Incidence of kiaa1549-braf fusion gene in Egyptian pediatric low grade glioma
© Taha et al.; licensee Springer. 2015
Received: 13 November 2014
Accepted: 3 February 2015
Published: 3 March 2015
Low grade gliomas are the most common brain tumor in children. Tandem duplication involving the KIAA1549 and the BRAF kinase genes results in a gene fusion that has been recently characterized in a subset of low grade glioma While there is no clear evidence that the KIAA1549-BRAF gene fusion has an effect on prognosis, it is an attractive target for therapy development and as a diagnostic tool.
In the current study we examine the prevalence of KIAA1549-BRAF gene fusion in pediatric patients diagnosed with low grade glioma in the Egyptian population and its relationship to clinical and histological subtypes. Sixty patients between the ages of 1 to 18 years were analyzed for the presence of KIAA1549-BRAF fusion gene products using reverse transcription-PCR and sequencing. The clinicopathologic tumor characteristics were then analyzed in relation to the different fusion genes.
KIAA1549-BRAF fusion genes were detected in 56.6% of patients. They were primarily associated with pilocytic astrocytoma (74.2%) and pilomyxoid astrocytoma (60%). Translocation 15–9 was the most common, representing (55.8%) of all positive samples followed by 16–9 (26.4%) and 16–11 (8.8%). Pilocytic astrocytomas presented primarily with 15–9 (32.2%), 16–9 (25.8%) and 16–11 (6.4%) while pilomyxoid astrocytomas presented with 15–9 (46.6%), 16–9 (6.6%) and 16–11 (6.6%) translocations.
Gene fusion is found to be significantly increased in cerebellar pilocytic astrocytoma tumors. Furthermore, 15–9 was found to have a higher incidence among our cohort compared to previous studies. While most of the gene fusion positive pilomyxoid astrocytomas were 15–9, we find the association none significant.
KeywordsGlioma BRAF Cancer Gene fusion KIAA1549
Pediatric brain tumors are the second most common childhood malignancy after leukemia accounting for 25% of cases. In the last two decades the overall survival rates have improved for childhood leukemia with a 5-year overall survival of over 80%. Despite advances in surgery, chemotherapy and radiotherapy, brain tumors continue to be the leading cause of cancer-related death in children. This is mainly attributed to cellular heterogeneity of these tumors with multiple cell of origin, lack of effective drugs that cross the blood brain barrier and the absence of molecular markers that could be used for targeted therapy.
Asrtrocytoms are the most common type of brain tumors seen in children compromising 53% of tumors . According to WHO criteria, which consider both pathological and clinical criteria, LGGs are classified into grade I or II. WHO grade I and II tumors are a heterogeneous group of tumors including pilocytic astrocytomas (PAs), pilomyxoid astrocytomas (PMAs), pleomorphic xanthoastrocytoma (PXA), diffuse or fibrillary astrocytoma (DA), subependymal giant cell astrocytoma (SEGA) and low grade glioneuronal tumors (LGGNs) . LGG tumors are generally associated with good prognosis with a 87% over all long term survival of 20 years. Surgical excision is primary treatment method for low grade glioma within accessible parts of the brain where chemotherapy and radiotherapy are used for inoperable and or partially removed tumors [3,4]. For several years the tumor suppressor gene NF1 which is either inherited as an autosomal disorder in patients with neurofibromatosis 1 or occur as a de novo mutation and tuberous sclerosis, were the only genetic factors associated with LGGs [5,6]. In recent years, several genomic alterations have been identified in sporadic low grade gliomas . Deregulation of the RAF gene leading to constitutive activation of the MAPK pathway is emerging as a common mechanism for oncogenesis in sporadic LGG [8,9]. The activation of RAF has been shown to occur either through an activating point mutation (BRAF V600E), or much more frequently, through genomic alteration on 7q34 which creates a tandem duplication between the KIAA1549 gene and the BRAF gene kinase domain [10,11]. As a result of this translocation the auto inhibitory domain of BRAF is lost and the MAPK/ERK pathway is constitutively activated in these tumors. Later studies confirmed the presence of these gene fusions, primarily in 65-75% of PAs and PMAs. Several break points were identified leading to gene fusion between KIAA1549 exon 16 with BRAF exon 9 (16–9) in 60% of the cases, KIAA1549 exon 15 with BRAF exon 9 (15–9) in 25% of the cases and KIAA1549 exon 16 with BRAF exon 11 (16–11) in 10-15% of the cases . While other fusions have been reported between exons 15–11 and 17–10, they only represented 1% of the cases. Gene fusions between BRAF and FAM131B and even less common fusions between RAF1 and SRGAP3 have also been reported . An activating mutation at codon 600 converting valine to glutamic acid (V600E), is another mechanism for activating BRAF without upstream RAS phosphorylation in LGG. V600E mutation exist in diverse tumors, and have been recently identified primarily in PXA and GG and less common among PAs and PMAs tumors .
