Skip to main content
NCBI home page
Brain Pathology logoLink to Brain Pathology
. 2017 Jan 6;27(5):580–589. doi: 10.1111/bpa.12444

Genetic alterations related to BRAF‐FGFR genes and dysregulated MAPK/ERK/mTOR signaling in adult pilocytic astrocytoma

Pankaj Pathak 1, Anupam Kumar 2, Prerana Jha 1, Suvendu Purkait 1, Mohammed Faruq 3, Ashish Suri 2, Vaishali Suri 1, Mehar C Sharma 1, Chitra Sarkar 1,✉
PMCID: PMC8029314  PMID: 27608415

Abstract

Pilocytic astrocytomas occur rarely in adults and show aggressive tumor behavior. However, their underlying molecular‐genetic events are largely uncharacterized. Hence, 59 adult pilocytic astrocytoma (APA) cases of classical histology were studied (MIB‐1 LI: 1%–5%). Analysis of BRAF alterations using qRT‐PCR, confirmed KIAA1549‐BRAF fusion in 11 (19%) and BRAF‐gain in 2 (3.4%) cases. BRAF‐V600E mutation was noted in 1 (1.7%) case by sequencing. FGFR1‐mutation and FGFR‐TKD duplication were seen in 7/59 (11.9%) and 3/59 (5%) cases, respectively. Overall 36% of APAs harbored BRAF and/or FGFR genetic alterations. Notably, FGFR related genetic alterations were enriched in tumors of supratentorial region (8/25, 32%) as compared with other locations (P = 0.01). The difference in age of cases with FGFR1‐mutation (Mean age ± SD: 37.2 ± 15 years) vs. KIAA1549‐BRAF fusion (Mean age ± SD: 25.1 ± 4.1 years) was statistically significant (P = 0.03). Combined BRAF and FGFR alterations were identified in 3 (5%) cases. Notably, the cases with more than one genetic alteration were in higher age group (Mean age ± SD: 50 ± 12 years) as compared with cases with single genetic alteration (Mean age ± SD: 29 ± 10; P = 0.003). Immunopositivity of p‐MAPK/p‐MEK1 was found in all the cases examined. The pS6‐immunoreactivity, a marker of mTOR activation was observed in 34/39 (87%) cases. Interestingly, cases with BRAF and/or FGFR related alteration showed significantly lower pS6‐immunostatining (3/12; 25%) as compared with those with wild‐type BRAF and/or FGFR (16/27; 59%) (P = 0.04). Further, analysis of seven IDH wild‐type adult diffuse astrocytomas (DA) showed FGFR related genetic alterations in 43% cases. These and previous results suggest that APAs are genetically similar to IDH wild‐type adult DAs. APAs harbor infrequent BRAF alterations but more frequent FGFR alterations as compared with pediatric cases. KIAA1549‐BRAF fusion inversely correlates with increasing age whereas FGFR1‐mutation associates with older age. Activation of MAPK/ERK/mTOR signaling appears to be an important oncogenic event in APAs and may be underlying event of aggressive tumor behavior. The findings provided a rationale for potential therapeutic advantage of targeting MAPK/ERK/mTOR pathway in APAs.

Keywords: adult pilocytic astrocytoma, BRAF, FGFR1, low grade glioma, MAPK/ERK, mTOR

Introduction

Pilocytic astrocytomas (PA) are the most common pediatric glial tumor comprising about 40% of all childhood brain tumors 14. In the updated 2016 World Health Organization (WHO) classification of CNS tumors, PAs are classified as Grade I astrocytic tumors with an indolent course in most cases, and a small proportion with aggressive clinical behavior 30. PAs in adults are associated with higher mortality than in pediatric and survival tends to decrease with increasing age in adults 8, 18, 44, 45. Characteristically, tandem duplication of 7q34 leading to various KIAA1549‐BRAF novel oncogenic fusions are known to be the most frequently observed genetic alteration, occurring in about 60%–70% of pediatric PAs 1, 6, 9, 15, 22. Other genetic alterations include the activating BRAF (v‐Raf murine sarcoma viral oncogene homolog B1) mutation namely BRAFV600E occurring in about 9% of PAs 32. In certain PAs, neurofibromatosis type 1 (NF1) inactivation mediates hyperactivation of the oncogene KRAS 16. Overall, majority of PAs are driven by aforementioned genetic alterations, leading to the mitogen‐activated protein kinase/extracellular signal‐regulated kinase (MAPK/ERK) pathway activation 17, 19. Additionally, activation of mTOR has also been reported in about 50% of PAs and its activation linked with histological aggressiveness 13, 40.

Recently, next generation sequencing (NGS) studies including exome and RNA sequencing defined the genetic background of PA to a great extent in pediatric patients. Recurrent activating FGFR1 (Fibroblast growth factor receptor 1) hotspot mutation, FGFR1 encoding the tyrosine kinase domain duplication (FGFR‐TKD) and FGFR1‐TACC1 fusion were identified in a small proportion of PAs with no BRAF related genetic alterations 20, 52. Functional study showed FGFR‐TKD induced FGFR1 autophosphorylation and upregulation of both MAPK/ERK and PI3K pathways 52. Furthermore, Brokinkel et al, also reported FGFR1 mutation in primary PAs and found it exclusively in BRAFV600E and KIAA1549‐BRAF wild type patients 3. However, in this study FGFR‐TKD was not examined. More recently, in a study on predominantly pediatric PAs, Becker et al reported a high frequency of KIAA1549:BRAF fusion associated with a better outcome whereas FGFR1 mutation was rare and found to be associated with worse outcome 2. Remarkably, these authors found that immunohistochemical expression of FGFR1 was present in 74% of PAs, regardless of the histology or BRAF/FGFR related genetic alterations.

Pilocytic astrocytoma in adults is rare and these present more clinical aggressive phenotype, with high rate of recurrence and morbidity 8, 18, 44, 45. The reported overall survival rate among adults is 89.5% at 24 months and 83.8% at 60 months 18. Most adverse outcome is reported in older adults (>60 years) with 5‐year survival rate of 52% 18. Similar to pediatric, constitutive activation of Ras/RAF/MAPK signaling appears to be underlying oncogenic event in sporadic adult PAs 3. However, the only genetic alteration reported in adult PA is KIAA1549‐BRAF fusion found in 20%–32% of cases and very rare or absent BRAFV600E mutation 9, 40, 45. In a recent published study, Brokinkel et al, and Jones et al, reported FGFR1 mutation in few adult PAs; however, the exact frequency of this mutation is unclear among adults 3, 20. Combining all of the genetic studies published on adult PAs, more than two‐thirds remain genetically uncharacterized.

