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Adult recurrent pilocytic astrocytoma: Clinical, histopathological and molecular study
Neurochirurgie 61 (2015) 392–397
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Clinical case
Adult recurrent pilocytic astrocytoma: Clinical, histopathological and molecular study Astrocytome pilocytique récidivant de l’adulte : étude clinique, histopathologique et moléculaire S. Trabelsi a,∗ , N. Mama b , M. Ladib c , S. Popov d , A. Burford d , M. Mokni e , K. Tlili b , H. Krifa c , M. Varella-Garcia f , C. Jones d , M. Tahar Yacoubi e , A. Saad a , D. H’mida Ben Brahim a a
Department of Cytogenetics, Molecular genetics and Reproductive Biology, Farhat Hached University Hospital, Street Ibn Eljazzar, 4000 Sousse, Tunisia Department of Imagery, Sahloul University Hospital, Sousse, Tunisia Department of Neurosurgery, Sahloul University Hospital, Sousse, Tunisia d Divisions of Molecular Pathology and Cancer Therapeutics, the Institute of Cancer Research, Sutton, United Kingdom e Department of Cytopathology, Farhat Hached University Hospital, Sousse, Tunisia f University of Colorado Denver School of Medicine, Division of Medical Oncology, Colorado, USA b c
a r t i c l e
i n f o
Article history: Received 14 March 2015 Received in revised form 9 June 2015 Accepted 29 July 2015 Available online 17 November 2015 Keywords: Pilocytic astrocytoma Adult Recurrence KIAA1549:BRAF fusion
a b s t r a c t Background. – PA is a grade I glial tumor that mostly occurs in children. However, although apparently similar to paediatric PA, adult PA presents a different clinical follow-up that could arise from specific molecular alterations. A variety of genetic alterations have been identified as diagnostic or prognostic glioma molecular markers. Material and methods. – We describe a right infratentorial tumor that occurred in a 58-year-old man. Neuroimaging and neuropathological examination suggested PA as an initial diagnosis. The tumor was completely resected. Unexpectedly, two years later, a rapidly growing tumor on the operative site was observed with a second location in the pineal region. Immunohistochemical reactions (IHC), Multiplex ligation probe amplification (MLPA) and fluorescence in situ hybridization (FISH) was performed in both primary and relapse tumor. Results. – Neuroimaging and neuropathological examinations suggested an unusual diagnosis for adult patients: a recurrent PA. Both MLPA and FISH analysis contribute to diagnostic confirmation by KIAA1549: BRAF fusion detection. Additional genetic results revealed interesting findings that justified the tumor aggressivity. Conclusion. – Molecular analysis of adult PA cases should be routinely combined with histopathological and neuroimaging examination to further refine prognostic diagnoses. © 2015 Elsevier Masson SAS. All rights reserved.
r é s u m é Mots clés : Astrocytome pilocytique Adulte Récidive Gène de fusion KIAA1549:BRAF
Introduction. – L’astrocytome pilocytique (AP) est une tumeur gliale de grade 1, survenant le plus souvent à un âge pédiatrique. Chez les adultes, en dépit de la similarité histologique, l’AP est de moins bon pronostic. Cette évolution défavorable est sous-tendue par un profil génétique spécifique. En effet, un grand nombre d’altérations moléculaires de valeurs diagnostiques, pronostiques et théranostiques seraient à l’origine de cette différence pronostique. Matériels et méthodes. – Dans cette étude, nous rapportons un cas de tumeur gliale infratentorielle chez un patient adulte de 58 ans. Les données d’imagerie et de neuropathologie suggèrent l’AP comme premier diagnostic. Deux ans après une résection complète, nous avons observé une récidive bifocale au siège initial de la tumeur avec une évolution rapide, accompagnée d’une deuxième localisation
∗ Corresponding author. E-mail address: [email protected] (S. Trabelsi). http://dx.doi.org/10.1016/j.neuchi.2015.07.002 0028-3770/© 2015 Elsevier Masson SAS. All rights reserved.
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dans la région pinéale. Aussi bien la tumeur initiale que la récidive ont été explorées par techniques d’immunohistochimie (IHC), d’amplification et ligation multiplexes de sondes (MLPA) et d’hybridation in situ fluorescente (FISH). Résultats. – Les données d’imagerie et neuropathologie suggèrent un diagnostic rare d’AP récidivant de l’adulte. Les analyses moléculaires par MLPA et FISH apportent la confirmation diagnostique en révélant la présence du gène de fusion KIAA1549:BRAF. D’autres altérations génétiques pouvant justifier l’agressivité de la tumeur ont également été détectées. Conclusion. – L’analyse moléculaire en matière d’AP de l’adulte s’avère complémentaire de l’histopathologie et de l’imagerie. En plus de sa valeur diagnostique connue, elle serait d’un grand apport pronostique. Elle devrait ainsi intégrer le protocole routinier du diagnostic des PA adultes. © 2015 Elsevier Masson SAS. Tous droits réservés.
