Analysis of KIAA1549 BRAF fusion gene expression and IDH1/ IDH2 mutations in low grade pediatric astrocytomas

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1 J Neurooncol (2014) 117: DOI /s LABORATORY INVESTIGATION Analysis of KIAA1549 BRAF fusion gene expression and IDH1/ IDH2 mutations in low grade pediatric astrocytomas Gabriela Rampazzo Cruz Indhira Dias Oliveira Laís Moraes Mário Del Giudice Paniago Maria Teresa de Seixas Alves Andrea Maria Capellano Nasjla Saba-Silva Sérgio Cavalheiro Janete Maria Cerutti Silvia Regina Caminada Toledo Received: 6 May 2013 / Accepted: 31 January 2014 / Published online: 17 February 2014 Ó Springer Science+Business Media New York 2014 Abstract Low-grade astrocytomas comprise about 30 % of the central nervous system tumors in children. Several investigations have searched a correlation between the BRAF gene fusions alterations and mutations at IDH1 and IDH2 genes in low grade pediatric astrocytomas. This study identified the expression of KIAA1549 BRAF fusion gene and BRAF V600E mutation, mutations at exon 4 of the IDH1 and IDH2 genes in samples of pilocytic astrocytomas (PA) and grade-ii astrocytomas (A-II) pediatric patients. The correlation between these alterations and the clinical profile of the patients was also evaluated. Eightytwo samples of low-grade astrocytomas (65 PA and 17 A-II) were analyzed by PCR and sequencing for each of the Electronic supplementary material The online version of this article (doi: /s ) contains supplementary material, which is available to authorized users. G. R. Cruz I. Dias Oliveira M. Del Giudice Paniago M. T. de Seixas Alves A. M. Capellano N. Saba-Silva S. Cavalheiro S. R. C. Toledo (&) Department of Pediatrics, Pediatrics Oncology Institute- GRAACC (Grupo de Apoio ao Adolescente e à Criança com Câncer), UNIFESP (Federal University of São Paulo), Rua Botucatu, no 743, Floor 8 Genetics Laboratory, Vila Clementino, São Paulo, SP , Brazil silviatoledo@graacc.org.br G. R. Cruz gabi.rampazzo@gmail.com I. Dias Oliveira indhiradias@graacc.org.br M. Del Giudice Paniago mariopaniago@graacc.org.br M. T. de Seixas Alves mtseixas@patologia.epm.br A. M. Capellano deacapellano@uol.com.br targets identified. We identified the KIAA1549 BRAF fusion transcript in 45 % of the samples. BRAF V600E and BRAFins598T mutations were detected in 7 and 1 % of the samples, respectively. Mutations in the R132/R172 residues of the IDH1/IDH2 genes were detected in only two samples, and the G105G polymorphism (rs :c[t) was identified in ten patients. Additionally, we observed two mutations out of the usual hotspots at IDH1 and IDH2 genes. We observed a smaller frequency of mutations in IDHs genes than previously described, but since the prior studies were composed of adult or mixed (adults and children) samples, we believe that our results represent a relevant contribution to the growing knowledge in low grade childhood astrocytomas. Keywords Astrocytoma KIAA1549 BRAF IDH1 IDH2 Gene expression Sequencing N. Saba-Silva nasjlasaba@bol.com.br S. Cavalheiro iscava@uol.com.br G. R. Cruz I. Dias Oliveira L. Moraes J. M. Cerutti S. R. C. Toledo Division of Genetics, Department of Morphology and Genetics, Federal University of São Paulo, São Paulo, SP, Brazil lais.moraes@gmail.com J. M. Cerutti j.cerutti@unifesp.br M. T. de Seixas Alves Department of Pathology, Federal University of São Paulo, São Paulo, SP, Brazil S. Cavalheiro Departament of Neurology, Federal University of São Paulo, São Paulo, SP, Brazil