In the current study we investigated the prevalence of KIAA1549-BRAF fusion genes in a cohort of Egyptian pediatric LGG. Apart from one study which correlated BRAF gene fusion with poor prognosis , the presence of BRAF gene fusions have not been associated with disease outcome. However, the characterization of these fusion genes serve as a molecular biomarker for LGG subtypes and may help in selecting patients for MAPK directed therapy . Our results confirm the presence of gene fusion in PAs and PMAs and to lesser extent in GG. While 16–9 gene fusion is the most common gene fusion, our results identify 15–9 KIAA-BRAF gene fusion as the most common in our cohort accounting for 32.2% of the positive cases where the majority of 15–9 gene fusion are seen among PMA histological subtype. Furthermore, 11% of the cases presented with both KIAA-BRAF 16–9 and 15–9 gene fusions in the same patient.
KIAA1549 and BRAF fusion genes primer sequences
5′ - CGTCCTGGACAAGACCAAGT - 3′
5′ - CTCGTCTTTCTCCTGCTCGT - 3′
5′ - ACTCCTGACTGCATGGAAGC - 3′
5′ - GTCCACAGCTGCTTTTCCAC - 3′
5′ - CGTCCTGGACAAGACCAAGT - 3′
5′ - CTCGTCTTTCTCCTGCTCGT - 3′
5′ - ACTCCTGACTGCATGGAAGC - 3′
5′ - GTCCACAGCTGCTTTTCCAC - 3′
RNA isolation and RT-PCR
Different primer pairs with associated product size for different gene fusions
Validation of gene fusion by Sanger sequencing
PCR products were examined on 2% agarose gels to confirm the presence of a single band corresponding to the excepted size prior to sequencing. PCR products were then purified before sequencing by using ThermoScientific PCR purification kit (ThermoScientific Rockford, Illinois, USA) according to the manufacturer’s protocols. Purified products were sequenced in both directions using BigDye Terminator v3.1 Cycle Sequencing kit (Applied BioSystems, Life Technologies, California, USA)where each reaction contained 8 μl BigDye terminator mix, 5-10 ng template DNA, 3.2 μl of primer (either forward or reverse) at a final concentration of 3.2pmol, and sterile water to a final volume of 20 μl. The thermal conditions were an initial ramp to 96°C by 2.5°C per second, 96°C for 1 minute followed by 24 cycles of (96°C for 10 seconds, ramp to optimum annealing temperature for specific PCR by 1°C/second then hold annealing temperature for 5 seconds, ramp to 60°C at 1°C/second followed by 60°C for 4 minutes), ending at 12°C. Sequence reaction products were then purified by CentriSep (Princeton Separations, Inc, Nj, USA) before obtaining DNA chromatograms on a 3130 DNA Analyzer (Applied BioSystems, Life Technologies, California, USA).
For clinical characteristics and genetic factors, analysis was performed with Fisher’s exact test (dichotomous factors). General data analysis was conducted with R programming language. All P values were based on a two-sided hypothesis, P < 0.05 was considered to have statistical significance.
