Ganglioglioma associated with alterations of NBN gene. A case report

Introduction
Gangliogliomas (GGs) are mixed glio-neuronal neoplasms, composed of neoplastic ganglion cells in combination with a neoplastic glial element. GGs represent about 1.3% of all brain tumours [3]. Most of them correspond to WHO grade I or II [3]. Some gangliogliomas exhibit anaplastic glial components and are considered to be grade III according to WHO criteria. Low-grade gangliogliomas are more frequently recognized in children and adolescents. These tumours are well-differentiated, slowly growing neoplasms causing intractable epilepsy. The majority of them are localized in the temporal lobe, but other locations such as frontal or parietal lobes, optic nerves, cerebellum and spinal cord have also been encountered. Most supratentorial GGS involving the temporal lobe are usually associated with medically intractable epilepsy in children and young adults [3,14]. Computed tomography (CT) may demonstrate a circumscribed solid mass or cyst with a mural nodule. MRI shows a T1-weighted hypointense, T2-weighted hyperintense circumscribed solid or cystic mass [18]. Microscopically gangliogliomas are composed of two histologically different elements: glial and neuronal [3,10]. The glial component usually exhibits astroglial origin and might reveal features of pilocytic astrocytoma, fibrillary astrocytoma, pleomorphic xanthoastrocytoma or anaplastic astrocytoma. The most common glial element of paediatric GGs is pilocytic astrocytoma with Rosenthal fibres and eosinophilic granular bodies. The neuronal component consists of large, sometimes bi- or multinucleated ganglion cells. Additional histopathological features of GGs include calcifications, extensive perivascular lymphoid infiltrates, and an abundant capillary network. Mitotic figures are rare. Reported Ki-67/MIB-1 labelling index, involving only the glial component, has ranged from 1.1 to 2.7% [3,14].
Ganglioglioma of the optic nerve has been noted in a patient with neurofibromatosis type 1 [15]. The most frequent cytogenetic alterations of GGs is the gain of chromosome 7 and partial loss of chromosome 9p [26]. CDKN2A deletion was observed in 2/3 of anaplastic gangliogliomas [26]. The glial component of some gangliogliomas demonstrates alterations in the TSC2 gene, including polymorphisms in intron 4 and exon 41 and a somatic mutation in intron 32 [1,2] Low-grade GGs with recurrences revealed TP53 mutation [1-3,26]. New genes potentially involved in pathogenesis of ganglioglioma are still being sought. One of them appeared to be the NBN (previously named NBS1) gene [5,11,13,27]. Mutations in this gene result in Nijmegen breakage syndrome (NBS) – a rare, autosomal recessive chromosomal instability disorder [6,22,25]. NBS is clinically characterized by microcephaly, dysmorphic features, immunodeficiency, radiosensitivity, and increased risk for cancer (mainly lymphomas and other neoplasms, including brain tumours). The most common NBN mutation (c.657_661del5) is found in over 90% of NBS patients (founder effect). The NBN gene (8p21) encodes nibrin, one of the nuclear Mre11/Rad50/NBN (MRN) complex components, playing an important role in the detection of double-strand breaks [5,13,19,30]. It is well documented that relatives of NBS patients carrying a mutation on one NBN allele may also develop diverse neoplasms [22,24]. Many studies have shown an increased risk in heterozygous carriers of NBN gene mutations of malignant melanoma, non-Hodgkin’s lymphomas, acute lymphoblastic leukaemia, stomach and colorectal cancer, breast and ovarian cancers, rhabdomyosarcoma, and medulloblastoma [6-8,11,16,17,20,21,24].
These observations prompted us to study the potential involvement of the most frequently identified NBN gene mutations (c.657_661del5, c.511A>G, and c.643C>T) in pathogenesis of paediatric brain tumours. The presented patient was selected from a group of children with brain tumours screened for the most common molecular variants in the NBN gene (exons 5 and 6). The study was part of a project assessing the frequency of heterozygote carriers of NBS1 gene mutations in Polish children with defined malignancy, particularly central nervous system tumours.