2 236 J Neurooncol (2014) 117: Introduction Brain tumors are the second largest group of childhood cancer, comprising % of cancer cases, and are the leading cause of cancer-related death in children. About 30 % of the central nervous system tumors in children are low-grade astrocytomas [1]. In this heterogeneous group, the most common is pilocytic astrocytomas (PA; WHO grade I), typically circumscribed and slow growing lesions with a favorable prognosis. In contrast, low-grade diffuse astrocytomas (A-II; WHO grade II) show extensive, diffuse infiltration into the brain parenchyma and stabilization of this disease depends more on adjuvant therapy than on surgical resection [2]. Until recently, little was known about the genetic background of pediatric low-grade gliomas; DNA copy number changes were rarely identified and when detected, were inconsistent, the most common abnormality being a gain of chromosome 7 or 7q [3, 4]. In the last years, studies have demonstrated that the mitogen-activated protein kinase (MAPK) signaling pathway is affected in the vast majority of PA [5 7]. This pathway is a conserved signaling cascade which utilizes a series of kinases to transduce signals from the cell membrane to the nucleus, thereby mediating cell growth, cell survival, and cell differentiation [2]. A new mechanism of MAPK pathway activation was identified after microarray analyses of pediatric low-grade astrocytomas that showed a discrete copy number gain of approximately 2 Mb at the chromosomal region 7q34, which reflects a tandem duplication [3, 5 7]. More detailed evaluation of this duplicated region revealed recurrent start and end points in the genes KIAA1549 and BRAF. Different fusion variants were identified, all resulting in an in-frame gene fusion [5 7]. Reports showed that the fusion gene KIAA1549 BRAF was present in about % cases of pediatric low-grade astrocytomas [3, 5 15]. The different KIAA1549 BRAF fusion variants are expected to be functionally similar because they all possess the BRAF kinase domain, whereas the NH2 terminus (including the BRAF auto-regulatory domain) is replaced by KIAA1549, which suggests that the fusion protein is constitutively active [16]. BRAF is an intracellular serine/ threonine kinase component of the MAPK pathway [17]. This pathway normally begins with activation of a transmembrane receptor tyrosine kinase, which binds, phosphorylates, and activates RAS, which in turn activates a RAF kinase and MEK1/2, leading to transcription of the ERK1/2 complex. Like most signaling pathways, the MAPK cascade has a wide range of effects, some of which seem contradictory. For example, MAPK activation can result in proliferation, survival, and tumorigenesis but can also trigger cell differentiation and senescence. This duality of the MAPK pathway might help explain why most gliomas with BRAF activation are low grade and tend to stay that way unless other genetic alterations also occur [18]. In gliomas, there is another way to turn on BRAF, the constitutively active valine-to-glutamate (V600E) point mutation, which activates MEK without first needing upstream RAS phosphorylation [18, 19]. BRAF V600E is not specific to PA, and is also found at high frequencies in gangliogliomas, pleomorphic xanthoastrocytomas, and diffuse astrocytomas. The BRAF V600E mutation it is likely that mimics the phosphorylation of the activating amino acids threonine and serine, thereby leading to constitutive activation of the BRAF protein [15]. This activation affects overall cell proliferation, differentiation, and survival. Another activating mutation, BRAFins598T involving a 3-bp insertion of a threonine residue is able to induce constitutive kinase activity and was described in tumor samples of children with PA [15, 20]. Ramkissoon et al. [22] identify recurrent focal amplification of MYBL1 exclusively in A-II samples in 5/18 (28 %) samples with no MYB deletion, of which none had BRAF duplication and 36 % had BRAF V600E mutations. They propose that these findings provide genetic and functional evidence of a role of MYBL1 and MYB protooncogene family of transcription factors in low-grade gliomagenesis. The MYB family members share extensive homology in their DNA-binding domains; however, they differ in their C-terminal domains. They suggest that truncations affecting the C terminus of MYB family transcription factors may be sufficient to drive oncogenesis into a discrete molecular subclass of A-II by acting as gain-offunction mutations. Zhang et al. [22] used whole-genome sequencing to identify genetic alterations and intragenic duplications of the portion of FGFR1 encoding the tyrosine kinase domain (TKD) and rearrangements of MYB or MYBL1. These