Overall, scarce genetic studies have been performed in adult PAs and their underlying genetic events that may lead to more aggressive clinical course and high morbidity is poorly understood. The recent genetic alterations reported in pediatric PAs hold promise to genetically characterize the adult tumors. The aggressive clinical course in adult PAs indicates possible genetic heterogeneity and necessitates comprehensive genetic studies to evolve with potential therapeutic target. Hence, we evaluated the clinicopathological and genetic alterations of adult pilocytic astrocytomas in an attempt to determine the underlying biology for their reported adverse clinical course. In the present study, we studied the frequency of different BRAF and FGFR related genetic alterations and also examined the MAPK/ERK and mTOR pathway activation in adult PAs. Notably, we found infrequent BRAF alterations but enriched FGFR alterations in adults as compared with that reported in pediatric PAs. In addition, coexistent BRAF and FGFR alterations and a significant association of FGFR alterations with age and tumor location were noted. The activation of MAPK/ERK and mTOR signaling was present in most of the cases suggesting that these pathways could be potential therapeutic targets in adult PAs.

Materials and Methods

Study design and tumor samples

A retrospective study was conducted after obtaining necessary ethical clearance from institute ethics committee for study on human subjects. Samples were selected on the basis of the availability of Formalin fixed paraffin embedded (FFPE, stored at room temperature) and/or frozen specimens (stored at −80°C) with adequate tumor tissue from All India Institute of Medical Sciences, New Delhi, India. The original hematoxylin and eosin (H&E) slides were re‐evaluated independently by three experienced neuropathologists (C.S., M.C.S. and V.S.). All the cases were histologically classified and diagnosis was reconfirmed following the guidelines of updated World Health Organization 2016 classification. Age, sex and other available clinical information of all patients were noted and compiled. Overall, this study was conducted on 59 sporadic adult PAs (diagnosed over a period of 10 years; 2006–2015) and 24 adult diffuse astrocytomas cases (diagnosed over a period of 4 years; 2012–2015). Among adult PAs, only the cases showing compacted piloid cells with rosenthal fibers, loose textured microcystic area with multipolar cells, all termed as classical PA were included. Cases with any proportion of oligodendroglial or ganglioglioma components were excluded.

Nucleic acid isolation and cDNA synthesis

DNA and RNA were extracted from frozen tissues as per the manufacturer's instructions using QIAmp DNA mini kit (Qiagen, Hilden, Germany) and miRvana kit (Invitrogen, Carlsbad, CA), respectively. From FFPE samples, DNA and RNA were extracted using RecoverAll nucleic acid extraction kit using manufacturer's protocol (Invitrogen, Carlsbad, CA). DNA and RNA were quantitatively and qualitatively examined using Qubit (Invitrogen, Carlsbad, CA) and agarose gel. For real time RT‐PCT reactions, cDNA synthesis was performed using 2 µg of total RNA using superscript VILO cDNA synthesis kit (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol.

qRT‐PCR for detection of KIAA1549‐BRAF and MYB‐QKI fusion

All three most abundant KIAA1549‐BRAF fusion transcripts were identified using hydrolysis probe assays described by Tian et al 47, and Kumar et al 26. Two MYB‐QKI fusion transcripts were analyzed using qRT‐PCR. The primer and probe sequences used in these assays are given in Supporting Information Table 1. Quantitative RT‐PCR (q‐RT‐PCR) was performed using 25 ηg of cDNA. RT‐PCR reaction was carried out in light cycler (LC480, Roche Diagnostics) and fluorescence was recorded and cycles to threshold (C T) were captured. All qRT‐PCR assays were performed in triplicate and PCR products were also examined on agarose gel.

Validation of KIAA1549‐BRAF fusion by sequencing

Sequencing reaction was setup with the cDNAs for the samples using pairs of primers (Supporting Information Table 1) flanking the fusion point between the KIAA1549 and BRAF. Bidirectional sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Courtaboeuf, France) using the ABI 3130xL sequencer (Applied Biosystems, Foster City, CA).

Mutation analysis of IDH1, H3F3A, BRAF‐V600E and FGFR1

For all the cases, 20 ηg of total DNA was PCR amplified for detection of mutation in IDH1, H3F3A, BRAF (codon 600) gene and hotspots codons for Asn546 and Lys656 within the kinase domain of FGFR1. The primer sequences for IDH1, H3F3A and BRAF (codon 600) were taken from elsewhere and for FGFR1, generated by Primer3 software and listed in Supporting Information Table 1. Sequencing was performed as described above.

Evaluation of BRAF gain and FGFR‐TKD duplication

BRAF‐gain and FGFR‐TKD duplication were assessed by qRT‐PCR. CF (7q31.2) and GAPDH (2p13.31) were used as reference sequences for this copy number/amplification analyzes. The Primer sequences used are provided in Supporting Information Table 1. The calculation of copy‐number change/amplification was performed using comparative Ct (threshold cycle) method, as described by Kim et al 24 The relative copy number/amplification of the target in the sample was then determined with the formula 2 × 2 − δδCt, where δδCt = δCt (sample) − δCt (normal control DNA). Control DNAs were obtained from normal brain tissues (n = 7). Tumor samples were considered to be positive for BRAF‐gain and FGFR‐TKD duplication when PCR reactions using both of the two references (GAPDH and CF) showed more than 4 fold copy‐number gain/amplification of genes/gene‐domain.

Immunohistochemical examination

Immunohistochemical analysis was performed on serial 5 µm thick FFPE tissues as described by Kumar et al 26. All the antibodies used were already proven for immunohistochemical examinations and used in several peer reviewed studies 13, 20, 26 delivering satisfactory sensitivity and specificity. For examination of MAPK/ERK and mTOR pathways activation, antibodies applied were directed against pMAPK (4376, Cell Signaling Technology, The United States), pMEK1 (2338, Cell Signaling Technology, The United States) and pS6 (residue 273; Cell Signaling Technology, The United States) and details are listed in Supporting Information Table 2. Positive controls included: breast carcinoma for pMEK1, colon carcinoma for pMAPK and pS6. Omission of secondary antibody from IHC protocol was used as negative control. Cytoplasmic staining for pMAPK and pMEK1 and nuclear/cytoplasmic staining for pS6 were scored. The labeling indices for all antibodies were calculated in a four‐tiered scale (0 = negative, 1 = 1%–10% positive cells, 2 = >10%–50% positive cells and 3 = >50% positive cells) as also described before 13.

Statistical analysis

Analysis of data was performed using SPSS 16.0 version. Associations were analyzed with two‐sided Fisher exact test, Student's t‐tes or Chi square. A P value of <0.05 was considered significant.