Case report
A 13-year-old girl presented in 2002 with grand mal seizures. Brain MRI scan showed a lesion in the frontal/parietal right lobes, with weak enhancement after contrast administration. The patient underwent surgery in the Department of Neurosurgery, The Children’s Memorial Health Institute. The lesion was only partially resected and biopsy material was scarce. Pathological examination was inconclusive and revealed abnormal neuronal cells, and glial tissue with no evidence of malignancy. The patient was subsequently followed with serial brain MRI scans. Two years later (2004) MRI showed an increase in size of the residual right frontal lesion. The second surgery and pathological examination were performed in another centre. The lesion was partially resected and histopathological diagnosis of diffuse astrocytoma was suggested. No further treatment was advised. Nine months later she deteriorated neurologically. The number of seizures increased and left hemiparesis was observed. Brain MRI scan revealed tumour progression, suggesting the development of malignant glioma. At this time she was consulted in the Department of Oncology, The Children’s Memorial Health Institute. Due to the size of the tumour and involvement of more than one lobe neurosurgical treatment was not recommended. Pathology specimens from two surgeries were reassessed and the final diagnosis of ganglioglioma WHO grade I was established in the Department of Pathology, The Children’s Memorial Health Institute. Chemotherapy consisting of cisplatinum and temozolomide was implemented but due to tumour progression it was discontinued after 4 courses. Radiotherapy was then administered. She received a total dose of 54 cGy to the tumour with margins. Radiotherapy was followed by maintenance chemotherapy resulting in disease stabilization.
Pathological findings
Microscopically, the tumour exhibited complex structure and consisted of glial and neuronal elements. The glial component was predominantly composed of elongated astroglial cells with oval nuclei. The neoplastic tissue showed the presence of numerous Rosenthal fibres (Fig. 1) and eosinophilic granular bodies. Mitoses and necrosis were absent. The majority of the glial element revealed features of pilocytic astrocytoma. The neuronal component comprised large, atypical neurons with multiple processes and bi- or multi-nucleated ganglion cells (Fig. 2). Neuronal nuclei were large and round with prominent nucleoli. Perivascular lymphocytic infiltrates were occasionally observed.
Immunohistochemical examination revealed immunoreactivity for glial fibrillary acid protein (GFAP) and S-100 protein in neoplastic astrocytic cells (Fig. 3). Neoplastic neuronal cells were positive for synaptophysin (Fig. 4), neurofilament protein (NF), neuronal specific enolase (NSE), and chromogranin A. Labelling index of Ki67 was about 2%. The histopathological features and immunohistochemistry allowed the diagnosis of ganglioglioma WHO grade I to be established. The immunoexpression of nibrin was assessed due to alteration of the NBN gene in this patient. The neoplastic cells of both glial and neuronal components were positive for nibrin. These cells showed nuclear and cytoplasmic staining. Strong cytoplasmic staining was especially found in neuronal elements (Fig. 5). In control tissue samples taken from paediatric ependymoma without alteration of the NBN gene neoplastic cells revealed strong nuclear staining of nibrin (Fig. 6).
Molecular study
Molecular studies were conducted on genomic DNA extracted from the patient’s dried blood spot. Searching for the most common molecular variants in NBN gene exons 5 and 6 (c.511A>G, c.657_661del5, and c.643C>T) revealed the presence of c.511A>G (p.Ile171Val) substitution on one allele (Fig. 7). Loss of heterozygosity (LOH) analysis was not performed due to lack of a frozen tumour sample from the patient.
Discussion
Gangliogliomas are well-differentiated, slowly growing biphasic neuroepithelial tumours composed of neoplastic ganglion cells and a neoplastic glial component. In children, various glioneuronal tumours and cortical dysplastic abnormalities are a frequent cause of intractable epilepsy [3,9,12]. Surgery is the best method of treatment of GGs, and in most cases, residual tissue grows slowly [3]. The role of adjuvant therapy remains controversial. Malignant transformation of gangliogliomas as in other glioneuronal tumours is rarely observed [4,14]. One can speculate that the unfortunate clinical course of our patient might have been associated with the presence of alteration in the NBN gene.
Cytogenetic abnormalities of GGs have appeared inconsistent. The most frequently diagnosed cytogenetic alterations were the gain of chromosome 7 and partial loss of chromosome 9p [26]. Approximately 65% of anaplastic gangliogliomas presented with CDKN2A deletion [26]. The glial component of GGs demonstrated various alterations, including splice-site specific polymorphisms in intron 4 and exon 41, and a somatic mutation in intron 32 of the TSC2 gene [1-3,23,26]. The TP53 mutation was reported in the recurrence of a low-grade ganglioglioma [26] .
Ganglioglioma has been reported in neurofibromatosis type 1 and 2, Turcot syndrome, and in
a Peutz-Jeghers patient with a germline mutation of the cyclin-dependent kinase CDK5 [1,3,15].