Results and discussion
Clinicopathological correlation with KIAA1549-BRAF fusion status in patients with LGG
Distribution of gene fusions among different LGG subtypes
DA and PXA
15 - 9
15 - 11
16 - 9
16 - 11
23 of 31(74.2%)
9 of 13 (60%)
2 of 9 (22%)
34 of 60 (56.6%)
Cerebellar tumors were found to be most commonly positive for the presence of the BRAF fusion. Among the positive cerebellar tumors 22 (84.6%) were PAs and 4 (15.3%) were PMAs. Furthermore, 11 (42.3%) tumors were 16–9, 12 (46.15%) were 15–9 and 3 (11.5%) were 16–11. No significant difference between gender was identified with respect to BRAF gene fusion positive and negative patients.
Traditionally brain tumor diagnosis and classification have relied mainly on histological and clinical criteria. The use of molecular markers are growing as a diagnostic tool in brain tumors due to their potential to overcome limitations inherent to both pathological and clinical assessments . The identification of the BRAF gene fusion highlighted the use of this genetic alteration as a potential prognostic marker and for MAPK targeted therapy [18,19]. While recent studies, investigated the effect of first generation BRAF inhibitors as a single agent both in vitro and phase II clinical trial were found to be not effective [20,21]. A phase I clinical trial where BRAF inhibitor Sorafenib, combined with mTOR inhibitor temsirolimus and VEGF inhibitor bevacizumab in spindle cell neoplasm showed a 25% tumor reduction in KIAA1549-BRAF positive PTEN null spindle cell neoplasm, suggesting that combined therapy may be more effective as a therapeutic alternative . Several studies have investigated the significance of BRAF gene fusion, however despite the uncertainty for its use as a prognostic marker, the detection of BRAF gene fusion in low grade tumors can potentially serve as a diagnostic tool for pilocytic or pilomyxoid subtypes as it was found to be associated with these histologies in a high percentage of cases.
In the current study we aimed at identifying the incidence of KIAA1549-BRAF gene fusion in a cohort of pediatric Egyptian patients. Our results are in agreement with previous studies identifying KIAA1549-BRAF gene fusions as characteristic of both PAs and PMAs in the cerebellum. In contrast to previous reports, the15-9 gene fusion was found to be the most common finding, accounting for 55.8% of positive samples followed by 26.4% for 16–9 and 8.8% for 16–11. Furthermore, 70.2% (p value = 0.008) of cerebellar tumors were positive for the KIAA1549-BRAF fusion genes. The difference in distribution of the 15–9 fusion from previously reported percentages could be attributed to the population being studied [8,10]. 7 out 9 of the PMAs positive cases were positive for the 15–9 gene fusion. Although this association was found to be none significant, further investigation with larger cohort of PMAs cases is required to determine if such association exist. Three samples (8.8%) had both 15–9 and 16–9 fusion genes identified in the same tumor. These samples were tested at least twice and examined using fusion specific primer for confirmation. While duplication events and exon skipping were suggested as possible reasons for the presence of multiple junctions , it is not clear yet what the significance of these result are on patient prognosis nor how it may differ from single gene fusion. Histological assessment of PAs positive tumors show a difference between 16–9 and 15–9 with the former displaying a classic PA histology while 15–9 positive PAs were characterized by few Rosenthal fibers and higher MIB-1 index. Furthermore, 15–9 and 16–9 positive PAs were closer in histology to 15–9 PAs.
The use of RT-PCR for the detection for KIAA1549-BRAF gene fusions using two different sets of primers that spans exon 15 in the KIAA1549 gene and exon 9 in the BRAF gene was found to be a reliable and rapid method that can provide confirmatory diagnosis. While FISH can provide a reliable method for the identification of BRAF gene fusion, it cannot distinguish between different fusion. On the other hand, RT-PCR coupled with sequencing can clearly distinguish between different fusions if needed which can be further confirmed by primer specific fusions thus allowing for more precise analysis.