Results

Clinical data

Fifty nine sporadic adult pilocytic astrocytoma patients including 31 frozen tumor tissues with corresponding FFPE tumor tissue blocks and 28 FFPE blocks with adequate tumor tissues fulfilled the inclusion criteria. The patients represented 31 males and 28 females (sex ratio = 1.1:1) with median age of the patients at diagnosis 30 years (range 19–69 years). Among 59 patients, all were primary tumors except one recurrent tumor. Tumor proliferation labeling indices MIB‐1 ranged from 1% to 5% among all the cases. Tumor site was available for 55 patients and this comprised of 25 supratentorial (median age at diagnosis: 28 years), 20 infratentorial including six cerebellar and seven brainstem (median age at diagnosis: 31 years), five optic (median age at diagnosis: 24 years) and five spinal (median age at diagnosis: 35 years). Further, none of the adult PA cases showed IDH1 or H3F3A mutation and summary of the various clinicopathological parameters along with molecular features examined in this study is detailed in Figure 1.

Figure 1.

Figure 1

Open in a new tab

Clinicopathological and molecular genetic features for the 59 adult pilocytic astrocytoma cases with the different genetic and immunohistochemical analyses. (B‐K fusion: BRAF‐KIAA fusion).

BRAF related genetic alterations

We analyzed KIAA1549‐BRAF fusion, BRAF gain and BRAF‐V600E mutation in all the patients. Among 59 cases, 11 (19%) showed qRT‐PCR based evidence of KIAA1549‐BRAF fusion gene transcripts. This comprised of KIAA1549‐Ex16 –BRAF‐Ex9 (7 cases), KIAA1549‐Ex15_BRAF‐Ex9 (3 cases) and none of KIAA1549‐Ex16_BRAF‐Ex11 fusion transcripts. One of the cases showed both KIAA1549‐Ex16_BRAF‐Ex9 and KIAA1549‐Ex15_BRAF‐Ex9 fusion transcripts. Representative samples with different fusion transcripts were also confirmed by sanger sequencing. KIAA1549‐BRAF fusion transcripts were present in 6/20 (30%) of infratentorial, 4/25 (16%) of supratentorial, 1/5 (20%) spinal and 0/5 (0%) of optic tumors respectively. Overall, KIAA1549‐BRAF fusion was more abundant in infratentorial locations (6/20, 30%) as compared with other tumor sites (5/35, 14.2%) (Figure 1), however it did not reach statistical significance (P = 0.1).qRT‐PCR revealed BRAF copy number gain in 2/59 (3.4%) and BRAF mutation analyzed by direct sequencing in only 1/59 (1.7%) cases. However BRAF ins598T was not identified in any case. Interestingly, all of the three cases with BRAF gain and BRAF mutation were in supratentorial location and none of the tumors with other locations showed these alterations (P = 0.01). Further, correlation of genetic alterations with sex showed no association (Figure 1). Notably, two cases with KIAA1549‐BRAF fusion also showed FGFR‐TKD duplication and FGFR1 mutation, respectively.

FGFR related genetic alterations

We examined FGFR1 hotspot mutations in exon 12 and 14 in all the 59 cases using direct sequencing. FGFR1 mutation was found in 7/59 (11.9%) cases. Five patients showed only one mutation (T658P, N546K, K656E, K656E and N546K, respectively) while two patients demonstrated two and three mutations [(K656M, T658P) and (N546K, T658P, K656E)], respectively. Next, FGFR1 mutations were found in 6/25 (24%) of supratentorial and 1/20 (5%) of infratentorial APAs. None of spinal and optic tumors showed FGFR1 mutations. Based on qRT‐PCR analyses of FGFR1 tyrosine kinase domain (exon 15 and 19), FGFR‐TKD duplication was identified in 3/59 (5%) cases. Among three cases in which FGFR‐TKD duplication was identified, 2 were located in supratentorial and one in infratentorial region. Therefore, notably FGFR related genetic alterations were significantly more commonly present in supratentorial region (8/25, 32%) as compared with other tumor locations (P = 0.01) (Figure 1). No association of sex with FGFR related genetic alterations were noted. Furthermore, one case with FGFR1 mutation also showed BRAF V600E mutation. However, both KIAA1549‐BRAF fusion and BRAF gain showed dependency and mutually exclusive occurrence with FGFR1 mutation to reach a statistical significance (P = 0.002 and P = 0.02, respectively; Fisher's exact test).

Age related associations with different genetic alterations

The frequency of KIAA1549‐BRAF fusion was found to decrease with age while, on the contrary occurrence of FGFR1 mutation increased with age (Figure 2A). BRAF gain was found in a patient of 35 years and other of 58 years while FGFR‐TKD was seen only in cases aged between 20 and 40 years (Figure 2B). On analysis of the age related associations, a higher median age (37 ± SD 15.5 year) was noted for the cases with FGFR1 mutation than for the cases with KIAA1549‐BRAF fusion (28.5 ± SD 8.8 years), but the difference showed only a trend of statistical significance (P = 0.08). The association of age with respect to FGFR1 mutant (Mean age ± SD: 37.2 ± 15 years) vs. cases with KIAA1549‐BRAF (Mean age ± SD: 25.1 ± 4.1 years) reached to statistical significance after adjustment for cases with co‐existent genetic alterations (P = 0.03; Unpaired t‐test). Notably, the cases with more than one genetic alteration (two cases with KIAA1549‐BRAF fusion + FGFR‐TKD duplication and one case with FGFR1 + BRAF V600E mutation) were in higher age group (Mean age ± SD: 50 ± 12 years) as compared with cases with single genetic alteration (Mean age ± SD: 29 ± 10 years) (P = 0.003; Unpaired t‐test).

Figure 2.

Figure 2

Open in a new tab

(A) Distribution of KIAA1549‐BRAF fusion transcript and FGFR1 mutation in pilocytic astrocytomas according to age group. (B) FGFR‐TKD and BRAF gain in different age group. Vertical bars illustrating percentage of patients with indicated genetic alterations.

