Detection of KIAA1549-BRAF Fusion Transcripts in Formalin-Fixed Paraffin-Embedded Pediatric Low-Grade Gliomas

Abstract

Alterations of BRAF are the most common known genetic aberrations in pediatric gliomas. They frequently are found in pilocytic astrocytomas, where genomic duplications involving BRAF and the poorly characterized gene KIAA1549 create fusion proteins with constitutive B-Raf kinase activity. BRAF V600E point mutations are less common and generally occur in nonpilocytic tumors. The development of BRAF inhibitors as drugs has created an urgent need for robust clinical assays to identify activating lesions in BRAF. KIAA1549-BRAF fusion transcripts have been detected in frozen tissue, however, methods for FFPE tissue have not been reported. We developed a panel of FFPE-compatible quantitative RT-PCR assays for the most common KIAA1549-BRAF fusion transcripts. Application of these assays to a collection of 51 low-grade pediatric gliomas showed 97% sensitivity and 91% specificity compared with fluorescence in situ hybridization or array comparative genomic hybridization. In parallel, we assayed samples for the presence of the BRAF V600E mutation by PCR pyrosequencing. The data further support previous observations that these two alterations of the BRAF, KIAA1549 fusions and V600E point mutations, are associated primarily with pilocytic astrocytomas and nonpilocytic gliomas, respectively. These results show that fusion transcripts and mutations can be detected reliably in standard FFPE specimens and may be useful for incorporation into future studies of pediatric gliomas in basic science or clinical trials.

Pilocytic astrocytoma with anaplastic features presenting good long-term clinical course after surgery alone: a case report

“…The BRAF rearrangement might not show as strong correlation as other prognostic markers for the clinical outcome of PAs. A lot of papers have reported that there are not still more reliable predictive factors for clinical aggressiveness of PAs than histological findings [4,7,8,12,14,17]. In the present case, nuclear atypia, increased MIB-1 labeling index, marked necrosis with [13]; however, some reports of anaplastic PA did not always meet the criteria [1,18].…”

Papillary glioneuronal tumors: histological and molecular characteristics and diagnostic value of SLC44A1-PRKCA fusion

“…Hydrolysis probe assays to detect K-B exon junctions were designed according to Tian and colleagues and purchased from Life Technologies (Darmstradt, Germany) [15]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as internal control.…”

Emerging insights into the molecular and cellular basis of glioblastoma

“…Likewise, BRAF fusions (Horbinski et al 2010;Hawkins et al 2011) and V600E mutants characterize additional astrocytoma subsets with clinical relevance (Dias-Santagata et al 2011;Schindler et al 2011;Tian et al 2011). In contrast to the mutant IDH/G-CIMP + tumors, glioblastomas appear surprisingly distinct but significantly more complex genomically, making diagnostic subclassification schemes more challenging.…”