With the identification of new molecular markers, treatment strategies are becoming more refined. The ERK/MAPK pathway is up regulated in about a third of all cancers, in most cases this is contributed to by an activating point mutation in RAS genes [23,24]. Over the last decade, it has become apparent that activation of components of the MAPK pathway in pediatric LGG can be achieved through several distinct genetic events highlighting the importance of this pathway in LGG development. Activation of the MAPK pathway was first implicated in the development of LGG specifically in optic pathway glioma . Patients with the tumor-predisposition syndrome Neurofibromatosis type 1 (NF-1) harbor a germ-line inactivating mutation in the NF-1 tumor suppressor gene product neurofibromin which act as a negative regulator of the RAS pathway [26-28]. Inactivation of neurofibromin can also occur due to de novo mutation in NF-1 that have been also identified in sporadic PAs . Another common way of activating the MAPK pathway is through the BRAF V600E mutation, which is detected in wide range of tumors such as melanomas , thyroid cancer , craniopharyngioma  and LGG, especially PXA and GG [13,33]. The BRAF V600E mutation induces a conformational change in the kinase domain which in turn results in constitutive activation of the pathway without RAS activation. Fusion genes involving BRAF are generated through tandem duplication and have been identified in many different LGGs, especially PAs and PMAs. In these cases, the auto inhibitory domain of BRAF is lost while the kinase domain is retained leading to downstream activation of the MAPK/ERK pathway . These findings highlights the importance of the MAPK pathway in LGG initiation or progression and the significance of identifying patients with these genetic alterations for potential targeted therapy.
Low grade glioma
Acidophilic globular bodies
Dysembryoplastic neuroepithelial tumors
Subependymal giant cell astrocytoma
Low grade glioneuronal
Neurofibromatosis type 1
Glial fibrillary acidic protein
Epithelial membrane antigen
We gratefully thank Dr. Mark Kieran for manuscript revision and technical advice. This study was supported by L'Oreal UNESCO Women in Science Fellowship Award.
- Manuscript A, Gliomas PL. NIH Public Access. 2010;24:1397–408. doi:10.1177/0883073809342005.Pediatric.
- Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, et al. The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol. 2007;114:97–109. doi:10.1007/s00401-007-0243-4.View ArticlePubMed CentralPubMedGoogle Scholar
- Sievert AJ, Fisher MJ. Pediatric low-grade gliomas. J Child Neurol. 2009;24:1397–408. doi:10.1177/0883073809342005.View ArticlePubMed CentralPubMedGoogle Scholar
- Bandopadhayay P, Bergthold G, London WB, Goumnerova LC, Morales LMC, Marcus KJ, et al. Long-term outcome of 4,040 children diagnosed with pediatric low-grade gliomas: an analysis of the Surveillance Epidemiology and End Results (SEER) database. Pediatr Blood Cancer. 2014;61:1173–9. doi:10.1002/pbc.24958.View ArticlePubMedGoogle Scholar
- Gutmann DH, McLellan MD, Hussain I, Wallis JW, Fulton LL, Fulton RS, et al. Somatic neurofibromatosis type 1 (NF1) inactivation characterizes NF1-associated pilocytic astrocytoma. Genome Res. 2013;23:431–9. doi:10.1101/gr.142604.112.View ArticlePubMed CentralPubMedGoogle Scholar
- Kamiryo T, Shinojima N, Ushio Y. Preliminary observations on genetic alterations in pilocytic astrocytomas associated with neurofibromatosis 1 1. 2003;228–234. doi:10.1215/S1152
- Zhang J, Wu G, Miller CP, Tatevossian RG, Dalton JD, Tang B, et al. Whole-genome sequencing identifies genetic alterations in pediatric low-grade gliomas. Nat Genet. 2013;45:602–12. doi:10.1038/ng.2611.View ArticlePubMed CentralPubMedGoogle Scholar
- Pfister S, Janzarik WG, Remke M, Ernst A, Werft W, Becker N, et al. BRAF gene duplication constitutes a mechanism of MAPK pathway activation in low-grade astrocytomas. 2008;118:1739–1749. doi:10.1172/JCI33656DS1
- Forshew T, Tatevossian RG, Lawson ARJ, Ma J, Neale G, Ogunkolade BW, et al. Activation of the ERK / MAPK pathway : a signature genetic defect in posterior fossa pilocytic astrocytomas. 2009;172–181. doi:10.1002/path.