Immunohistochemical analysis of MAPK/ERK and mTOR pathway

Activation of mTOR pathway was evaluated using mTORC1 component p‐S6 immunopositivity in 39 cases. In total, pS6 immunoreactivity of any degree was observed in 34/39 (87%) of cases irrespective of tumor site (Figure 3C,D). However, moderate (10%–50%) to strong (>50%) pS6 immunostaining was seen in 19/39 (49%) of cases. Interestingly cases with BRAF and/or FGFR related alteration showed lower frequency of moderate to strong pS6 immunostatining (3/12, 25%) as compared with BRAF and/or FGFR wild type (16/27, 59%) and this was statistically significant (P = 0.04). Further, we tried to examine whether mTOR activation had any association with tumor site. pS6 immunostaining (moderate to strong) was seen in 2/3 (67%) optic, 3/5 (60%) spinal, 6/13 (46%) infratentorial and 6/15 (40%) supratentorial adult PAs respectively. Although there was indication of differences in mTOR activation among different tumors based on site, but statistically the comparison was not significant (P > 0.05). Activation of MAPK/ERK pathway was evaluated by immunohistochemistry using phospho specific antibodies for phospho (p)‐MAPK1 and phospho (p)‐MEK1 in both the cases with and without BRAF and FGFR related genetic alterations. Overall, moderate to strong p‐MAPK/p‐MEK1 immunopositivity corresponding to MAPK/ERK pathway activation was found in all the 45 cases examined (Figure 3E,F).

Figure 3.

Figure 3

Open in a new tab

(A) Representative photomicrographs of Pilocytic astrocytoma showing compacted piloid cells with Rosenthal fibers ((hematoxylin and eosin ×100). (B) loose textured microcystic area with multipolar cells (hematoxylin and eosin ×200). (C) Representative photomicrograph showing immunopositivity for phospho‐S6 (×200). (D) photomicrograph with immunonegativity for phospho‐S6 (×200). (E) photomicrograph showing immunopositivity for phospho‐MEK (×200). (F) photomicrograph with immunopositivity for phospho‐MAPK (×200).

Genetic and histopathological findings in IDH wild‐type adult diffuse astrocytoma (DA)

Among 24 adult DAs analyzed for IDH1 mutation, 7 cases were found to be IDH wild‐type. Further, FGFR1 hotspot mutation (exon 12 and 14) and FGFR‐TKD duplication were examined to evaluate whether these 7 cases had any genetic similarity with adult PAs. Notably, one patient showed FGFR1 mutation (T658P, age: 38 years) and other 2 cases identified with FGFR‐TKD duplication (mean age 42.5 years). Thus, overall 3/7 (42.9%) of IDH‐wt adult DA cases showed FGFR related genetic alteration (Supporting Information Table 3). However, out of 17 IDH1‐mutant adult DAs, 2 cases (11.8%) showed FGFR‐TKD alterations and no case was identified with FGFR1 mutation. Next, we analyzed all 7 IDH‐wt adult DAs for BRAF‐V600E mutation and MYB‐QKI fusion and none of the cases showed these alterations. Further, re‐examination of the histopathological diagnosis in each of these 7 IDH‐wild‐type adult DA cases was done. None of the cases showed morphology of either pleomorphic xanthoastrocytoma (PXA), ganglioglioma (GG), dysembryoplastic neuroepithelial tumor (DNET) or angiocentric glioma (AG) or pilocytic astrocytomas with a more diffuse growth pattern. All these 7 cases were predominantly composed of fibrillary neoplastic astrocytes showing mild nuclear atypia and a low cellular density. No mitotic figures, vascular proliferation or necrosis was observed.

Discussion

Pilocytic astrocytoma in adults accounts for only 4%–6% of all adult brain tumors. Moreover, PA in adults is found to be associated with high mortality as compared with childhood and juvenile patients irrespective of extent of resection 18. Therefore it is important to explore the genetic makeup of adult PAs to understand the possible mechanisms leading to this aggressive disease course. Hence, in order to explore the genetic characteristics and associated signaling pathways as well as identify possible pharmacologic targets in APAs, this study was undertaken. All the 59 patients in the present study showed median age, age range, tumor site and MIB‐LI nearly similar to the previous studies on adult PAs 14, 18, 33, 40, 44.

Overall, 19% of our patients showed KIAA1549‐BRAF fusion, which is consistent with previous report of Theeler et al, (9/45; 20%), but relatively lower than study of Hasselblatt et al, (9/37; 24%) and Rodriquez et al, (12/38; 32%, when excluding the cases with BRAF polysomy) 9, 40, 45. Relative occurrence of KIAA1549‐BRAF fusion transcripts and extremely low frequency of BRAF‐V600E mutation as observed in our study was similar to previous reports 2, 9, 20, 41, 51. BRAF gain/amplification is a frequently seen genetic alteration in majority of pediatric PAs ranging from 50% to 80% 11, 22, 25, 34. Our study also highlights that, like KIAA1549‐BRAF fusion, BRAF gain is also relatively uncommon in adults. Since we used BRAF specific primers for BRAF‐gain qRT‐PCR assay, the findings are true representation of more sensitive and specific BRAF copy number gain/amplification. In a study by Laviv et al, qRT‐PCR assay based BRAF gain was observed in 50% (15/30) of pediatric PAs and, when compared with our data, it was found to be significantly lower (3.4%) in adult PAs (P = 0.0) 27.

Among FGFR related genetic alterations, 12% of our cases harbored this genetic aberration. Although, Hasselblatt et al, evaluated FGFR1 mutation in adults but exact frequency was not clear. However, the authors reported FGFR1 mutations in 10/97 (10%) patients which include patients with median age of 16 years (range: 1–74 years) 20. A very recent study also reported FGFR1 mutation in 6.6% of PAs 2. This study also included 5 adult PAs but none showed presence of FGFR1 mutation. On comparison with our adult PA data, a low frequency of FGFR1 mutation (2%–5%) was reported in pediatric PAs 20, 52. Interestingly, FGFR1 mutation T658P was found in adults only and no pediatric case is reported with this alteration in any study; however this may also be a coincidental finding. Next, we examined FGFR1‐TKD duplication for the first time in adult PAs and found this to be infrequent (5%), nonetheless higher than pediatric PAs; reported in 2% of cases in two different NGS studies (2/88 and 2/90, cases respectively) 20, 52. Overall, both FGFR related alteration (FGFR1 mutation and FGFR‐TKD) were significantly higher in adults (10/59; 17%) as compared with 6/88 (7%) and 4/90 (4%) pediatric PAs, identified in two previous studies (P = 0.04 and P = 0.01, respectively) 20, 52. FGFR related genetic alterations were significantly more enriched in adult tumors of supratentorial region. However, Hasseblatt et al, did not find this association in their study, probably caused by relatively small number of adult cases examined 3. Jones et al, also reported that FGFR1 mutant tumors in pediatric PAs were mostly extra‐cerebellar and midline in location 20, thus complementing our observations. Overall, FGFR related alterations were more confined to adult PAs, and supratentorial locations. Similarly BRAF mutation identified in our study was located in supratentorial tumor, similar to established observations of BRAF‐V600E mutation in PAs and its association with extra‐cerebellar location 3, 20.