events were recurrent and mutually exclusive in 53 % (21/ 39) of A-II. Duplication of the FGFR1 TKD or MYB overexpression show activation of the MAPK/ERK and PI3 K pathways, thus presenting an immunoassay profiles that are similar to those of pilocytic astrocytomas with KIAA1549 BRAF fusions and suggesting potential of the benefit therapeutic intervention of targeting upregulation of the MAPK/ERK and PI3K pathways in a disease that causes considerable morbidity and early mortality. Alteration of cellular metabolism has been proposed as a novel oncogenetic mechanism in diverse malign tumors, including gliomas. The genes encoding, cytoplasmic and mitochondrial, isocitrate dehydrogenases IDH1 and IDH2, respectively, are recurrently mutated in cancer [24]. These metabolic enzymes, which integrate to cellular respiration, have subsequently unveiled a fascinating and complex

3 J Neurooncol (2014) 117: biology underlying the dysregulation and functional consequences of metabolites in cancers [24]. In mitochondria, this reaction is part of the tricarboxylic-acid cycle (also known as the Krebs cycle). The function of all isocitrate dehydrogenases is to catalyse the oxidative decarboxylation of isocitrate into a-ketoglutarate with oxalosuccinate formed as an intermediate product. During the enzymatic reaction, nicotinamide adenine dinucleotide phosphate (NADP? ) functions as the electron acceptor [25]. Mutant IDH1 and IDH2 proteins are not inactive, once they adopt a new active mutated site [25, 26]. A series of biochemical experiments confirmed that mutant IDH1 and IDH2 have a fascinating neomorphic enzymatic, acquiring capacity to metabolize a-ketoglutarate (a-kg) into 2-hydroxy-glutarate (2-HG), oncometabolite the accumulation of which promotes tumorigenesis though inhibiting a cancer-associated transcription factor such as hypoxiainduced factor [27]. These findings suggest that the normal physiology of cellular respiration and metabolism is profoundly altered in IDH1 or IDH2 mutant cancer cells [25, 26]. A genome-wide sequencing study identified somatic, missense, heterozygous mutations in the IDH1 gene, mainly affecting the codon 132 in glioblastoma multiforme (GBM) tumors [25, 26]. Subsequent studies showed that some gliomas without cytosolic IDH1 mutations have mutations in IDH2 that affect R172, the residue analogous to IDH1 R132, and R140 [27, 28]. The incidence of IDH1 mutation is high in most adult diffuse gliomas and mutation in the homologous IDH2 gene and also occurs though not so often as in IDH1. In grade-ii and grade-iii adult gliomas, IDH1 is the most commonly mutated gene, with an incidence of up to 75 %, and is associated with overall and progression-free survival in cases of gliomas, including GBM, which suggests that future clinical trials might need stratification by IDH mutation status [25]. Although several investigations have shown the correlation of BRAF gene fusion and mutations with the clinicpathological aspects of PA in childhood and many other studies are seeking the role of mutations of the genes IDH1 and IDH2 in astrocytomas, no studies have addressed both aspects in a population composed exclusively of pediatric patients. This study aims to analyze the expression of the fusion gene KIAA1549 BRAF, BRAF V600E and mutations of genes IDH1 (R132) and IDH2 (R172/R140) in samples of PA and A-II pediatric patients, seeking to validate the findings of the literature in our population and its correlation with the clinical profile of patients. Materials and methods Eighty-two frozen astrocytoma samples, 65 PA and 17 A-II, were obtained after informed consent was signed by patients/guardians attending the IOP-GRAACC/UNIFESP (Pediatric Oncology Institute Grupo de Apoio à Criança e ao Adolescente com Câncer /Federal University of São Paulo) and according to the University s IRB (0475/11). Total RNA and DNA were isolated from tumor samples. RNA from each sample was synthesized into doublestranded cdna. The KIAA1549 BRAF fusion variants and BRAF V600E mutation were amplified by RT-PCR (reverse transcriptase polymerase chain reaction), using