1. Introduction Pilocytic astrocytoma (PA) is a circumscribed glioma that primarily occurs in the cerebellum in children [1]. Clinical presentation depends on tumor location. Headache, neck pain, vomiting and visual abnormalities are usually reported with the cerebellum location [2]. Histopathological description includes biphasic pattern, elongated and rounded cells, Rosenthal fibers, and eosinophilic granular bodies [2]. PA is classified as grade I by the World Health Organization (WHO) [3] and usually reported with a good prognosis. In most cases, only surgical resection is considered to be curative [4]. Very few recurrent adult PA cases have been reported with malignant progression [4–6]. In such unusual presentations, features like microvascular proliferation, necrosis and nuclear atypia may occur. Differential diagnoses as high-grade gliomas could then be suspected with a considerably worse prognosis [7]. Furthermore, unlike paediatric PA, the adult cases seem to present a poor prognosis [8]. The correct diagnosis has great importance in the treatment choice and prognosis of patients. 2. Material and methods 2.1. Patient A 58-year-old man with no previous family medical history of cancer was referred for severe headache, neck stiffness and difficulty in walking. The physical examination showed signs of cerebellar dysfunction. MRI revealed a well-circumscribed lesion extended into the 4th ventricle (Fig. 1a, b). The patient underwent a complete tumor resection without evidence of any residual or recurrent mass at 6-months follow-up by MRI. Two years later, the patient was readmitted with increased intracranial pressure, a cerebellar syndrome, vertical/horizontal strabismus and VI and IX right nerve palsy. There was no previous family medical history or clinical signs of neurofibromatosis [9]. The MRI showed a bifocal tumor located in the cerebellar region at the operative site with a second location in the pineal region (Fig. 1c, d). The masses presented with cystic and intensely enhanced solid components. The patient was readmitted for a second tumor resection. The pineal component was unresectable and the tumor was partially removed in the cerebellar location alone. The patient died shortly after the operation. 2.2. Histological Immunohistochemical study Routine sections (4 mm) of both primary and relapse tumor were stained with haematoxylin and eosin. Immunohistochemical analysis was performed on 4 mm of formalin-fixed, paraffinembedded sections using a panel of polyclonal antibodies (Table 1). Slides were dewaxed in xylene and rehydrated through a descending ethanol series. Antigen retrieval was performed at 98 ◦ C with
DAKO antigen retrieval solution during 20 min (Table 1). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol. The detection system used was Novolink Polymer (Leica Microsystems, Newcastle Ltd.) with diaminobenzidine as chromogen. Slides were counterstained with Mayer’s haematoxylin. HE and IHC results were separately reviewed by two pathologists. 2.3. DNA extraction Primary tumor Genomic DNA was purified from FFPE sections using an Qiagen kit (Qiamp DNA FFPE Tissue Kit). Recurrent tumor genomic DNA was purified from fresh tumoral tissue according to phenol–chloroform protocol. 2.4. Multiplex ligation probe amplification (MLPA) Both primary and recurrent tumoral DNAs were analysed using a multiplex ligation probe amplification (MLPA) reaction through SALSA MLPA Kits P105, P370 and P088 (MRC Holland, Amsterdam, The Netherlands) according to manufacturers protocol. DNA from healthy tissue was adopted as MLPA control sample. MLPA kits enabled us to analyse multiple chromosomal aberrations located at 1p36, 7q3, 9p21, 10q23, 11p11, 17p13 and 19q13 (Table 2). Additional point mutations may be detected using the P370 MLPA kit (IDH1 R132H, IDH1 R1321C, IDH2 R172K, IDH2 R172M and BRAF V600E). PCR products were analysed on ABI 310 (Applied Biosystems, Foster City, CA) using as an internal size standard the ROX-500 “Genescan® ” (ABI 401734). Data analysis was performed with “GeneMarker® ” software V1.91 (SoftGenetics). Intra normalization for sample data was initially performed on control probes, and then on healthy control samples. Normal ratio limits were set at 0.75 and 1.3 [10]. For more accuracy, we considered that displaying 2 or more probes, adjacent to each other on a chromosomal region, exhibiting the ratio below 0.45 or above 2 were accepted respectively as deleted or gained. 2.5. Fluorescence in situ hybridization (FISH) FISH assay was performed in both the primary tumor and relapse to look for presence of the KIAA1549:BRAF fusion gene. A dual-colour FISH probe set was designed to identify the chromosomal duplication that generates the KIAA1549:BRAF fusion, using human genomic sequences from the RP4-726N20 clone that encompass BRAF gene labelled in Spectrum Red and the using human genomic sequences from the RP11-355D18 clone that encompass KIAA1549 labeled in Spectrum Green. Probe preparation and FISH assays were performed as reported elsewhere [11].
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Fig. 1. a, b: first tumor: well-circumscribed lesion extended through the 4th ventricle; c, d: recurrence: bifocal tumor located on the cerebellar and pineal regions. Double cystic and Intensely enhanced tissular components. a, b : tumeur initiale : lésion bien circonscrite étendue au 4e ventricule ; c, d : récidives : tumeur bifocale située sur la région cérébelleuse et pinéale. Une double composante kystique avec un rehaussement tissulaire intense.
3. Results 3.1. Histolopathology and immunohistochemistry results First tumor macroscopic examination described 2.5 cm white crumbly fragments. Histological examination demonstrated round and spindle cells with eosinophilic cytoplasm, fibrillary areas and rich vascularity. Occasional mitoses and vascular proliferation were also observed (Fig. 2a). Immunohistochemical (IHC) description of the primary tumor demonstrated a positive staining for GFAP (Dako Clone Poly ZCG23, 1/300 dilution) and negative staining for EMA (Dako Clone E29,
1/100 dilution) (Fig. 2). Based on this description, pilocytic astrocytoma was highly suspected (Table 1), with anaplastic astrocytoma as a differential diagnosis. Macroscopic examination of the recurrence showed a 3.5 × 3 × 2 cm white fragment with focal necrosis. Haematoxylin and eosin (H&E) staining showed a biphasic pattern, with heterogeneous cellular density. Tumor cells were pleomorphic with atypia and hyper-chromatin nuclei. A prominent vascularity as well as necrotic area was observed (Fig. 2). The recurrent tumor was positive for GFAP and negative for synaptophysine and neurofilament (NF), with weak P53 (Dako Clone DO7, diluted 1/50) and KI67 (Dako Clone MIB1,
Table 1 List of antibodies and IHC staining results. Liste des anticorps et des résultats du marquage en IHC. Antigen
Antibody and manufacturers
Dilution
Antigen Retrieval
First tumor
Relapse
S100 NSE Synaptophysine NF GFAP EMA P53 KI67 Olig2 IDH R132H
Dako clone Dako Clone BBS/NC/V1H14 Neomarker clone Rb-SP111-RM Dako clone 2f11 Dako Clone Poly ZCG23 Dako clone E29 Dako clone DO7 Dako clone MIB1 Millipore AB9610 Dianova clone H09
1:300 1:50 1:200 1:100 1:300 1:100 1:50 1: 300 1:100 01:20
/ EDTA PH6 EDTA PH6 EDTA PH6 EDTA PH6 EDTA PH6 EDTA PH6 EDTA PH6 EDTA PH9 EDTA PH 6
+ ND ND ND + − 5% 1% + −
+ + − − + − 5% 1% + −
NSE: neuron-specific enolase; NF: neurofilament; GFAP: glial fibrillary antigen protein; EMA: epithelial membrane antigen; /: without antigen retrieval; +: positive staining; −: negative staining; ND: not determined. NSE : neuron-specific enolase ; NF : neurofilament ; GFAP : glial fibrilary antigen protein ; EMA : epithelial membrane antigen ; / : sans démasquage antigénique ; + : marquage positif staining ; − : marquage négatif ; ND : non determiné.