Interestingly, we observed an inverse trend of KIAA1549‐BRAF fusion and FGFR1 mutation with age. Occurrence of KIAA1549‐BRAF fusion decreased with age and FGFR1 mutation increased with age. Although Hasselblatt et al, reported that FGFR1 mutation was found more in elderly patients but it was not statistically significant 3. However, we established this fact that FGFR1 mutation is associated with higher age (Mean age: 37 years) as compared with patients with KIAA1549‐BRAF fusion (Mean age: 25 years) in adult PAs. FGFR‐TKD duplication was also observed in patients with mean age of 27 years, thus in slightly elder patients as compared with patients with KIAA1549‐BRAF fusion (Mean age: 25 years).

In this series, we found three cases with co‐existent BRAF and FGFR genetic alterations. On contrary, Hasselblatt et al, have not found any duplicate alteration of MAPK in their series of PAs (Age range 1–74 years) 3. Rarely, duplicate alterations of MAPK pathway have been identified. Although there exist reports of such observation, as one patient with coincident mutations of FGFR1, NF1 and KRAS 20 and one case with BRAFV600E and KIAA1549:BRAF fusion 37 had been identified. Interestingly, both of these cases were aggressive tumor, one oligoastrocytoma and other optic nerve PA. Notably, in our study, the cases with cofounding genetic alterations were in higher age group. Thus acquisition of mutation with increasing age could have led to multi‐hit MAPK pathway. Moreover, such genetic heterogeneity could be inherent to adult PAs as no previous study has performed comprehensive genetic study on adult PAs.

MAPK/ERK pathway activation was seen in all of our cases, with and without BRAF and FGFR related genetic alterations, a consistent observation also seen in other studies on PAs 20, 22, 23, 34, 40. The MAPK/ERK pathway activation was also consistent irrespective of cases with single or multiple genetic alterations, age or tumor location. Activation of mTOR pathway has been reported in large series of pediatric PA and in a small subset of adults 13, 40. Recently Zhang et al, also showed role of mTOR pathway in pediatric low grade gliomas characterized by KIAA1549‐BRAF fusion and FGFR1 duplication 52. Thus we analyzed our cases for mTOR activation as our adult PAs were enriched with FGFR related genetic alteration. Overall, we found pS6 immunoreactivity (+1 to +3) of any extent in 87% of cases. Interestingly, Cabezas et al, also reported similar findings (81%) in PAs 13. Further, we found a significantly lower pS6 (moderate to strong) immunostaining in patients harboring BRAF and/or FGFR related alteration (25%) compared with wild types (59%) (P = 0.04). This finding is in agreement with earlier report in pediatric PAs as well 13. However, this association may be false positive as most of our cases showed mTOR activation of any degree (+1 to +3) otherwise certain other uncharacterized genetic alteration may have contributed to activation of mTOR signaling. A more likely phenomenon could be recently reported FGF2 overexpression in PAs which was not restricted to only FGFR1‐mutant or ‐wild‐type tumors 20. The other likely reason could be another recent observation of strong immunohistochemical FGFR1 expression in 51/69 (74%) of PAs despite of rare occurrence of FGFR1 mutation and FGFR1 low copy number gain 2. These findings are suggestive of ligand‐mediated pathway activation leading to probably MAPK and mTOR activation and resulting in tumorigenesis.

One drawback of our study is that we have not been able to directly establish the prognostic significance of genetic alterations as we had follow up of insufficient number of patients with any genomic alteration. However, Hasselblatt et al, have not found any prognostic significance of BRAF fusion, BRAF V600E or FGFR1 mutation in their study, encompassing both pediatric and adult PAs 3. Theeler et al, were also not able to show any difference in prognosis in adult patients with or without KIAA1549‐BRAF fusion 18. Moreover, several contrary reports of association of clinical outcome of patients with and without KIAA1549‐BRAF fusion are present in pediatric PAs 10, 11, 12, 28, 48. Nevertheless, recently, Becker et al, reported prognostic significance of KIAA1549‐BRAF fusion and FGFR1 mutations in PAs 2. They found a significant association of genetic alteration in FGFR with shorter overall survival (OS) and event‐free survival (EFS) whereas better outcome associated with KIAA1549‐BRAF fusion in pediatric PAs. Therefore, because of the fact that FGFR alterations are associated with poor clinical outcome in terms of OS and EFS, presence of frequent FGFR related genetic alteration in our adult PA patient cohort mechanistically link the poor survival outcome in adults.

Importantly, patients with cerebellar PAs typically show high frequency of KIAA1549‐BRAF fusion and greater OS than other locations. Translating these findings to adults, a probable reason of aggressiveness and high mortality of adult PAs could be low frequency of KIAA1549‐BRAF fusion and their confinement mostly to non‐cerebellar tumor locations. The tumors located in these sites harbor FGFR1 mutations, FGFR‐TKD and BRAFV600E, reported to be associated with poor survival outcome. Zhang et al, identified FGFR‐TKD duplication in 24% of diffuse WHO grade II gliomas, also seen in 5% of our adult PAs 52. Importantly, FGFR1‐TKD duplication has been shown to rapidly generate high grade astrocytic tumor. Similarly, FGFR1 amplification has been also reported in breast and lung cancers 46, 50. Reports of FGFR1 mutations and FGFR1‐TACC1 or FGFR3‐TACC3 gene fusion also exist in adult glioblastoma 4, 36, 43 and, this partly explains aggressive tumor behavior of APAs as they are enriched with FGFR related genetic alterations. Additionally, there were 3 patients (5%) in our cohort that showed multiple genetic alterations, a very rare incidence reported till now in PAs and has been more frequently observed in aggressive tumors 21, 42, 52.

