Pilocytic astrocytomas: BRAFV600E and BRAF fusion expression patterns in pediatric and adult age groups

Abstract

Purpose

Pilocytic astrocytomas (PCAs) are characterized by two dominant molecular alterations of the BRAF gene, i.e., BRAFV600E mutation and KIAA1549-BRAF fusions which show a differential pattern of frequency across different age-groups.

Methods

Formalin-fixed paraffin-embedded tissues of 358 (pediatric 276 and adult 82) consecutive PCAs were evaluated for BRAFV600E mutation by Sanger sequencing and KIAA1549:BRAF fusion transcripts (KIAA1549:BRAF 16-9, KIAA1549:BRAF 15-9, and KIAA1549:BRAF 16-11) by reverse transcriptase polymerase chain reaction, which were correlated with different clinicopathological features.

Results

BRAFV600E mutation was detected in 8.9% pediatric and 9.75% adult PCAs, whereas 41.1% and 25.7% of pediatric and adult cases showed KIAA1549-BRAF fusions respectively. BRAFV600E did not show any statistically significant correlation with any of the clinical parameters (age, location, and gender). KIAA1549:BRAF fusions showed a significant statistical association with the pediatric age group and cerebellar location. KIAA1549-BRAF 16-9 was the commonest variant and was predominantly associated with cerebellar location than non-cerebellar whereas fusion variant 15-9 negatively correlated with cerebellar locations.

Conclusions

The present study showed overall frequency of 53.5% and 37.3% BRAF alterations in pediatric and adult PCA cases respectively. BRAF fusion in PCA cases showed a different distribution pattern across age groups and locations; while no such differential pattern was observed for BRAFV600E.

Appendix. Detailed methodology of nucleic acid extraction, PCR with subsequent sequencing for BRAFV600E and RT-PCR for BRAF fusions

DNA and RNA extraction

Selected FFPE blocks were subjected to genomic DNA and total RNA extraction from four sections each of 10 μm thickness. Sections were deparaffinized with limonene (Sigma Aldrich, USA) followed by overnight digestion. DNA was extracted using QIAamp DNA mini kit (Qiagen) as per manufacturer’s instructions. Extracted DNA was checked for quality (260:280 ratio) and quantity by Nanodrop (Thermo Scientific, USA). The integrity of the DNA was assessed by PCR for beta-actin (ACTB-208 bp) housekeeping gene (Table 1) and the positive samples were then subjected to PCR for BRAF Exon 15. Total RNA extraction was performed using RecoverAll total nucleic acid isolation kit (Ambion, Thermo Scientific, USA). RNA was quantitated using Nanodrop (Thermo Scientific, USA) and complementary DNA (cDNA) was synthesized from 100 ng RNA using RevertAid™ H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, USA). The quality of cDNA was determined using ACTB housekeeping gene PCR and cDNA amplifiable for ACTB gene were subjected to RT-PCR for KIAA1549-BRAF fusion.

BRAF polymerase chain reaction and sequencing

Amplification was carried out in a total volume of 20 μl reaction containing 10 μl of 2× master mix (Thermo Scientific, USA), 1 μl each of 10 pmol forward and reverse primer (Table 1), and 100 ng of template DNA. PCR conditions were initial denaturation at 94 °C for 3 min, 35 cycles of 94 °C for 30 s, annealing at 54 °C for 45 s, 72 °C for 45 s, and a final extension at 72 °C for 10 min. PCR products were electrophoresed on 1.5% agarose gel and purified with EXOSAP-IT (USB, Affymetrix). Direct DNA sequencing was performed on the purified PCR products using BigDye v3.1 cycle sequencing kit (Thermo Scientific, USA) followed by purification with BigDye × terminator kit. The purified products were sequenced on ABI3500 Genetic Analyzer (Thermo Scientific, USA) and sequences analyzed using Chromas Lite software and compared with the reference sequence for BRAFV600E mutation and other adjoining mutations.

KIAA1549-BRAF fusions: reverse transcriptase–polymerase chain reaction

RT-PCR was performed for 3 different fusion transcripts; KIAA1549-BRAF 16-9 (124 bp), KIAA1549-BRAF 15-9 (159 bp) and KIAA1549-BRAF 16-11 (182 bp). PCR reaction contained 2 μl of template cDNA, 5 μl of 2X PCR master mix (Thermo Scientific, USA), and 0.5 μl of forward and reverse primers specific for each fusion transcript (Table 1) in a total volume of 10 μl. PCR program was as follows; 94 °C for 3 min followed by 40 cycles of 94 °C for 45 s, 57–61 °C for 45 s, and 72 °C for 45 s, and completed with an extension step at 72 °C for 10 min. Annealing temperature for KIAA1549-BRAF 16-9 (124 bp), KIAA1549-BRAF 15-9 (159 bp) andKIAA1549-BRAF 16-11 (182 bp) fusion transcripts were 57 °C, 59 °C, and 61 °C respectively. PCR products were separated on 10% polyacrylamide gel, stained with ethidium bromide, and visualized under UV illumination (Alpha Imager, Bioscreen, USA).