- Jones DTW, Kocialkowski S, Liu L, Pearson DM, Bäcklund LM, Ichimura K, et al. Tandem duplication producing a novel oncogenic BRAF fusion gene defines the majority of pilocytic astrocytomas. Cancer Res. 2008;68:8673–7. doi:10.1158/0008-5472.CAN-08-2097.View ArticlePubMed CentralPubMedGoogle Scholar
- Lawson ARJ, Hindley GFL, Forshew T, Tatevossian RG, Jamie GA, Kelly GP, et al. RAF gene fusion breakpoints in pediatric brain tumors are characterized by significant enrichment of sequence microhomology. Genome Res. 2011;21:505–14. doi:10.1101/gr.115782.110.View ArticlePubMed CentralPubMedGoogle Scholar
- Jones DTW, Kocialkowski S, Liu L, Pearson DM, Ichimura K, Collins VP, et al. Oncogenic RAF1 rearrangement and a novel BRAF mutation as alternatives to KIAA1549: BRAF fusion in activating the MAPK pathway in pilocytic astrocytoma. Oncogene. 2009;28:2119–23. doi:10.1038/onc.2009.73.View ArticlePubMed CentralPubMedGoogle Scholar
- Dias-Santagata D, Lam Q, Vernovsky K, Vena N, Lennerz JK, Borger DR, et al. BRAF V600E mutations are common in pleomorphic xanthoastrocytoma: diagnostic and therapeutic implications. PLoS One. 2011;6:e17948. doi:10.1371/journal.pone.0017948.View ArticlePubMed CentralPubMedGoogle Scholar
- Hawkins C, Walker E, Mohamed N, Zhang C, Jacob K, Shirinian M, et al. BRAF-KIAA1549 fusion predicts better clinical outcome in pediatric low-grade astrocytoma. Clin Cancer Res. 2011;17:4790–8. doi:10.1158/1078-0432.CCR-11-0034.View ArticlePubMedGoogle Scholar
- Ichimura K, Nishikawa R, Matsutani M. Molecular markers in pediatric neuro-oncology; 2012.90–99
- Tian Y, Rich BE, Vena N, Craig JM, MacConaill LE, Rajaram V, et al. Detection of KIAA1549-BRAF fusion transcripts in formalin-fixed paraffin-embedded pediatric low-grade gliomas. J Mol Diagn. 2011;13:669–77. doi:10.1016/j.jmoldx.2011.07.002.View ArticlePubMed CentralPubMedGoogle Scholar
- Komotar RJ, Mocco J, Carson BS, Sughrue ME, Zacharia BE, Sisti AC, et al. Pilomyxoid astrocytoma: a review. MedGenMed. 2004;6:42.PubMed CentralPubMedGoogle Scholar
- Hegi ME, Murat A, Lambiv WL, Stupp R. Brain tumors: molecular biology and targeted therapies. Ann Oncol. 2006;17 Suppl:1:x191–7. doi:10.1093/annonc/mdl259.
- Manuscript A. NIH Public Access. 2012;16:103–19. doi:10.1517/14728222.2011.645805.Targeting.