A recent landmark multi‐center study from TCGA on very large sample‐set of over 1000 diffuse gliomas profiled using multiple high throughput platforms robustly described clinically relevant, molecularly discrete groups of glioma 5. Remarkably, analysis of IDH‐wild‐type gliomas separated into three DNA methylation clusters, two of which resemble the previously defined molecular subgroups, particularly classic‐like and mesenchymal‐like 49. Interestingly, a third group also emerged with similar genomic and epigenetic profile to pilocytic astrocytoma (WHO grade I) and clinically exhibiting an excellent prognosis. This group encompassed a larger number of LGG when compared with other two IDH‐wild‐type molecular subgroups and occurred in younger patients (mean 37.6 vs. 50.8 years). Subsequently, Ceccarelli et al 5 found that the frequency of mutations, fusions and amplifications in eight PA‐associated genes (BRAF, NF1, NTRK1, NTRK2, FGFR1 and FGFR2) was 52% (13/25) in PA‐like LGG. The PA‐like group was characterized by relatively low frequency of typical GBM alterations in genes such as EGFR, CDKN2A/B, and PTEN and displayed euploid DNA copy number profiles. The authors independently re‐reviewed these cases and confirmed the presence of the histologic features of diffuse glioma (grade II or grade III) in 23 of the 26 cases in the cluster. The remaining three cases were re‐named as PA (grade I). Overall, the analysis of the IDH‐wild‐type group of adult glioma revealed the existence of a novel subgroup sharing genetic and DNA methylation features with PA. We have also examined 7 IDH wild‐type adult DAs for FGFR related genetic alterations and about 43% cases showed these aberrations. Further, the histopathological and genetic analysis (BRAF‐V600E mutation: commonly found in GG/PXA/DNET and associated with PAs with a more diffuse growth pattern, and MYB‐QKI fusion: characteristic genetic feature of AGs) was carried out to rule out any misdiagnosis of these cases as PXA, GG, DNET, AG or PAs. All 7 cases showed typical diffuse astrocytoma morphology and none of them showed BRAF mutation or MYB‐QKI fusion. These observations to a certain extent also ruled out these cases to be molecularly defined GG, PXA, DNET or PAs with a more diffuse growth pattern. Pathogenic FGFR1 mutations and FGFR‐TKD have been identified in majority of pediatric DNETs or diffuse oligodendroglial tumors and rarely in GGs 35. In our study, 3/7 (43%) adult IDH‐wt DAs were identified with FGFR alterations and similarly Zhang et al, 52 and Qaddoumi et al, 35 also reported FGFR alterations in 25% and 12% of pediatric DAs, respectively, and all the cases were IDH‐wt. Taken together, these findings emphasize the need for integration of molecular signatures into clinical classification for this subgroup of patients.

Therefore we can assume that despite PA histology, there exist a genetic and epigenetic similarity of a subgroup of adult PAs with a subgroup of IDH‐wild‐type adult diffuse grade II and III astrocytomas. Thus a more aggressive clinical course pertaining to genetic manifestations are more likely observed in subgroup of adult PAs. Now, upgraded WHO 2016 classification has included genetic alteration based classification of CNS tumors as it gives better overview of risk stratification, prognostic and therapeutic advantage 29, 30, 39. Recently, IDH mutant diffuse and anaplastic astrocytomas have been shown to have similar age at presentation and little difference in survival 38, 39. Similarly an identical approach could be adapted for astrocytoma grading in adult PAs and DAs and it may suggest a possible re‐stratification of adult PAs with aggressive behavior and increased morbidity as they share common genetic alteration with a subgroup of IDH‐wild‐type adult DAs. However, more studies based on genetic and epigenetic characterization of adult IDH‐wild type DAs and adult PAs are required to identify their precise genetic and epigenetic similarity for possible integrative classification.

Overall, we genetically characterized more than one third of adult PAs harboring BRAF and FGFR genetic alterations and partly resolved the alternative MAPK pathway alterations in these tumors. Adult PAs are constituted more of non‐cerebellar tumor, and these tumor locations have been also identified with PTPN11 mutation and NTRK2 fusions in pediatric PAs 20. Additionally, five different BRAF partnering fusions, KRAS and PIK3CA mutation were also reported in pediatric and/or adult PAs 18, 20, 52. Thus analysis of these genetic alterations could have helped us to genetically characterize more of our cases and identify the causal events of MAPK‐ERK and mTOR activation. FGFR related genetic alterations are enriched in adult PAs and we analyzed two such most important genomic alterations. Nevertheless, pediatric PA has also been identified with FGFR1‐TACC1 fusion in one case 52, thus it is more likely that adult PAs which harbor frequent FGFR related alterations may show a significant number of cases with FGFR partnering fusions namely FGFR3‐TACC1/TACC3. Hence, the molecular heterogeneity in adults observed in present study and reported by other workers offers hope of developing molecularly targeted therapeutic measures.

In conclusion, the present study represents the largest reported adult PA patient cohort molecularly profiled to date and provides a unique opportunity to estimate subgroup‐specific molecular alterations within the seemingly more aggressive adult PAs, as compared with pediatric. We were able to find at least one genetic alteration in more than one third of the cases. Notably, in addition to the only known genetic alteration, namely KIAA1549‐BRAF fusion reported till date in adults, we identified the frequency of FGFR mutation; FGFR‐TKD duplication and BRAF gain in adult PAs. Therefore, the combined activation of constitutive BRAF and FGFR related MAPK/ERK and PI3K/mTOR signaling appears to be an important oncogenic event in sporadic adult PAs and this may be the underlying event of aggressive behavior of these tumors. Co‐existent BRAF and FGFR genetic alteration, in addition to significant inverse correlation of KIAA1549‐BRAF fusion with increasing age and association of FGFR1‐mutation with older age were our other notable findings. On the whole, this study provides a rationale for potential therapeutic advantage of targeting candidate genetic signature of individual adult PA patient. Further studies should be carried out to expand on these findings to define the genetic and biological features of adult PAs. Our present findings and recent reports also suggest that adult PAs are possibly a variant of, and genetically similar to, IDH‐wild‐type adult DAs. Besides, the role of BRAF and FGFR related genetic alteration and MAPK‐ERK/mTOR pathway activation in pathogenesis and clinical course of adult PAs, relevance of these molecular aberrations to prognosis need to be further established.

Supporting information

Additional Supporting Information may be found in the online version of this article at the publisher's web‐site:

Table S1. Primer sequences used for real time RT‐PCR, gene amplification/copy number and sequencing analysis

Table S2. List of primary antibodies with dilution and antigen retrieval methods used for immunohistochemistry for analysis of MAPK/ERK and mTOR pathway activation

Table S3. Clinical and genetic features of seven IDH1 wild‐type adult diffuse astrocytomas analysed for FGFR related genetic alterations

Click here for additional data file. (64.5KB, doc)

Acknowledgment

We are thankful to Neuro Sciences Centre and Department of Pathology, All India Institute of Medical Sciences, New Delhi for funding of this work. This study was also supported by Department of Science and Technology ‐ J C Bose Fellowship, to CS and SERB‐National Post Doctoral Fellowship to PP. The authors are also thankful to Technical staff of Neuropathology Laboratory for their help in immunohistochemistry.