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Fig. 2. First tumor haematoxylin and eosin stain: a: round and spindle eosinophilic cells (× 40); b: cells with fibrillary areas with occasional mitosis (× 200); c: rich vascularity (× 200); d: endothelial proliferation (× 200). Relapse hematoxylin and eosin stain: e: pleomorphic tumor cells with atypia and hyper-chromatic nuclei (× 40); f: biphasic pattern with necrotic areas (× 12.5); g: prominent vascularity (× 40). IHC stain: h: IDH R132H negative (× 40); i: first tumor GFAP positive (× 100); j: Relapse GFAP positive (× 40); k: first tumor olig2 positive (× 40); l: relapse tumor olig2 positive (× 40). Coloration HE de la tumeur initiale : a : des cellules rondes et fusiformes éosinophiles (× 40) ; b : des cellules avec des zones fibrillaires et mitoses occasionnelles (× 200) ; c : une riche vascularisation (× 200) ; d : une prolifération endothéliale (× 200). Coloration HE de la récidive : e : les cellules tumorales pléomorphes avec atypies nucléaires et noyaux hyper-chromatiques (× 40) ; f : un aspect bi-phasique avec des zones nécrotiques (× 12,5) ; g : une vascularisation importante (× 40). Coloration IHC : h : IDH R132H négative (× 40); i : tumeur initiale: GFAP positive (× 100) ; j : récidive : GFAP positive (× 40) ; k : tumeur initiale: Olig2 positif (× 40) ; l : récidive : Olig2 positif (× 40).
diluted 1/300) labelling index respectively at 5 and 1% (Fig. 2, Table 1). 3.2. Genetic results MLPA profiles showed that both primary tumor and relapse were wild type for IDH1 (R132H, R132C) IDH2 (R172K, R172 M) and BRAF (V600E) mutations (Table 2). The IDH1 R132H mutation negative staining was reproduced by IHC (Dianova Clone H09 diluted 1:20) (Fig. 2e). MLPA analysis showed amplification on KIAA1549 as well as BRAF probes that interestingly retained the BRAF kinase domain in both first tumor and relapse. The KIAA1549:BRAF gene fusion was identified by FISH as red and green doublet signals partially overlapped, while the normal copies of the genes were identified as single copies of red and green signals separated by a small gap. For this assay, specimens are classified as positive for the KIAA1459:BRAF fusion when exhibiting positive
signals in ≥15% of tumor cells (Fig. 3). FISH assays succeeded only in the relapse sample and clearly confirmed the amplification and fusion of BRAF and KIAA1549. MLPA analysis revealed additional genetic alterations: CDKN2A/P14 gene promoter loss and EGFR focal amplification. PTEN gene was found to be lost only in first tumor (Table 2).
4. Discussion According to the clinical findings, there was no strong evidence of PA. Thus, differential diagnosis of high -grade astrocytic tumors should also be considered. However, the posterior fossa location of the primary tumor and the cystic components are likely compatible with a pilocytic astrocytoma diagnosis. Histopathological assessment strongly suggested that the primary tumor was pilocytic astrocytoma with a biphasic pattern and eosinophilic granular bodies.
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Fig. 3. FISH analysis for the KIAA1549-BRAF fusion: tissue section shows numerous signals of tandem duplication for BRAF in red (A), KIAA1549 in green (B) and fusion result of these loci in yellow with merged signal (C). Analyse par FISH de la fusion KIAA1549 : BRAF. La section de tissu analysée présente de nombreux signaux deduplication en tandem pour le locus BRAF en rouge (A), KIAA1549 en vert (B) ainsi que le résultat de la fusion des deux loci avec un signal de superposition en jaune (C).
Table 2 Genetic alterations in primary and recurrent tumor detected by MLPA. Les altérations génétiques détectées par MLPA au niveau de la tumeur initiale et la récidive. MLPA Kit references
Region (gene)
First tumor
Relapse
P370
7q34 (KIAA1549 B-RAF) 2q33 (IDH1 p.R132H, IDH1 p.R132C) 9p21 (CDKN2A)a BRAFV600E 9p21 (CDKN2A)a 10q23 (PTEN) 11p11 (EGFR)
Gain Absent
Gain Absent
Loss Absent Loss Loss Gain
Loss Absent Loss NS Gain
P105
NS: normal statues. NS : statut normal. a CDKN2A gene is detected by 2 separate MLPA kit P370 and P105. a Gène CDKN2A est analysé par deux kits MLPA séparés P370 et P105.
Two years later, the patient presented with an unexpected tumor relapse. Histopathological examination noted high cellularity with small atypical cells, and broad necrosis. Despite the patient’s age and the tumor location, the histopathological description was also strongly suggestive of a diagnosis of PA. Moreover, histopathological description suggested the differential diagnosis of ganglioglioma very possible [12]. The absence of IDH mutations in MLPA and IHC investigations provides an additional argument for a diagnosis of pilocytic astrocytoma, with IDH mutation occurring frequently in diffuse astrocytoma but rarely in PA [13–15]. BRAF V600E mutation has been described only in 9% of PA and was also absent in both primary and relapse specimens [16,17]. Our patient’s primary tumor revealed the presence of the KIAA1549:BRAF fusion gene, typical of pilocytic astrocytoma [14,18] in children. This fusion was also present two years later in the recurrent tumor supporting the diagnosis of PA, even with histological atypias and extra cerebellar location [19].
Despite the WHO description of pilocytic astrocytoma as low grade tumor, this case seems to be a clear exception, with a recurrence after total resection, and eventual death due to their disease. Moreover, the simultaneous presence of many arguments are in the opposite of the differential diagnosis of ganglioglioma: absence of IDH mutation, absence of BRAFV600E mutation, negative IHC staining of neuro-markers (NF and Synaptophysin) and KIAA1549:BRAF fusion in both first and relapse tumor. Here, the adult patient presented a PA with an anaplastic pattern in the bifocally recurrent tumor. One hypothesis could be suggested: During tumor resection and due to their motility nature, residual astrocytoma cells were likely competitively selected to escape and migrate to the pineal and cerebellar regions. In addition, previous data suggest that subtotal resection of this tumor subtype carries a high risk of recurrence [20]. Such behaviour is closely associated with gene-expression and molecular profiles [21,22]. This poor clinical outcome was well reflected in the molecular findings. CDKN2A/P14 loss noticed in both tumors is already known to confer an aggressive behaviour to tumoral cells and had been currently associated with a poor outcome [23–25]. These types of molecular alterations in primary PA can switch tumor grading from apparently benign to highly malignant potential tumor. This upgrading would justify an aggressive therapeutic approach that could improve patient’s outcome. Previously adult PA was thought to be a begin tumor, such as the paediatric PA [13,21,26,27]. Recently, a series of adult PA with malignant transformation and recurrence [28], as well as PA with unusual anaplastic pattern [21,29] were reported. In those previous studies the diagnosis of anaplastic adult cases of PA was mainly based on histopathology and imaging [5,10,21,30–32].