- Karajannis MA, Legault G, Fisher MJ, Milla SS, Cohen KJ, Wisoff JH, et al. Phase II study of sorafenib in children with recurrent or progressive low-grade astrocytomas. Neuro Oncol. 2014;16:1408–16. doi:10.1093/neuonc/nou059.View ArticlePubMedGoogle Scholar
- Sievert AJ, Lang S-S, Boucher KL, Madsen PJ, Slaunwhite E, Choudhari N, et al. Paradoxical activation and RAF inhibitor resistance of BRAF protein kinase fusions characterizing pediatric astrocytomas. Proc Natl Acad Sci U S A. 2013;110:5957–62. doi:10.1073/pnas.1219232110.View ArticlePubMed CentralPubMedGoogle Scholar
- Subbiah V, Westin SN, Wang K, Araujo D, Wang W-L, Miller VA, et al. Targeted therapy by combined inhibition of the RAF and mTOR kinases in malignant spindle cell neoplasm harboring the KIAA1549-BRAF fusion protein. J Hematol Oncol. 2014;7:8. doi:10.1186/1756-8722-7-8.View ArticlePubMed CentralPubMedGoogle Scholar
- Prior IA, Lewis PD, Mattos C. A comprehensive survey of Ras mutations in cancer. Cancer Res. 2012;72:2457–67. doi:10.1158/0008-5472.CAN-11-2612.View ArticlePubMed CentralPubMedGoogle Scholar
- Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer. 2011;11:761–74. doi:10.1038/nrc3106.View ArticlePubMed CentralPubMedGoogle Scholar
- Rodriguez FJ, Ligon AH, Horkayne-szakaly I, Rushing J, Ligon KL, Vena N, et al. in Gliomas of the Optic Nerve Proper. 2012;71:789–794. doi:10.1097/NEN.0b013e3182656ef8.BRAF
- Bajenaru ML, Zhu Y, Hedrick NM, Donahoe J, Parada L, Gutmann DH, et al. Astrocyte-Specific Inactivation of the Insufficient for Astrocytoma Formation Astrocyte-Specific Inactivation of the Neurofibromatosis 1 Gene ( NF1) Is Insufficient for Astrocytoma Formation. 2002. doi:10.1128/MCB.22.14.5100.Google Scholar
- Yunoue S, Tokuo H, Fukunaga K, Feng L, Ozawa T, Nishi T, et al. Neurofibromatosis type I tumor suppressor neurofibromin regulates neuronal differentiation via its GTPase-activating protein function toward Ras. J Biol Chem. 2003;278:26958–69. doi:10.1074/jbc.M209413200.View ArticlePubMedGoogle Scholar
- Cichowski K, Santiago S, Jardim M, Johnson BW, Jacks T, et al. . Dynamic regulation of the Ras pathway via proteolysis of the NF1 tumor suppressor. Genes Dev. 2003;17:449–54. doi:10.1101/gad.1054703.View ArticlePubMed CentralPubMedGoogle Scholar
- Jentoft M, Giannini C, Cen L, Scheithauer BW, Hoesley B, Sarkaria J, et al. Phenotypic variations in NF1-associated low grade astrocytomas: possible role for increased mTOR activation in a subset. 2011;4:43–57
- Hutchinson KE, Lipson D, Stephens PJ, Otto G, Lehmann BD, Lyle PL, et al. BRAF fusions define a distinct molecular subset of melanomas with potential sensitivity to MEK inhibition. Clin Cancer Res. 2013;19:6696–702. doi:10.1158/1078-0432.CCR-13-1746.View ArticlePubMedGoogle Scholar
- Frasca F, Nucera C, Pellegriti G, Gangemi P, Attard M, Stella M, et al. BRAF(V600E) mutation and the biology of papillary thyroid cancer. Endocr Relat Cancer. 2008;15:191–205. doi:10.1677/ERC-07-0212.View ArticlePubMedGoogle Scholar
- Brastianos PK, Taylor-Weiner A, Manley PE, Johns RT, Dias-Santagata D, Thorner AR, et al. Exome sequencing identifies BRAF mutations in papillary craniopharyngiomas. Nat Genet. 2014;46:161–5. doi:10.1038/ng.2868.View ArticlePubMed CentralPubMedGoogle Scholar
- Schindler G, Capper D, Meyer J, Janzarik W, Omran H, Herold-Mende C, et al. Analysis of BRAF V600E mutation in 1,320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma, ganglioglioma and extra-cerebellar pilocytic astrocytoma. Acta Neuropathol. 2011;121:397–405. doi:10.1007/s00401-011-0802-6.View ArticlePubMedGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.