The authors declare that there is no conflict of interest

References

  • 1. Bar EE, Lin A, Tihan T, Burger PC, Eberhart CG (2008) Frequent gains at chromosome 7q34 involving BRAF in pilocytic astrocytoma. J Neuropathol Exp Neurol 67:878–887. [DOI] [PubMed] [Google Scholar]
  • 2. Becker AP, Scapulatempo‐Neto C, Carloni AC, Paulino A, Sheren J, Aisner DL (2015) KIAA1549: BRAF gene fusion and FGFR1 hotspot mutations are prognostic factors in pilocytic astrocytomas. J Neuropathol Exp Neurol 74:743–754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Brokinkel B, Peetz‐Dienhart S, Ligges S, Brentrup A, Stummer W, Paulus W (2015) A comparative analysis of MAPK pathway hallmark alterations in pilocytic astrocytomas: age‐related and mutually exclusive. [corrected]. Neuropathol Appl Neurobiol 41:258–261. [DOI] [PubMed] [Google Scholar]
  • 4.Cancer Genome Atlas Research Network (2008) Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 455:1061–1068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Ceccarelli M, Barthel FP, Malta TM, Sabedot TS, Salama SR, Murray BA (2016) Molecular profiling reveals biologically discrete subsets and pathways of progression in diffuse glioma. Cell 164:550–563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Collins VP, Jones DT, Giannini C (2015) Pilocytic astrocytoma: pathology, molecular mechanisms and markers. Acta Neuropathol 129:775–788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Dutt A, Ramos AH, Hammerman PS, Mermel C, Cho J, Sharifnia T (2011) Inhibitor‐sensitive FGFR1 amplification in human non‐small cell lung cancer. PLoS One 6:e20351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Ellis JA, Waziri A, Balmaceda C, Canoll P, Bruce JN, Sisti MB (2009) Rapid recurrence and malignant transformation of pilocytic astrocytoma in adult patients. J Neurooncol 95:377–382. [DOI] [PubMed] [Google Scholar]
  • 9. Hasselblatt M, Riesmeier B, Lechtape B, Brentrup A, Stummer W, Albert FK (2011) BRAF‐KIAA1549 fusion transcripts are less frequent in pilocytic astrocytomas diagnosed in adults. Neuropathol Appl Neurobiol 37:803–806. [DOI] [PubMed] [Google Scholar]
  • 10. Hawkins C, Walker E, Mohamed N, Zhang C, Jacob K, Shirinian M (2011) BRAF‐KIAA1549 fusion predicts better clinical outcome in pediatric low‐grade astrocytoma. Clin Cancer Res 17:4790–4798. [DOI] [PubMed] [Google Scholar]
  • 11. Horbinski C, Hamilton RL, Nikiforov Y, Pollack IF (2010) Association of molecular alterations, including BRAF, with biology and outcome in pilocytic astrocytomas. Acta Neuropathol 119:641–649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Horbinski C, Nikiforova MN, Hagenkord JM, Hamilton RL, Pollack IF (2012) Interplay among BRAF, p16, p53, and MIB1 in pediatric low‐grade gliomas. Neuro Oncol 14:777–789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Hutt‐Cabezas M, Karajannis MA, Zagzag D et al (2013) Activation of mTORC1/mTORC2 signaling in pediatric low‐grade glioma and pilocytic astrocytoma reveals mTOR as a therapeutic target. Neuro Oncol 15:1604–1614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Ishkanian A, Laperriere NJ, Xu W et al (2011) Upfront observation versus radiation for adult pilocytic astrocytoma. Cancer 117:4070–4079. [DOI] [PubMed] [Google Scholar]
  • 15. Jacob K, Albrecht S, Sollier C et al (2009) Duplication of 7q34 is specific to juvenile pilocytic astrocytomas and a hallmark of cerebellar and optic pathway tumours. Br J Cancer 101:722–733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Janzarik WG, Kratz CP, Loges NT et al (2007) Further evidence for a somatic KRAS mutation in a pilocytic astrocytoma. Neuropediatrics 38:61–63. [DOI] [PubMed] [Google Scholar]
  • 17. Jeuken JW, Wesseling P (2010) MAPK pathway activation through BRAF gene fusion in pilocytic astrocytomas; a novel oncogenic fusion gene with diagnostic, prognostic, and therapeutic potential. J Pathol 222:324–328. [DOI] [PubMed] [Google Scholar]
  • 18. Johnson DR, Brown PD, Galanis E, Hammack JE (2012) Pilocytic astrocytoma survival in adults: analysis of the surveillance, epidemiology, and end results program of the national cancer institute. J Neurooncol 108:187–193. [DOI] [PubMed] [Google Scholar]
  • 19. Jones DT, Gronych J, Lichter P, Witt O, Pfister SM (2012) MAPK pathway activation in pilocytic astrocytoma. Cell Mol Life Sci 69:1799–1811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Jones DT, Hutter B, Jager N, Korshunov A, Kool M, Warnatz HJ (2013) Recurrent somatic alterations of FGFR1 and NTRK2 in pilocytic astrocytoma. Nat Genet 45:927–932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Jones DT, Jager N, Kool M, Zichner T, Hutter B, Sultan M (2012) Dissecting the genomic complexity underlying medulloblastoma. Nature 488:100–105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Jones DT, Kocialkowski S, Liu L et al (2008) Tandem duplication producing a novel oncogenic BRAF fusion gene defines the majority of pilocytic astrocytomas. Cancer Res 68:8673–8677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Jones DT, Kocialkowski S, Liu L, Pearson DM, Ichimura K, Collins VP (2009) Oncogenic RAF1 rearrangement and a novel BRAF mutation as alternatives to KIAA1549:BRAF fusion in activating the MAPK pathway in pilocytic astrocytoma. Oncogene 28:2119–2123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Kim YH, Nonoguchi N, Paulus W, Brokinkel B, Keyvani K, Sure U (2012) Frequent BRAF gain in low‐grade diffuse gliomas with 1p/19q loss. Brain Pathol 22:834–840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Korshunov A, Meyer J, Capper D, Christians A, Remke M, Witt H (2009) Combined molecular analysis of BRAF and IDH1 distinguishes pilocytic astrocytoma from diffuse astrocytoma. Acta Neuropathol 118:401–405. [DOI] [PubMed] [Google Scholar]
  • 26. Kumar A, Pathak P, Purkait S, Faruq M, Jha P, Mallick S (2015) Oncogenic KIAA1549‐BRAF fusion with activation of the MAPK/ERK pathway in pediatric oligodendrogliomas. Cancer Genet 208:91–95. [DOI] [PubMed] [Google Scholar]
