INTRODUCTION: Pilocytic astrocytomas (WHO grade I) are the most common paediatric gliomas. The FISH test for KIAA1549-BRAF fusions is most commonly used, but this is difficult to interpret. We aimed to develop a reverse-transcriptase and real-time PCR (RT-PCR) test as a first-line screen for the three most common KIAA1549-BRAF fusion variants. METHODS: A RT-PCR method for detecting KIAA1549-BRAF fusions from formalin-fixed paraffin-embedded (FFPE) brain tumour tissues (pilocytic astrocytoma) was developed based on Tian et al 2011. The three most common fusion variants are detected using this assay: exon 16 of KIAA1549 fused to exon 9 of BRAF, exon 15 of KIAA1549 fused to exon 9 of BRAF and exon 16 of KIAA1549 fused to exon 11 of BRAF fusion. GAPDH expression was used as a control. The three KIAA1549-BRAF fusion variants account for 96% of all the reported BRAF fusions in pilocytic astrocytomas; the 16-9 variant is the most common accounting for 63%, the 15-9 fusion accounts for 23% and the 16-11 fusion accounts for 10% (Jones et al 2008, Tian et al 2011, Lawson et al 2011, Cin et al 2011). RESULTS: The RT-PCR assay was initially validated on 12 samples previously tested by FISH or RT-PCR in a different laboratory. The RT-PCR assay had a sensitivity of 89% (8/9 - one sample tested positive by FISH but negative on RT-PCR) and a specificity of 100% (2/2). The failure rate was 8.3% (1/12). Sensitivity experiments showed that the fusion can be detected when present at a least 5% of the total cDNA content. 51 Neuropathology diagnostic FFPE samples from 42 pilocytic astrocytoma patients were then tested using the BRAF fusion RT-PCR assay. The overall pick-up rate was 54% (20/37 patients) Of the positive patients (20), 55% (11/20) had the 16-9 fusion and 45% (9/20) had the 15-9 fusion. Two patients had multiple fusions (2/20 positive patients, 10%) showing the 16-9 fusion and a low-level 16-11 fusion. No patients exclusively had the 16-11 fusion. Multiple biopsy samples were available for 8 patients. In 7/8 of the patients, the results from the multiple biopsies were identical. In one patient, both biopsies sampled had the 16-9 fusion but the 16-11 fusion was detected at a very low level in only one of the biopsies. This could be due to the very low expression of the 16-11 fusion in the tumour samples. The failure rate of the assay was 12% (5/42 patients). The failed samples were very small tumour samples and so RNA extraction from these biopsies was not successful. However the assay was successful on archived material, some of which were over 20 years old. CONCLUSIONS: We propose undertaking RT-PCR for BRAF fusion as a first-line test in all pilocytic astrocytoma referrals. If the result is negative or the RT-PCR fails, then FISH can be offered as a second-line test. This strategy would more than halve the number of FISH tests, reducing the burden of interpreting difficult and ambiguous cases.