A Multiplex Quantitative Reverse Transcription Polymerase Chain Reaction Assay for the Detection of KIAA1549-BRAF Fusion Transcripts in Formalin-Fixed Paraffin-Embedded Pilocytic Astrocytomas

Mol Diagn Ther. 2019 Aug;23(4):537-545. doi: 10.1007/s40291-019-00403-3.


Background and objective: Genomic duplications and fusion involving BRAF and KIAA1549 that create fusion proteins with constitutive B-RAF kinase activity are a hallmark of pilocytic astrocytomas (PAs). The detection of KIAA1549-BRAF fusion transcripts is of paramount importance to classify these tumors and to identify patients who could benefit from BRAF inhibitors. In a clinical setting, the available material for molecular analysis from these pediatric tumors is often limited to formalin-fixed paraffin-embedded (FFPE) tissue. The aim of the present study was to develop a new method to detect the three most frequent KIAA1549-BRAF fusion transcripts, 15-9, 16-11, and 16-9, where numbers refer to the exons fused together, using a FFPE-compatible multiplex quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Methods: We compared performance of the assay to a reference singleplex method on a collection of 46 FFPE PAs.

Results: The results showed that both methods are comparable. The multiplex method had an overall 97% sensitivity and 100% specificity compared to the singleplex method, and agreement between the two techniques was almost perfect (Cohen's kappa: 0.97). There was no evidence of a significant difference between the qRT-PCR efficiencies of the multiplex technique and of the singleplex assay for all fusion transcripts and for GAPDH, the latter used as a reference gene. The multiplex method consumed four times less complementary DNA (cDNA), cost less, and required half the hands-on technical time.

Conclusion: The results show that it could be beneficial to implement the multiplex method in a clinical setting, where samples presenting low quantity of degraded RNA are not unusual.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Astrocytoma / diagnosis
  • Astrocytoma / genetics*
  • Biopsy
  • Cost-Benefit Analysis
  • Female
  • Gene Frequency
  • Humans
  • Male
  • Multiplex Polymerase Chain Reaction* / methods
  • Neoplasm Grading
  • Oncogene Proteins, Fusion / genetics*
  • Paraffin Embedding
  • Real-Time Polymerase Chain Reaction* / methods
  • Reproducibility of Results


  • BRAF-KIAA1549 fusion protein, human
  • Oncogene Proteins, Fusion