5. Conclusion Even if apparently benign, adult PA could present a poor outcome arising from tumor molecular alterations. Combined with pathologic and neuroimaging examination, genetic investigations
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should be required for that particular PA subset to provide a better disease management. Disclosure of interest The authors declare that they have no competing interest. Acknowledgement We would like to thank the patient for agreeing to participate in this study. We also thank the technical help given by the Division of Medical Oncology at the School of Medicine, University of Colorado, USA. We would like also to acknowledge the efforts of GETUC Tunisian group members ,“Groupe d’étude des tumeurs cérébrales”. We also thank glioma team in the division of molecular pathology and cancer therapeutics at The Institute of Cancer Research, Sutton, UK. Ethical standards: the manuscript was approved by the responsible authorities of our laboratory and by the ethics committee of Farhat Hached University Hospital of Sousse, Tunisia. We have no substantial direct or indirect commercial or financial incentive associated with publishing the article. References [1] Shibahara I, Kawaguchi T, Kanamori M, Yonezawa S, Takazawa H, Asano K, et al. Pilocytic astrocytoma with histological malignant features without previous radiation therapy – case report. Neurol Med Chir (Tokyo) 2011;51(2):144–7. [2] 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(2):97–109. [3] Li HM, Hsu SS, Wang JS, Weng MJ, Fu JH, Chen CK, et al. Cerebral pilocytic astrocytoma with spontaneous intracranial hemorrhage in adults. J Chin Med Assoc 2008;71(11):587–93. [4] Ellis JA, Waziri A, Balmaceda C, Canoll P, Bruce JN, Sisti MB. Rapid recurrence and malignant transformation of pilocytic astrocytoma in adult patients. J Neurooncol 2009;95(3):377–82. [5] Stuer C, Vilz B, Majores M, Becker A, Schramm J, Simon M. Frequent recurrence and progression in pilocytic astrocytoma in adults. Cancer 2007;110(12):2799–808. [6] Sioutos PJ, Hamilton AJ, Narotam PK, Weinand ME. Unusual early recurrence of a cerebellar pilocytic astrocytoma following complete surgical resection. Case report and review of the literature. J Neurooncol 1996;30(1):47–54. [7] Azad A, Deb S, Cher L. Primary anaplastic pilocytic astrocytoma. J Clin Neurosci 2009;16(12):1704–6. [8] Johnson DR, Brown PD, Galanis E, Hammack JE. Pilocytic astrocytoma survival in adults: analysis of the surveillance, epidemiology, and end results program of the National Cancer Institute. J Neurooncol 2012;108(1):187–93. [9] Tonsgard JH. Clinical manifestations and management of neurofibromatosis type 1. Semin Pediatr Neurol 2006;13(1):2–7. [10] Franco-Hernandez C, Martinez-Glez V, de Campos JM, Isla A, Vaquero J, Gutierrez M, et al. Allelic status of 1p and 19q in oligodendrogliomas and glioblastomas: multiplex ligation-dependent probe amplification versus loss of heterozygosity. Cancer Genet Cytogenet 2009;190(2):93–6. [11] Minuti G, Cappuzzo F, Duchnowska R, Jassem J, Fabi A, O’Brien T, et al. Increased MET and HGF gene copy numbers are associated with trastuzumab failure in HER2-positive metastatic breast cancer. Br J Cancer 2012;107(5):793–9.
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[12] Collins VP, Jones DT, Giannini C. Pilocytic astrocytoma: pathology, molecular mechanisms and markers. Acta Neuropathol 2015;129(6):775–88. [13] Balss J, Meyer J, Mueller W, Korshunov A, Hartmann C, von Deimling A. Analysis of the IDH1 codon 132 mutation in brain tumors. Acta Neuropathol 2008;116(6):597–602. [14] Korshunov A, Meyer J, Capper D, Christians A, Remke M, Witt H, et al. Combined molecular analysis of BRAF and IDH1 distinguishes pilocytic astrocytoma from diffuse astrocytoma. Acta Neuropathol 2009;118(3):401–5. [15] Mukasa A, Takayanagi S, Saito K, Shibahara J, Tabei Y, Furuya K, et al. Significance of IDH mutations varies with tumor histology, grade, and genetics in Japanese glioma patients. Cancer Sci 2011;103(3):587–92. [16] Schindler G, Capper D, Meyer J, Janzarik W, Omran H, Herold-Mende C, et al. Analysis of BRAF V600E mutation in 1320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma, ganglioglioma and extra-cerebellar pilocytic astrocytoma. Acta Neuropathol 2011;121(3):397–405. [17] Basto D, Trovisco V, Lopes JM, Martins A, Pardal F, Soares P, et al. Mutation analysis of B-RAF gene in human gliomas. Acta Neuropathol 2005;109(2):207–10. [18] Jones DT, Kocialkowski S, Liu L, Pearson DM, Backlund LM, Ichimura K, et al. Tandem duplication producing a novel oncogenic BRAF fusion gene defines the majority of pilocytic astrocytomas. Cancer Res 2008;68(21):8673–7. [19] Cykowski MD, Allen RA, Kanaly AC, Fung KM, Marshall R, Perry A, et al. The differential diagnosis of pilocytic astrocytoma with atypical features and malignant glioma: an analysis of 16 cases with emphasis on distinguishing molecular features. J Neurooncol 2013;115(3):477–86. [20] Wade A, Hayhurst C, Amato-Watkins A, Lammie A, Leach P. Cerebellar pilocytic astrocytoma in adults: a management paradigm for a rare tumour. Acta Neurochir (Wien) 2013;155(8):1431–5. [21] Giese A, Bjerkvig R, Berens ME, Westphal M. Cost of migration: invasion of malignant gliomas and implications for treatment. J Clin Oncol 2003;21(8):1624–36. [22] Hu B, Thirtamara-Rajamani KK, Sim H, Viapiano MS. Fibulin-3 is uniquely upregulated in malignant gliomas and promotes tumor cell motility and invasion. Mol Cancer Res 2009;7(11):1756–70. [23] Riemenschneider MJ, Jeuken JW, Wesseling P, Reifenberger G. Molecular diagnostics of gliomas: state of the art. Acta Neuropathol 2010;120(5):567–84. [24] Schiffman JD, Hodgson JG, VandenBerg SR, Flaherty P, Polley MY, Yu M, et al. Oncogenic BRAF mutation with CDKN2A inactivation is characteristic of a subset of pediatric malignant astrocytomas. Cancer Res 2010;70(2):512–9. [25] Kraus JA, Glesmann N, Beck M, Krex D, Klockgether T, Schackert G, et al. Molecular analysis of the PTEN, TP53 and CDKN2A tumor suppressor genes in long-term survivors of glioblastoma multiforme. J Neurooncol 2000;48(2):89–94. [26] von Deimling A, Korshunov A, Hartmann C. The next generation of glioma biomarkers: MGMT methylation, BRAF fusions and IDH1 mutations. Brain Pathol 2010;21(1):74–87. [27] Lin A, Rodriguez FJ, Karajannis MA, Williams SC, Legault G, Zagzag D, et al. 