primers as per sequences listed at Table 1 of Supplementary Table, based on previously published protocols [7, 29]. PCR products were analyzed by electrophoresis with 2.0 % agarose gels and stained with Gel Red (Biotium, Hayward, CA, USA). Exon 4 genome DNA isolated was amplified by PCR amplification for exon 4 of both IDH1 and IDH2 genes, using primers as per sequences are listed at Table 1 of Supplementary Table. The PCR amplicons were visualized in 2.0 % agarose gel. Our PCR products from IDH1, IDH2 and KIAA1549 BRAF PCR were sequenced to identify mutations. Data analysis was performed using GraphPad Prism version 5 (San Diego, California, USA). Clinical variables tested in the presence of the fusion gene and gene mutations were: gender, age at diagnosis, tumor grade, primary site, and occurrence of relapse. Amino acid sequence of IDH1 was aligned as described by Hermely et al. [30]. Additional methods are detailed in full in Supplementary Methods. Results Clinical data A summary of the the clinicopathological parameters analyzed in this study is detailed in Table 1. The clinicopathological parameters analyzed were age at diagnosis, gender, relapse, tumor location, and histological type. RT- PCR for detection of KIA1549 BRAF fusion gene and PCR followed by DNA sequencing for identification of IDH1 and IDH2 mutations on 82 astrocytoma samples from 82 patients. KIAA1549 BRAF fusion gene Out the 82 samples, 37 (45 %) had the KIAA1549 BRAF fusion gene while in 45 (55 %) it was not detected (Table 2). The sequence identities were confirmed using the BLAST platform (Pubmed) and showed that there were 2 types of fusion genes. The first group consisted of 600 bp corresponding to the fusion gene between exon 16 of KIAA1549 and exon 9 of gene BRAF (KIAA149ex16- BRAFex9) it was observed in 35 of the 37 positive samples (96 %). The other group consisted of only 2 samples (4 %)

4 238 J Neurooncol (2014) 117: Table 1 Clinic-pathological parameters of patients evaluated Parameters Classes N (%) Gender Male 37 (45 %) Female 45 (55 %) Histology PA 65 (79 %) A-II 17 (21 %) Status Alive 56 (68 %) In treatment 6 (7 %) Dead 20 (24 %) Relapse Yes 9 (11 %) No 73 (89 %) Age at diagnosis (years) (34 %) (36 %) (24 %) (4 %) with 750 bp corresponding to the fusion gene between exon 15 of KIAA1549 and exon 9 of gene BRAF (KIAA149ex15-BRAFex9). In our group of 37 positive samples, 34 (92 %) were from PA and 3 were from A-II (8 %). Eight patients (22 %) died from disease progression, 24 (65 %) are free of disease and 4 (11 %) are still in treatment (Table 4 of Supplementary Table). The presence of the KIAA1549 BRAF fusion gene did not correlate with clinic-pathological parameters. Overall survival analysis did not demonstrate significant difference between PA patients with positive and negative KIAA1549 BRAF tumor samples (Fig. 1a). In the A-II group, the presence of this gene fusion showed a worse impact on the patients survival. However, this result is not significant as the group with positive KIAA1549 BRAF/A-II consisted of three patients only (Fig. 1b). Additionally, we analyzed a subgroup of 28 patients (the clinically relevant group according to Hawkins et al., 2011) which included patients with noncerebellar, non neurofibromatosis 1, and sub totally resected or inoperable PA. The survival analysis of this subgroup demonstrated that those with the KIAA1549 BRAF fusion had a better survival (5-year overall survival) than those who did not present this gene fusion; however this difference did not significant (p = ) (Fig. 2). BRAF mutation A mutational analysis of the BRAF gene was performed in all 82 samples. BRAF mutations were present in 7/82 (8.5 %), 6 of which showed V600E and only one showed BRAF ins598t. BRAF V600E was present in 5/65 (8 %) of PA samples and in 1/17 (6 %) of A-II samples. BRA- Fins598T was identified in one (Tables 2, 4 of Supplementary Table). Table 2 Frequency of IDH mutations, KIAA1549 BRAF fusion gene and BRAF mutations in PA and II-A samples Histology IDH1/2 alterations KIAA1549 BRAF BRAF V600E BRAF T599_V600insT IDH1 mutation IDH1 polymorphism IDH2 mutation Frequency Type Frequency Type Frequency Type PA (65 samples) 1/65 (1,6 %) c.326g[a 8/65 (12,3 %) G105G 2/65 (3,1 %) R172 and c.429g[c 34/65 (52,3 %) 5/65 (7,7 %) 0 A-II (17 samples) 1/17 (5,8 %) R132 2/17 (11,7 %) G105G 0 3/17 (17,6 %) 1/17 (6 %) 1/17 (6 %) Total (82 samples) 6/82 (7,31 %) 1/82 (1.2 %)

