Comprehensive transcriptomic profiling of fusions and abnormal variant transcripts in pilocytic astrocytoma using NanoString nCounter technology

Abstract

Background Pilocytic astrocytoma (PA), a common pediatric and young adult low-grade brain tumor, exhibits MAPK pathway alterations, involving recurrent fusion and duplication events. These genetic changes are crucial for diagnosis, therapy guidance, and predicting patient survival. BRAF alterations are commonly detected using FISH and RT-PCR; however their cost is intensive and they lack multiplexing capacity. This study utilizes NanoString technology to provide a broader analysis of alterations in PA, aiming to improve diagnostic accuracy. Methodology Sixty histologically confirmed PA cases were analyzed using RT-PCR and FISH with 30 positive for KIAA1549-BRAF fusions. A customized NanoString fusion panel was developed to detect other possible fusions. RNA was isolated from FFPE samples, followed by gene fusion assessment using NanoString nCounter platform. Radiological and pathological features were analysed to evaluate their association with molecular alterations. Results Fusions were identified in 91 % of cases using the NanoString nCounter platform. KIAA1549-BRAF fusions were observed in majority of cases (74 %) with the most common fusion being KIAA1549-BRAF_e16-e9, present in 28 % of cases, followed by KIAA1549-BRAF_e15-e9 (22 %). Fusion events showed a significant correlation with anatomical location, particularly in the posterior fossa. However, no significant correlation was found between specific radiological features and fusion subtypes. The NanoString assay demonstrated 97 % sensitivity in detecting BRAF fusion variants, with 100 % specificity. Conclusion The NanoString nCounter platform demonstrated high sensitivity and specificity and identified additional fusion events, highlighting its potential as a powerful tool for high throughput analyses in clinical settings, particularly for the evaluation of PAs.