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 2011;71(1):66–72. [28] Burkhard C, Di Patre PL, Schuler D, Schuler G, Yasargil MG, Yonekawa Y, et al. A population-based study of the incidence and survival rates in patients with pilocytic astrocytoma. J Neurosurg 2003;98(6):1170–4. [29] Otero-Rodriguez A, Sarabia-Herrero R, Garcia-Tejeiro M, Zamora-Martinez T. Spontaneous malignant transformation of a supratentorial pilocytic astrocytoma. Neurocirugia (Astur) 2010;21(3):245–52. [30] Yong EX, McKelvie P, Murphy M, Wang YY. Anaplastic pilocytic astrocytoma. J Clin Neurosci 2014;21(11):1993–6. [31] Bell D, Chitnavis BP, Al-Sarraj S, Connor S, Sharr MM, Gullan RW. Pilocytic astrocytoma of the adult – clinical features, radiological features and management. Br J Neurosurg 2004;18(6):613–6. [32] Brown PD, Buckner JC, O’Fallon JR, Iturria NL, Brown CA, O’Neill BP, et al. Adult patients with supratentorial pilocytic astrocytomas: a prospective multicenter clinical trial. Int J Radiat Oncol Biol Phys 2004;58(4):1153–60.
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Clinical case
Adult recurrent pilocytic astrocytoma: Clinical, histopathological and molecular study Astrocytome pilocytique récidivant de l’adulte : étude clinique, histopathologique et moléculaire S. Trabelsi a,∗ , N. Mama b , M. Ladib c , S. Popov d , A. Burford d , M. Mokni e , K. Tlili b , H. Krifa c , M. Varella-Garcia f , C. Jones d , M. Tahar Yacoubi e , A. Saad a , D. H’mida Ben Brahim a a
Department of Cytogenetics, Molecular genetics and Reproductive Biology, Farhat Hached University Hospital, Street Ibn Eljazzar, 4000 Sousse, Tunisia Department of Imagery, Sahloul University Hospital, Sousse, Tunisia Department of Neurosurgery, Sahloul University Hospital, Sousse, Tunisia d Divisions of Molecular Pathology and Cancer Therapeutics, the Institute of Cancer Research, Sutton, United Kingdom e Department of Cytopathology, Farhat Hached University Hospital, Sousse, Tunisia f University of Colorado Denver School of Medicine, Division of Medical Oncology, Colorado, USA b c
a r t i c l e
i n f o
Article history: Received 14 March 2015 Received in revised form 9 June 2015 Accepted 29 July 2015 Available online 17 November 2015 Keywords: Pilocytic astrocytoma Adult Recurrence KIAA1549:BRAF fusion
a b s t r a c t Background. – PA is a grade I glial tumor that mostly occurs in children. However, although apparently similar to paediatric PA, adult PA presents a different clinical follow-up that could arise from specific molecular alterations. A variety of genetic alterations have been identified as diagnostic or prognostic glioma molecular markers. Material and methods. – We describe a right infratentorial tumor that occurred in a 58-year-old man. Neuroimaging and neuropathological examination suggested PA as an initial diagnosis. The tumor was completely resected. Unexpectedly, two years later, a rapidly growing tumor on the operative site was observed with a second location in the pineal region. Immunohistochemical reactions (IHC), Multiplex ligation probe amplification (MLPA) and fluorescence in situ hybridization (FISH) was performed in both primary and relapse tumor. Results. – Neuroimaging and neuropathological examinations suggested an unusual diagnosis for adult patients: a recurrent PA. Both MLPA and FISH analysis contribute to diagnostic confirmation by KIAA1549: BRAF fusion detection. Additional genetic results revealed interesting findings that justified the tumor aggressivity. Conclusion. – Molecular analysis of adult PA cases should be routinely combined with histopathological and neuroimaging examination to further refine prognostic diagnoses. © 2015 Elsevier Masson SAS. All rights reserved.
r é s u m é Mots clés : Astrocytome pilocytique Adulte Récidive Gène de fusion KIAA1549:BRAF
Introduction. – L’astrocytome pilocytique (AP) est une tumeur gliale de grade 1, survenant le plus souvent à un âge pédiatrique. Chez les adultes, en dépit de la similarité histologique, l’AP est de moins bon pronostic. Cette évolution défavorable est sous-tendue par un profil génétique spécifique. En effet, un grand nombre d’altérations moléculaires de valeurs diagnostiques, pronostiques et théranostiques seraient à l’origine de cette différence pronostique. Matériels et méthodes. – Dans cette étude, nous rapportons un cas de tumeur gliale infratentorielle chez un patient adulte de 58 ans. Les données d’imagerie et de neuropathologie suggèrent l’AP comme premier diagnostic. Deux ans après une résection complète, nous avons observé une récidive bifocale au siège initial de la tumeur avec une évolution rapide, accompagnée d’une deuxième localisation
∗ Corresponding author. E-mail address: [email protected] (S. Trabelsi). http://dx.doi.org/10.1016/j.neuchi.2015.07.002 0028-3770/© 2015 Elsevier Masson SAS. All rights reserved.
