Pilocytic astrocytoma (PAs) account for about 20 % of all childhood primary brain tumors and is the most common glioma in children. [1] These tumors typically occur in the ages of 5 to 10 years, with a decline in incidence with increasing age. [1] PAs can manifest in various anatomical locations within the brain, with cerebellum being the commonest site followed by optic nerve, optic chiasma/hypothalamus, brain stem, basal ganglia and spinal cord. [1,2] On imaging, PAs typically appear well-defined, with a mix of cystic and solid components, often showing contrast enhancement [3,4]. PAs generally have a favorable prognosis, with a 10-year survival rate exceeding 95 % [3,5]. The primary treatment approach is gross total surgical resection (GTR). [6] However, in adults, PAs often originate in critical locations were achieving GTR is more challenging. [6,7] In some cases, these tumors display characteristics that make them more likely to become aggressive, potentially leading to recurrences or leptomeningeal dissemination. [8]

The last decade of research revealed that majority of pediatric low-grade gliomas (PLGGs) are driven by a single genetic event causing upregulation of the RAS–mitogen-activated protein kinase (RAS/ MAPK) pathway, resulting in somatic events like BRAF alterations. [9] In PAs, a 2-Mbp tandem duplication at 7q34 was identified, causing loss of N-terminal regulatory domain of BRAF influencing downstream up-regulation of RAS/MAPK signaling pathway resulting in BRAF and KIAA1549 fusion [9,10] Seminal studies observed that KIAA1549::BRAF fusion is the most frequent and poignant molecular alteration in PAs (>60 %) and in some cases of diffuse leptomeningeal glioneuronal tumor (DLGNT) and high-grade astrocytoma with piloid features [11]. There are several KIAA1549::BRAF exon-exon fusions that have been reported in the following order of prevalence: 16;9 (49 %), 15;9 (35 %), 16;11, 18;10, and 19;9 all resulting in the loss of BRAF's regulatory domain [12]. Inhibitors of BRAF, some of which already are being evaluated in adult clinical trials, may be promising therapeutics for pediatric gliomas harboring BRAF fusions [13] As such, there is an emerging need for clinically robust technologies to identify tumors with activated forms of BRAF in individuals who might benefit from these targeted agents. WHO 2021 CNS5 classification recommends KIAA1549::BRAF fusion testing under essential criterion for PAs, however, they have not recommended optimal mode of detection and platform for the same. [14] Several institutions have incorporated next-generation sequencing (NGS) for molecular analysis of brain tumors in diagnostic settings. [15,16] However, most high throughput techniques require 2–4 weeks for the completion of results, challenges arise from the need for computational resources and technical expertise to analyse and clinically interpret the data. [11,15] Studies exhibit that RNA sequencing has been widely regarded as the benchmark for identifying gene fusions in diagnostic contexts [13,15], yet, it is not universally accessible and can present challenges when dealing with formalin-fixed paraffin-embedded samples (FFPE). The FISH assay is widely used for detecting gene fusions and copy number events in FFPEs and is considered as a gold standard in several Pathology laboratories. [16] Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), has also been increasingly adopted for diagnosing KIAA1549::BRAF fusion, often alongside or replacing FISH in diagnostic settings (15) Nonetheless, each platform has been associated with its unique set of advantages and limitations, which has been explored further in this study. We tested KIAA1549::BRAF fusions using FISH and qRT-PCR based TaqMan Assay (initially designed by Tian et al). [17]

Our study was involved in assessing the presence of KIAA1549::BRAF fusions and explored its potential associations with tumor location, demographic characteristics, radiological findings, and overall survival. Further, our goal was to evaluate the agreement between the FISH and qRT-PCR platforms, exploring their practical advantages, limitations, cost-effectiveness, and turn-around time within the context of routine clinical practice.