7%). Fusion events significantly correlated with anatomical location, particularly in the posterior fossa. The NanoString assay demonstrated 96.7% sensi- tivity and 100% specificity. CONCLUSION The study demonstrated the NanoString nCounter platform’s efficacy, detecting common (KIAA1549-BRAF, FGFR1-FGFR1b) and rare (YAP1-FAM118B) fusions in >90% of cases, with KIAA1549-BRAF e16-e9 as the most prevalent subtype in PAs. These fusions correlated with tumor location, suggesting potential biomarkers for localization. Novel fusions like YAP1-FAM118B were identified, with implications for targeted therapies. The platform exhibited high sensitivity, robustness, and cost-effectiveness, proving to be a valuable tool for molec- ular profiling, enhancing diagnostic precision and therapeutic strategies in CNS Tumors.