PATH-56. PYROSEQUENCING METHOD FOR RAPID DETECTION OF KIAA1549-BRAF FUSION GENES IN PEDIATRIC BRAIN TUMORS

Abstract

Abstract BACKGROUND Fusion genes driving tumorigenesis have drawn the attention of oncologists and researchers. Recently, independent genomic researchers have identified a tandem duplication of BRAF at 7q34 that leads to several KIAA1549-BRAF gene exon fusions in the majority of pilocytic astrocytoma (PA). Because KIAA1549-BRAF fusion transcripts are usually absent in diffuse astrocytoma, these genetic alterations may have diagnostic value. Despite the importance of such molecular alterations, there are no comprehensive reproducible methods to detect these fusion genes. METHODS Samples of brain tumors were selected retrospectively from our institution. 22 pediatric brain tumors of five types, namely PA, oligodendroglioma (OD), anaplastic astrocytoma (AA), glioblastoma (GBM) and, ganglioglioma (GG), were evaluated to detect KIAA1549-BRAF fusion genes using a pyrosequencing-based method following RNA isolation, cDNA synthesis and real-time polymerase chain reaction. We designed the primers to detect the three most common fusion variants: KIAA1549 ex15-BRAF ex9, KIAA1549 ex16-BRAF ex9, and KIAA1549 ex16-BRAF ex11. RESULTS Our method successfully detected KIAA1549-BRAF fusion in 16 out of 22 samples, that is, eleven PA, two OD, one AA, one GBM, and one GG. The entire procedure from RNA extraction to obtaining mutation data was performed within 2 hours. CONCLUSION A comprehensive method for detecting fusion genes in pediatric brain tumors was evaluated. This method identified KIAA1549–BRAF fusion variants quickly compared to other methods. Our results will be useful for studies on intraoperative diagnosis and classification of tumors for improving surgical decisions.