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dans la région pinéale. Aussi bien la tumeur initiale que la récidive ont été explorées par techniques d’immunohistochimie (IHC), d’amplification et ligation multiplexes de sondes (MLPA) et d’hybridation in situ fluorescente (FISH). Résultats. – Les données d’imagerie et neuropathologie suggèrent un diagnostic rare d’AP récidivant de l’adulte. Les analyses moléculaires par MLPA et FISH apportent la confirmation diagnostique en révélant la présence du gène de fusion KIAA1549:BRAF. D’autres altérations génétiques pouvant justifier l’agressivité de la tumeur ont également été détectées. Conclusion. – L’analyse moléculaire en matière d’AP de l’adulte s’avère complémentaire de l’histopathologie et de l’imagerie. En plus de sa valeur diagnostique connue, elle serait d’un grand apport pronostique. Elle devrait ainsi intégrer le protocole routinier du diagnostic des PA adultes. © 2015 Elsevier Masson SAS. Tous droits réservés.
1. Introduction Pilocytic astrocytoma (PA) is a circumscribed glioma that primarily occurs in the cerebellum in children [1]. Clinical presentation depends on tumor location. Headache, neck pain, vomiting and visual abnormalities are usually reported with the cerebellum location [2]. Histopathological description includes biphasic pattern, elongated and rounded cells, Rosenthal fibers, and eosinophilic granular bodies [2]. PA is classified as grade I by the World Health Organization (WHO) [3] and usually reported with a good prognosis. In most cases, only surgical resection is considered to be curative [4]. Very few recurrent adult PA cases have been reported with malignant progression [4–6]. In such unusual presentations, features like microvascular proliferation, necrosis and nuclear atypia may occur. Differential diagnoses as high-grade gliomas could then be suspected with a considerably worse prognosis [7]. Furthermore, unlike paediatric PA, the adult cases seem to present a poor prognosis [8]. The correct diagnosis has great importance in the treatment choice and prognosis of patients. 2. Material and methods 2.1. Patient A 58-year-old man with no previous family medical history of cancer was referred for severe headache, neck stiffness and difficulty in walking. The physical examination showed signs of cerebellar dysfunction. MRI revealed a well-circumscribed lesion extended into the 4th ventricle (Fig. 1a, b). The patient underwent a complete tumor resection without evidence of any residual or recurrent mass at 6-months follow-up by MRI. Two years later, the patient was readmitted with increased intracranial pressure, a cerebellar syndrome, vertical/horizontal strabismus and VI and IX right nerve palsy. There was no previous family medical history or clinical signs of neurofibromatosis [9]. The MRI showed a bifocal tumor located in the cerebellar region at the operative site with a second location in the pineal region (Fig. 1c, d). The masses presented with cystic and intensely enhanced solid components. The patient was readmitted for a second tumor resection. The pineal component was unresectable and the tumor was partially removed in the cerebellar location alone. The patient died shortly after the operation. 2.2. Histological Immunohistochemical study Routine sections (4 mm) of both primary and relapse tumor were stained with haematoxylin and eosin. Immunohistochemical analysis was performed on 4 mm of formalin-fixed, paraffinembedded sections using a panel of polyclonal antibodies (Table 1). Slides were dewaxed in xylene and rehydrated through a descending ethanol series. Antigen retrieval was performed at 98 ◦ C with
DAKO antigen retrieval solution during 20 min (Table 1). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol. The detection system used was Novolink Polymer (Leica Microsystems, Newcastle Ltd.) with diaminobenzidine as chromogen. Slides were counterstained with Mayer’s haematoxylin. HE and IHC results were separately reviewed by two pathologists. 2.3. DNA extraction Primary tumor Genomic DNA was purified from FFPE sections using an Qiagen kit (Qiamp DNA FFPE Tissue Kit). Recurrent tumor genomic DNA was purified from fresh tumoral tissue according to phenol–chloroform protocol. 2.4. Multiplex ligation probe amplification (MLPA) Both primary and recurrent tumoral DNAs were analysed using a multiplex ligation probe amplification (MLPA) reaction through SALSA MLPA Kits P105, P370 and P088 (MRC Holland, Amsterdam, The Netherlands) according to manufacturers protocol. DNA from healthy tissue was adopted as MLPA control sample. MLPA kits enabled us to analyse multiple chromosomal aberrations located at 1p36, 7q3, 9p21, 10q23, 11p11, 17p13 and 19q13 (Table 2). Additional point mutations may be detected using the P370 MLPA kit (IDH1 R132H, IDH1 R1321C, IDH2 R172K, IDH2 R172M and BRAF V600E). PCR products were analysed on ABI 310 (Applied Biosystems, Foster City, CA) using as an internal size standard the ROX-500 “Genescan® ” (ABI 401734). Data analysis was performed with “GeneMarker® ” software V1.91 (SoftGenetics). Intra normalization for sample data was initially performed on control probes, and then on healthy control samples. Normal ratio limits were set at 0.75 and 1.3 [10]. For more accuracy, we considered that displaying 2 or more probes, adjacent to each other on a chromosomal region, exhibiting the ratio below 0.45 or above 2 were accepted respectively as deleted or gained. 2.5. Fluorescence in situ hybridization (FISH) FISH assay was performed in both the primary tumor and relapse to look for presence of the KIAA1549:BRAF fusion gene. A dual-colour FISH probe set was designed to identify the chromosomal duplication that generates the KIAA1549:BRAF fusion, using human genomic sequences from the RP4-726N20 clone that encompass BRAF gene labelled in Spectrum Red and the using human genomic sequences from the RP11-355D18 clone that encompass KIAA1549 labeled in Spectrum Green. Probe preparation and FISH assays were performed as reported elsewhere [11].
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Fig. 1. a, b: first tumor: well-circumscribed lesion extended through the 4th ventricle; c, d: recurrence: bifocal tumor located on the cerebellar and pineal regions. Double cystic and Intensely enhanced tissular components. a, b : tumeur initiale : lésion bien circonscrite étendue au 4e ventricule ; c, d : récidives : tumeur bifocale située sur la région cérébelleuse et pinéale. Une double composante kystique avec un rehaussement tissulaire intense.
