Integrated molecular approach including single-cell FISH enables accurate detection of genetic rearrangements, classification, and identification of novel variants in pediatric brain tumors

INTRODUCTION: Pediatric brain tumors (PBTs) are a heterogeneous group of neoplasms. The WHO classification highlights the relevance of genetic and epigenetic alterations in defining molecular subtypes. This study investigated key rearrangements ( MYB , MYBL1 , ZFTA , YAP1 , MN1 ) and fusions ( KIAA1549::BRAF, FGFR1::TACC1, PRKCA::SLC44A1 ), which have diagnostic, prognostic, and therapeutic implications. MATERIALS AND METHODS We applied a novel single-cell fluorescence in situ hybridization (FISH) method in 50 PBTs, comparing results to whole-transcriptome sequencing (16/50) and DNA methylation profiling (23/50). Our FISH-based approach, using signal distance measurements and pattern analysis, aims to objectively distinguish normal from rearranged signals, improving accuracy in detecting structural variants and discriminating ambiguous cases. RESULTS FISH-based signal distance analysis identified rearrangements in MYB (5+/10), MYBL1 (3+/10), ZFTA (7+/12), YAP1 (3+/12), MN1 (4+/8), including novel unbalanced patterns. Fusion detection by pattern analysis revealed KIAA1549::BRAF (8+/10), FGFR1::TACC1 (4+/6), and PRKCA::SLC44A1 (1+/4). High concordance was observed with RNA-seq and methylation profiling. RNA-seq identified novel fusions: MYB::CA10 , YAP1::TYR , and PAX5::ATM . Concurrent structural variants were observed in single tumors, including KIAA1549::BRAF with MN1::BEND2 and ZFTA::RELA with YAP1::TYR , revealing genetic heterogeneity of PBTs. CONCLUSIONS Our single-cell FISH approach provides standardized, reproducible criteria for structural variant detection, minimizing interpretive variability. When combined with transcriptomic and epigenetic data, this approach supports accurate subtype classification and reveals clinically relevant intratumoral heterogeneity in PBTs.