5 J Neurooncol (2014) 117: survival survival apercent b p= Months IDH1 and IDH2 alterations PA KIAA1549-BRAF + PA KIAA1549-BRAF - AII KIAA1549-BRAF + AII KIAA1549-BRAF - p= Months Fig. 1 Overall survival of PA and A-II patients with KIAA1549 BRAF fusion gene. a Overall survival of PA patients classified by positive and negative tumors for KIAA1549 BRAF fusion gene. b Overall survival of A-II patients classified by positive and negative tumors for KIAA1549 BRAF fusion gene Percent survival KIAA1549-BRAF + KIAA1549-BRAF - p= Months Fig. 2 Overall survival of subgroup of clinically relevant patients classified for KIAA1549 BRAF fusion gene presence Out the 82 samples investigated, 11 (13.4 %) showed alterations in at least one of the IDHs tested (Table 2). The 11 patients were aged from 1 to 15 years (five 1 5, four 6 10, two years old). Two samples showed mutations at the IDH1 gene, one at the previously discussed R132H (c.395g[a) (supplementary figure 1A) and another at R109K (c.326g[a) (supplementary figure 1B), a mutation that had been first described in one sample of lung carcinoma at COSMIC database. We proceeded the alignment of the sequence to improve the data of this mutation, since no reference had done it before (supplementary figures 2). One sample showed a mutation at R172S (c.516g[c) of IDH2 gene (supplementary figure 1C). We also found a new synonymous variant of IDH2 exon 4, L143L (c.429g[c) (supplementary figure 1D). Additionally 10 samples showed polymorphism rs :c[t (G105G) at IDH1 gene. Both the sample carrying the mutation at R132S and the sample carrying the L143L exon variant showed the G105G polymorphism. In the total, we found three mutations in 63 PA samples (4,8 %) and 1 mutation in 21 A-II samples (4,8 %) (Table 4 of Supplementary Table). None of the studied samples showed mutations at the R140 of IDH2 gene. Statistical analyses did not show correlations between the presence of mutations/polymorphisms and the clinicopathological or survival parameters evaluated. KIAA1549 BRAF versus IDH1/2 mutations Of the 39 samples with positive KIAA1549 BRAF fusion gene, four (10 %) showed a simultaneous IDH1 G105G polymorphism, but no sample had a mutation at residues R132 of IDH1 and R172 or R140 of IDH2 gene. Discussion Several studies have investigated the importance of the MAPK signaling pathway activation in the development of low-grade pediatric astrocytomas, and recently some studies have shown that mutations of IDH1 and IDH2 genes are determining factors in establishing various types of cancers, including high-grade gliomas, in adults only [3, 5 15, 21 24]. Concomitant analysis of the presence of KIAA1549 BRAF gene fusion and BRAF V600E mutation and the identification of IDH1 and IDH2 mutations, in a group of PA and A-II pediatric astrocytomas may shed light on the understanding of this disease in children. In our study, we identify KIAA1549 BRAF gene fusions in 45 % of 82 low-grade glioma samples and 96 % of these transcripts corresponded to KIAA149ex16-BRAFex9 variant fusion, involving the exon 16 of KIAA1549 gene and exon 9 of BRAF gene. The remaining 4 % samples showed KIAA1549ex15-BRAFex9 variant fusion, involving the exon 15 of KIAA1549 and exon 9 of BRAF gene. Our results are in agreement with studies published on lowgrade gliomas, showing that % of patients with PA and about 22 % of patients with non pilocytic low-grade gliomas present the KIAA1549 BRAF fusion or BRAF copy number gain [3, 5 15]. Furthermore, the KIAA149ex16-BRAFex9 variant fusion was the most