3. Results 3.1. Histolopathology and immunohistochemistry results First tumor macroscopic examination described 2.5 cm white crumbly fragments. Histological examination demonstrated round and spindle cells with eosinophilic cytoplasm, fibrillary areas and rich vascularity. Occasional mitoses and vascular proliferation were also observed (Fig. 2a). Immunohistochemical (IHC) description of the primary tumor demonstrated a positive staining for GFAP (Dako Clone Poly ZCG23, 1/300 dilution) and negative staining for EMA (Dako Clone E29,
1/100 dilution) (Fig. 2). Based on this description, pilocytic astrocytoma was highly suspected (Table 1), with anaplastic astrocytoma as a differential diagnosis. Macroscopic examination of the recurrence showed a 3.5 × 3 × 2 cm white fragment with focal necrosis. Haematoxylin and eosin (H&E) staining showed a biphasic pattern, with heterogeneous cellular density. Tumor cells were pleomorphic with atypia and hyper-chromatin nuclei. A prominent vascularity as well as necrotic area was observed (Fig. 2). The recurrent tumor was positive for GFAP and negative for synaptophysine and neurofilament (NF), with weak P53 (Dako Clone DO7, diluted 1/50) and KI67 (Dako Clone MIB1,
Table 1 List of antibodies and IHC staining results. Liste des anticorps et des résultats du marquage en IHC. Antigen
Antibody and manufacturers
Dilution
Antigen Retrieval
First tumor
Relapse
S100 NSE Synaptophysine NF GFAP EMA P53 KI67 Olig2 IDH R132H
Dako clone Dako Clone BBS/NC/V1H14 Neomarker clone Rb-SP111-RM Dako clone 2f11 Dako Clone Poly ZCG23 Dako clone E29 Dako clone DO7 Dako clone MIB1 Millipore AB9610 Dianova clone H09
1:300 1:50 1:200 1:100 1:300 1:100 1:50 1: 300 1:100 01:20
/ EDTA PH6 EDTA PH6 EDTA PH6 EDTA PH6 EDTA PH6 EDTA PH6 EDTA PH6 EDTA PH9 EDTA PH 6
+ ND ND ND + − 5% 1% + −
+ + − − + − 5% 1% + −
NSE: neuron-specific enolase; NF: neurofilament; GFAP: glial fibrillary antigen protein; EMA: epithelial membrane antigen; /: without antigen retrieval; +: positive staining; −: negative staining; ND: not determined. NSE : neuron-specific enolase ; NF : neurofilament ; GFAP : glial fibrilary antigen protein ; EMA : epithelial membrane antigen ; / : sans démasquage antigénique ; + : marquage positif staining ; − : marquage négatif ; ND : non determiné.
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Fig. 2. First tumor haematoxylin and eosin stain: a: round and spindle eosinophilic cells (× 40); b: cells with fibrillary areas with occasional mitosis (× 200); c: rich vascularity (× 200); d: endothelial proliferation (× 200). Relapse hematoxylin and eosin stain: e: pleomorphic tumor cells with atypia and hyper-chromatic nuclei (× 40); f: biphasic pattern with necrotic areas (× 12.5); g: prominent vascularity (× 40). IHC stain: h: IDH R132H negative (× 40); i: first tumor GFAP positive (× 100); j: Relapse GFAP positive (× 40); k: first tumor olig2 positive (× 40); l: relapse tumor olig2 positive (× 40). Coloration HE de la tumeur initiale : a : des cellules rondes et fusiformes éosinophiles (× 40) ; b : des cellules avec des zones fibrillaires et mitoses occasionnelles (× 200) ; c : une riche vascularisation (× 200) ; d : une prolifération endothéliale (× 200). Coloration HE de la récidive : e : les cellules tumorales pléomorphes avec atypies nucléaires et noyaux hyper-chromatiques (× 40) ; f : un aspect bi-phasique avec des zones nécrotiques (× 12,5) ; g : une vascularisation importante (× 40). Coloration IHC : h : IDH R132H négative (× 40); i : tumeur initiale: GFAP positive (× 100) ; j : récidive : GFAP positive (× 40) ; k : tumeur initiale: Olig2 positif (× 40) ; l : récidive : Olig2 positif (× 40).
diluted 1/300) labelling index respectively at 5 and 1% (Fig. 2, Table 1). 3.2. Genetic results MLPA profiles showed that both primary tumor and relapse were wild type for IDH1 (R132H, R132C) IDH2 (R172K, R172 M) and BRAF (V600E) mutations (Table 2). The IDH1 R132H mutation negative staining was reproduced by IHC (Dianova Clone H09 diluted 1:20) (Fig. 2e). MLPA analysis showed amplification on KIAA1549 as well as BRAF probes that interestingly retained the BRAF kinase domain in both first tumor and relapse. The KIAA1549:BRAF gene fusion was identified by FISH as red and green doublet signals partially overlapped, while the normal copies of the genes were identified as single copies of red and green signals separated by a small gap. For this assay, specimens are classified as positive for the KIAA1459:BRAF fusion when exhibiting positive
signals in ≥15% of tumor cells (Fig. 3). FISH assays succeeded only in the relapse sample and clearly confirmed the amplification and fusion of BRAF and KIAA1549. MLPA analysis revealed additional genetic alterations: CDKN2A/P14 gene promoter loss and EGFR focal amplification. PTEN gene was found to be lost only in first tumor (Table 2).
4. Discussion According to the clinical findings, there was no strong evidence of PA. Thus, differential diagnosis of high -grade astrocytic tumors should also be considered. However, the posterior fossa location of the primary tumor and the cystic components are likely compatible with a pilocytic astrocytoma diagnosis. Histopathological assessment strongly suggested that the primary tumor was pilocytic astrocytoma with a biphasic pattern and eosinophilic granular bodies.
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Fig. 3. FISH analysis for the KIAA1549-BRAF fusion: tissue section shows numerous signals of tandem duplication for BRAF in red (A), KIAA1549 in green (B) and fusion result of these loci in yellow with merged signal (C). Analyse par FISH de la fusion KIAA1549 : BRAF. La section de tissu analysée présente de nombreux signaux deduplication en tandem pour le locus BRAF en rouge (A), KIAA1549 en vert (B) ainsi que le résultat de la fusion des deux loci avec un signal de superposition en jaune (C).