  • 27. Laviv Y, Toledano H, Michowiz S, Dratviman‐Storobinsky O, Turm Y, Fichman‐Horn S (2012) BRAF, GNAQ, and GNA11 mutations and copy number in pediatric low‐grade glioma. FEBS Open Biol 2:129–134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Lin A, Rodriguez FJ, Karajannis MA, Williams SC, Legault G, Zagzag D (2012) BRAF alterations in primary glial and glioneuronal neoplasms of the central nervous system with identification of 2 novel KIAA1549:BRAF fusion variants. J Neuropathol Exp Neurol 71:66–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Louis DN, Perry A, Burger P, Ellison DW, Reifenberger G, von Deimling A (2014) International society of neuropathology–Haarlem consensus guidelines for nervous system tumor classification and grading. Brain Pathol 24:429–435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella‐Branger D, Cavenee WK (2016) The 2016 world health organization classification of tumors of the central nervous system: a summary. Acta Neuropathol 131:803–820. [DOI] [PubMed] [Google Scholar]
  • 31. McBride SM, Perez DA, Polley MY, Vandenberg SR, Smith JS, Zheng S (2010) Activation of PI3K/mTOR pathway occurs in most adult low‐grade gliomas and predicts patient survival. J Neurooncol 97:33–40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Myung JK, Cho H, Park CK, Kim SK, Lee SH, Park SH (2012) Analysis of the BRAF(V600E) mutation in central nervous system tumors. Transl Oncol 5:430–436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Newton HB (2008) Do pilocytic astrocytomas have a benign course in adult patients?. Nat Clin Pract Neurol 4:296–297. [DOI] [PubMed] [Google Scholar]
  • 34. Pfister S, Janzarik WG, Remke M, Ernst A, Werft W, Becker N (2008) BRAF gene duplication constitutes a mechanism of MAPK pathway activation in low‐grade astrocytomas. J Clin Invest 118:1739–1749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Qaddoumi I, Orisme W, Wen J, Santiago T, Gupta K, Dalton JD (2016) Genetic alterations in uncommon low‐grade neuroepithelial tumors: BRAF, FGFR1, and MYB mutations occur at high frequency and align with morphology. Acta Neuropathol 131:833–845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Rand V, Huang J, Stockwell T, Ferriera S, Buzko O, Levy S (2005) Sequence survey of receptor tyrosine kinases reveals mutations in glioblastomas. Proc Natl Acad Sci U S A 102:14344–14349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Reis GF, Bloomer MM, Perry A, Phillips JJ, Grenert JP, Karnezis AN (2013) Pilocytic astrocytomas of the optic nerve and their relation to pilocytic astrocytomas elsewhere in the central nervous system. Mod Pathol 26:1279–1287. [DOI] [PubMed] [Google Scholar]
  • 38. Reuss DE, Mamatjan Y, Schrimpf D, Capper D, Hovestadt V, Kratz A (2015) IDH mutant diffuse and anaplastic astrocytomas have similar age at presentation and little difference in survival: a grading problem for WHO. Acta Neuropathol 129:867–873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Reuss DE, Sahm F, Schrimpf D, Wiestler B, Capper D, Koelsche C (2015) ATRX and IDH1‐R132H immunohistochemistry with subsequent copy number analysis and IDH sequencing as a basis for an “integrated” diagnostic approach for adult astrocytoma, oligodendroglioma and glioblastoma. Acta Neuropathol 129:133–146. [DOI] [PubMed] [Google Scholar]
  • 40. Rodriguez EF, Scheithauer BW, Giannini C, Rynearson A, Cen L, Hoesley B (2011) PI3K/AKT pathway alterations are associated with clinically aggressive and histologically anaplastic subsets of pilocytic astrocytoma. Acta Neuropathol 121:407–420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Schindler G, Capper D, Meyer J, Janzarik W, Omran H, Herold‐Mende C (2011) 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 121:397–405. [DOI] [PubMed] [Google Scholar]
  • 42. Schwartzentruber J, Korshunov A, Liu XY, Jones DT, Pfaff E, Jacob K (2012) Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma. Nature 482:226–231. [DOI] [PubMed] [Google Scholar]
  • 43. Singh D, Chan JM, Zoppoli P, Niola F, Sullivan R, Castano A (2012) Transforming fusions of FGFR and TACC genes in human glioblastoma. Science 337:1231–1235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Stuer C, Vilz B, Majores M, Becker A, Schramm J, Simon M (2007) Frequent recurrence and progression in pilocytic astrocytoma in adults. Cancer 110:2799–2808. [DOI] [PubMed] [Google Scholar]
  • 45. Theeler BJ, Ellezam B, Sadighi ZS, Mehta V, Tran MD, Adesina AM (2014) Adult pilocytic astrocytomas: clinical features and molecular analysis. Neuro Oncol 16:841–847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46. Theillet C, Adelaide J, Louason G, Bonnet‐Dorion F, Jacquemier J, Adnane J (1993) FGFRI and PLAT genes and DNA amplification at 8p12 in breast and ovarian cancers. Genes Chromosomes Cancer 7:219–226. [DOI] [PubMed] [Google Scholar]
  • 47. Tian Y, Rich BE, Vena N, Craig JM, Macconaill LE, Rajaram V (2011) Detection of KIAA1549‐BRAF fusion transcripts in formalin‐fixed paraffin‐embedded pediatric low‐grade gliomas. J Mol Diagn 13:669–677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48. Tihan T, Ersen A, Qaddoumi I, Sughayer MA, Tolunay S, Al‐Hussaini M (2012) Pathologic characteristics of pediatric intracranial pilocytic astrocytomas and their impact on outcome in 3 countries: a multi‐institutional study. Am J Surg Pathol 36:43–55. [DOI] [PubMed] [Google Scholar]
  • 49. Verhaak RG, Hoadley KA, Purdom E, Wang V, Qi Y, Wilkerson MD (2010) Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 17:98–110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50. Weiss J, Sos ML, Seidel D, Peifer M, Zander T, Heuckmann JM (2010) Frequent and focal FGFR1 amplification associates with therapeutically tractable FGFR1 dependency in squamous cell lung cancer. Sci Transl Med 2:62ra93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51. Yeo YH, Byrne NP, Counelis GJ, Perry A (2013) Adult with cerebellar anaplastic pilocytic astrocytoma associated with BRAF V600E mutation and p16 loss. Clin Neuropathol 32:159–164. [DOI] [PubMed] [Google Scholar]
  • 52. Zhang J, Wu G, Miller CP, Tatevossian RG, Dalton JD, Tang B (2013) Whole‐genome sequencing identifies genetic alterations in pediatric low‐grade gliomas. Nat Genet 45:602–612. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional Supporting Information may be found in the online version of this article at the publisher's web‐site:

Table S1. Primer sequences used for real time RT‐PCR, gene amplification/copy number and sequencing analysis

Table S2. List of primary antibodies with dilution and antigen retrieval methods used for immunohistochemistry for analysis of MAPK/ERK and mTOR pathway activation

Table S3. Clinical and genetic features of seven IDH1 wild‐type adult diffuse astrocytomas analysed for FGFR related genetic alterations

Click here for additional data file. (64.5KB, doc)

Articles from Brain Pathology are provided here courtesy of Wiley

RESOURCES