6 240 J Neurooncol (2014) 117: common variant observed in low grade astrocytoma samples [5 7]. KIAA1549 BRAF fusion gene is commonly found in PA and rarely in A-II. An earlier study had suggested that KIAA1549 BRAF fusion could be specific to PA [31]. In our study, we found this fusion gene in 34 (52 %) of the 65 PA samples and in 3 (18 %) of the 17 A-II samples analyzed, therefore the histological classification could not be determined. Lin et al. [8] showed that KIAA1549 BRAF fusions are found predominantly in PA, but can occur also in other low-grade gliomas. While the KIAA1549 BRAF fusion is the most common alteration in PA, it remains debatable whether it is a specific diagnostic marker for PA [15]. Previous studies reported activation of the MAPK pathway in PA by an alternative mechanism, such as the previously mentioned SRGAP3 RAF1 or punctual mutations as BRAF V600E [12]. Further experiments are necessary to verify whether the MAPK pathway is also activated in the remaining samples and, if so, which molecular mechanism is responsible for that alteration. There are conflicting reports about correlation of the KIAA1549 BRAF fusion with survival. The overall survival analysis of our cohort demonstrated that KIAA1549 BRAF fusion gene had no impact on the PA patient group. In the A-II group, patients carrying KIAA1549 BRAF positive tumors showed worse overall survival than their counterparts. However, the number of A-II positive patients was extremely low (only 3) and this result is not likely to be biologically significant. A recent study, involving a clinically relevant group of 70 children with neocerebellar, non neurofibromatosis 1, subtotally resected or inoperable PA demonstrated that 100 % of those with the KIAA1549 BRAF fusion had a 5-year overall survival compared with 88 % for those who did not have the fusion [13]. On the other hand, other studies used a similar cohort of clinically relevant PA, but there was no significant increase in the survival in the KIAA1549 BRAF fusion group [8, 18]. When we selected the same group of patients, using the same clinicopathological criteria, we also obtained the same results. Additionally, we performed BRAF mutational analysis looking for exon 15 point mutations and noticed that six samples (7.3 %) had BRAF V600E and one (1.2 %) BRAF ins598t mutations. BRAF V600E exists in diverse tumors and is present in % of pediatric A-II, but in\2 % of comparable adult gliomas. Both in children and adults, \10 % of all PAs underwent the mutation [18]. Although sometimes present in tumors with PA like morphology, BRAF V600E is more associated with other tumors, including % of pediatric and adult gangliogliomas and % of pleomorphic xanthoastrocytomas in both age groups [18]. As to BRAFins598T mutation causes a constitutive activity similar to the BRAF V600E and is found in approximately in 1 2 % of PA samples [32 34]. Occasionally, the mutation can occur in conjunction with a BRAF fusion in the same tumor. In our study, of five PA samples positive for the BRAF V600E mutation, three showed the KIAA1549 BRAF fusion gene. Hawkins et al. (2011) identified BRAF V600E mutation in three (2.7 %) PA samples, two of which had concomitant KIAA1549 BRAF fusion of the gene. Horbinski et al. (2012) observed six tumors with both BRAF rearrangement and BRAF V600E mutations occurring in the same tumor. Because such tumors were unusual, the authors suggested that it is difficult to prove whether they behave differently from other tumors. While the KIAA1549 BRAF fusion gene is the most common in pediatric PA, IDH1/IDH2 mutations are common in diffuse gliomas in adults [31, 32]. Contrarily to their evidence in adults, IDH mutations are rare in pediatric gliomas, regardless of histological type [31, 36, 37]. Our analyses have shown that only one of the 17 A-II samples investigated had a mutation at R132 of IDH1 gene (5.8 %). Pollack et al. (2011) examined the frequency of IDH mutations in a cohort of pediatric high-grade gliomas (III and IV) and found IDH1 mutations in 7 of the 43 (16.3 %) tumors, while no IDH2 mutations were observed [36]. Dougherty et al. [37] studied 31 low-grade gliomas (ganglioglioma, desmoplastic infantile ganglioglioma, dysembryoplastic neuroepithelial tumor and pleomorphic xanthoastrocytoma). There were no cases with a KIAA1549 BRAF fusion product, only