Table 2 Genetic alterations in primary and recurrent tumor detected by MLPA. Les altérations génétiques détectées par MLPA au niveau de la tumeur initiale et la récidive. MLPA Kit references
Region (gene)
First tumor
Relapse
P370
7q34 (KIAA1549 B-RAF) 2q33 (IDH1 p.R132H, IDH1 p.R132C) 9p21 (CDKN2A)a BRAFV600E 9p21 (CDKN2A)a 10q23 (PTEN) 11p11 (EGFR)
Gain Absent
Gain Absent
Loss Absent Loss Loss Gain
Loss Absent Loss NS Gain
P105
NS: normal statues. NS : statut normal. a CDKN2A gene is detected by 2 separate MLPA kit P370 and P105. a Gène CDKN2A est analysé par deux kits MLPA séparés P370 et P105.
Two years later, the patient presented with an unexpected tumor relapse. Histopathological examination noted high cellularity with small atypical cells, and broad necrosis. Despite the patient’s age and the tumor location, the histopathological description was also strongly suggestive of a diagnosis of PA. Moreover, histopathological description suggested the differential diagnosis of ganglioglioma very possible [12]. The absence of IDH mutations in MLPA and IHC investigations provides an additional argument for a diagnosis of pilocytic astrocytoma, with IDH mutation occurring frequently in diffuse astrocytoma but rarely in PA [13–15]. BRAF V600E mutation has been described only in 9% of PA and was also absent in both primary and relapse specimens [16,17]. Our patient’s primary tumor revealed the presence of the KIAA1549:BRAF fusion gene, typical of pilocytic astrocytoma [14,18] in children. This fusion was also present two years later in the recurrent tumor supporting the diagnosis of PA, even with histological atypias and extra cerebellar location [19].
Despite the WHO description of pilocytic astrocytoma as low grade tumor, this case seems to be a clear exception, with a recurrence after total resection, and eventual death due to their disease. Moreover, the simultaneous presence of many arguments are in the opposite of the differential diagnosis of ganglioglioma: absence of IDH mutation, absence of BRAFV600E mutation, negative IHC staining of neuro-markers (NF and Synaptophysin) and KIAA1549:BRAF fusion in both first and relapse tumor. Here, the adult patient presented a PA with an anaplastic pattern in the bifocally recurrent tumor. One hypothesis could be suggested: During tumor resection and due to their motility nature, residual astrocytoma cells were likely competitively selected to escape and migrate to the pineal and cerebellar regions. In addition, previous data suggest that subtotal resection of this tumor subtype carries a high risk of recurrence [20]. Such behaviour is closely associated with gene-expression and molecular profiles [21,22]. This poor clinical outcome was well reflected in the molecular findings. CDKN2A/P14 loss noticed in both tumors is already known to confer an aggressive behaviour to tumoral cells and had been currently associated with a poor outcome [23–25]. These types of molecular alterations in primary PA can switch tumor grading from apparently benign to highly malignant potential tumor. This upgrading would justify an aggressive therapeutic approach that could improve patient’s outcome. Previously adult PA was thought to be a begin tumor, such as the paediatric PA [13,21,26,27]. Recently, a series of adult PA with malignant transformation and recurrence [28], as well as PA with unusual anaplastic pattern [21,29] were reported. In those previous studies the diagnosis of anaplastic adult cases of PA was mainly based on histopathology and imaging [5,10,21,30–32].
5. Conclusion Even if apparently benign, adult PA could present a poor outcome arising from tumor molecular alterations. Combined with pathologic and neuroimaging examination, genetic investigations
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should be required for that particular PA subset to provide a better disease management. Disclosure of interest The authors declare that they have no competing interest. Acknowledgement We would like to thank the patient for agreeing to participate in this study. We also thank the technical help given by the Division of Medical Oncology at the School of Medicine, University of Colorado, USA. We would like also to acknowledge the efforts of GETUC Tunisian group members ,“Groupe d’étude des tumeurs cérébrales”. We also thank glioma team in the division of molecular pathology and cancer therapeutics at The Institute of Cancer Research, Sutton, UK. Ethical standards: the manuscript was approved by the responsible authorities of our laboratory and by the ethics committee of Farhat Hached University Hospital of Sousse, Tunisia. We have no substantial direct or indirect commercial or financial incentive associated with publishing the article. References [1] Shibahara I, Kawaguchi T, Kanamori M, Yonezawa S, Takazawa H, Asano K, et al. Pilocytic astrocytoma with histological malignant features without previous radiation therapy – case report. Neurol Med Chir (Tokyo) 2011;51(2):144–7. [2] 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(2):97–109. [3] Li HM, Hsu SS, Wang JS, Weng MJ, Fu JH, Chen CK, et al. Cerebral pilocytic astrocytoma with spontaneous intracranial hemorrhage in adults. J Chin Med Assoc 2008;71(11):587–93. [4] Ellis JA, Waziri A, Balmaceda C, Canoll P, Bruce JN, Sisti MB. Rapid recurrence and malignant transformation of pilocytic astrocytoma in adult patients. J Neurooncol 2009;95(3):377–82. [5] Stuer C, Vilz B, Majores M, Becker A, Schramm J, Simon M. Frequent recurrence and progression in pilocytic astrocytoma in adults. Cancer 2007;110(12):2799–808. [6] Sioutos PJ, Hamilton AJ, Narotam PK, Weinand ME. Unusual early recurrence of a cerebellar pilocytic astrocytoma following complete surgical resection. Case report and review of the literature. J Neurooncol 1996;30(1):47–54. [7] Azad A, Deb S, Cher L. Primary anaplastic pilocytic astrocytoma. J Clin Neurosci 2009;16(12):1704–6. [8] Johnson DR, Brown PD, Galanis E, Hammack JE. Pilocytic astrocytoma survival in adults: analysis of the surveillance, epidemiology, and end results program of the National Cancer Institute. J Neurooncol 2012;108(1):187–93. [9] Tonsgard JH. Clinical manifestations and management of neurofibromatosis type 1. Semin Pediatr Neurol 2006;13(1):2–7. [10] Franco-Hernandez C, Martinez-Glez V, de Campos JM, Isla A, Vaquero J, Gutierrez M, et al. Allelic status of 1p and 19q in oligodendrogliomas and glioblastomas: multiplex ligation-dependent probe amplification versus loss of heterozygosity. Cancer Genet Cytogenet 2009;190(2):93–6. [11] Minuti G, Cappuzzo F, Duchnowska R, Jassem J, Fabi A, O’Brien T, et al. Increased MET and HGF gene copy numbers are associated with trastuzumab failure in HER2-positive metastatic breast cancer. Br J Cancer 2012;107(5):793–9.
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