one IDH1 R132H mutation was detected, and in 14 out of 31, they found BRAF V600E mutation. Since our sample group was composed of only pediatric low-grade astrocytomas, we believe that our results represent a reliable distribution panel of IDH mutations. There is a growing evidence of the widespread presence of IDH1 and IDH2 mutations in different cancers and we analyzed the three most commonly reported mutation spots, having also found out two new mutations out besides R132/R172. A new study has shown that mutations in other spots of IDH genes can produce the oncometabolic 2-HG: IDH1 R100, as in adult glioma; IDH1 G97 mutated in colon cancer cell lines and pediatric glioblastomas, and IDH1 Y139 [4]. These findings corroborate the theory that deregulation in metabolic enzymes encoded by genes IDH1 and IDH2 can originate from changes other than R132/ R172 described in most studies. Recently, a synonymous single-nucleotide polymorphism (SNP) G105G (rs :c[t), located in the 5 0 region of exon 4 of IDH1 genes, has been associated with a worse prognosis for AML (acute myeloid leukemia) patients with normal karyotype [32, 33]. As to adult gliomas, this polymorphism was present in approximately 11 % of the samples and was associated with a negative

7 J Neurooncol (2014) 117: impact on prognosis of patients with high-grade gliomas [37]. In our study we found polymorphism G105G in 10 (15 %) samples, but statistical analyses show no correlation with the clinical features or prognosis of the patients. Sequence alignment of human IDH1 with that of different mammal species showed that the c.326g[a mutation is located in a highly conserved region, which suggests this mutation to be potentially affected by IDH1 s enzymatic activity. Further experiments should be performed to verify whether the enzymatic activity is really altered by this mutation. The formation of the KIAA1549 BRAF fusion gene and the IDH mutations have been considered to be two genetic events occurring, respectively, in a mutually exclusive manner in PAs and diffuse gliomas [31]. However, it is necessary to emphasize that these two types of neoplasm occur in different age groups, PA in children and diffuse gliomas in adults. Then, it is possible that the apparent mutual exclusion in presence of the KIAA1549 BRAF fusion gene or IDH mutation may merely reflect the existence of these different classes [35]. Moreover most of previous studies have investigated either BRAF alterations or IDH mutations in pediatric or adult populations. Our study addressed both IDH mutations and the presence of KIAA1549 BRAF fusion gene in a large series of pediatric gliomas. In 2012 a similar investigation had been carried out by Badiali et al. Their results showed IDH mutations and the presence of KIAA1549 BRAF fusion gene in 17 out of 180 (9.4 %). Our study showed that G105G polymorphism in IDH1 gene can be found simultaneously with the presence of KIAA1549 BRAF fusion gene, which may support their findings, indicating gene fusion presence detection and mutations of IDH genes are independent events. Future studies should expand on these observations and continue to define the genetic and biological features of pediatric low-grade gliomas, the role of BRAF alterations in the pathogenesis and clinical behavior of these tumors as well as the relevance of IDH mutations to prognosis. Knowing the reason for this heterogeneity may lead to better understanding of low-grade astrocytomas in children and may shed light on how they differ from their adult counterparts. Acknowledgments This work was supported by grants from FA- PESP (The State of São Paulo Research Foundation: 2011/ ; 2011/ ) and GRAACC (Grupo de Apoio ao Adolescente e Criança com Câncer). Conflict of interest of interest. The authors declare that they have no conflict Ethical Standards Samples from each Astrocytoma tumor were collected after informed consent was signed by patients/guardians according to the university s institutional review board (IRB/Federal University of São Paulo no 0475/11). References 1. 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