Our screening for the common NBN gene molecular variants revealed the presence of c.511A>G (p.Ile171Val) substitution on one allele. The mutation p.Ile171Val disturbs the structure and activity of nibrin. It is assumed to affect its BRCA1 carboxy-terminal (BRCT) domain, which is highly conserved in eukaryotic nuclear proteins engaged in cell cycle, gene regulation, and DNA repair pathways [5,13,19,25,30]. An increased incidence of tumourigenesis, associated with the presence of the germline p.Ile171Val mutation in the NBN gene, has been described among patients with lymphoid malignancies (acute lymphoblastic leukaemia, ALL), breast and ovarian cancers, and larynx cancers [16,17,20-22,24,28,29]. The above-mentioned findings could suggest the probability that this molecular variant of the NBN gene could be a general susceptibility factor for malignancies. Thus, we could not exclude the role of NBN gene mutation (probably in association with other genes) in pathogenesis of ganglioglioma in our patient. Recent studies have shown the co-presence of mutations in the NBN gene and TP53 gene in medulloblastoma tissue [11]. It has been suggested that mutation of the NBN gene may have caused genomic instability and subsequent molecular alterations of the TP53 gene [11,27]. Our present finding of alteration in the NBN gene in ganglioglioma requires further study associated with molecular alterations of other genes, including the TP53 gene.
Immunohistochemical analysis of the presented ganglioglioma revealed expression of nibrin in the glial and neuronal component. Astrocytic cells showed weak nuclear and cytoplasmic positivity for nibrin, whereas neoplastic ganglion cells revealed mainly cytoplasmic immunostaining. In contrast, the cells of ependymoma without abnormalities in the NBN gene demonstrated a very strong nuclear reaction. Recently, some studies have indicated a role of nibrin overexpression as a marker of aggressive head and neck cancer [28]. Analysis of nibrin immunoexpression in our case showed a slightly different pattern in comparison with control brain tumour tissue, probably due to identified alteration in the NBN gene. The correlation between the immunohistochemistry results of nibrin expression with mRNA and protein levels should be ascertained.
Acknowledgements
This study was supported by a grant from the Committee for Science Research, Poland (PBZ-KBN-090/P05/04-17).
References
1. Becker AJ, Klein H, Baden T, Aigner L, Normann S, Elger CE, Schramm J, Wiestler OD, Blumcke I. Mutational and expression analysis of the reelin pathway components CDK5 and doublecortin in gangliogliomas. Acta Neuropath 2002; 104: 403-408.
2. Becker AJ, Lobach M, Klein H, Normann S, Nothen MM, von Deimling A, Mizuguchi M, Elger CE, Schramm J, Wiestler OD, Blumcke I. Mutational analysis of TSC1 and TSC2 genes in gangliogliomas. Neuropathol Appl Neurobiol 2001; 27: 105-114.
3. Becker AJ, Wiestler OD, Figarella-Branger D, Blumcke I. Ganglioglioma and gangliocytoma. In: Louis ND, Ohgaki H, Wiestler OD, Cavenee WK (eds.). WHO Classification of Tumours of Central Nervous System. Lyon: IARC 2007: 74-78.
4. Biernat W, Zakrzewski K, Polis L, Liberski PR. Glioneuronal-mesenchymal tumour with malignant transformation. Folia Neuropathologica 2007; 45: 140-143.
5. Czornak K, Chughtai S, Chrzanowska KH. Mystery of DNA repair: the role of the MRN complex and ATM kinase in DNA damage repair. J Appl Genet 2008; 49: 383-396.
6. Chrzanowska KH, Piekutowska-Abramczuk D, Popowska E, Gładkowska-Dura M, Małdyk J, Syczewska M, Krajewska-Walasek M, Goryluk-Kozakiewicz B, Bubała H, Gadomski A, Gaworczyk A, Kazanowska B, Kołtan A, Kuźmicz M, Luszawska-Kutrzeba T, Maciejka-Kapuścińska L, Stolarska M, Stefańska K, Sznurkowska K, Wakulińska A, Wieczorek M, Szczepański T, Kowalczyk J. Carrier frequency of mutation 657del5 in the NBS1 gene in
a population of Polish pediatric patients with sporadic lymphoid malignancies. Int J Cancer 2006; 118: 1269-1274.
7. Dembowska-Baginska B, Perek D, Brozyna A, Wakulinska A, Olczak-Kowalczyk D, Gladkowska-Dura M, Grajkowska W, Chrzanowska KH. Non-Hodgkin lymphoma (NHL) in children with Nijmegen Breakage syndrome (NBS). Pediatr Blood Cancer 2009; 52: 186-190.
8. Gładkowska-Dura M, Dzierzanowska-Fangrat K, Dura WT, van Krieken JH, Chrzanowska KH, van Dongen JJ, Langerak AW. Unique morphological spectrum of lymphomas in Nijmegen breakage syndrome (NBS) patients with high frequency of consecutive lymphoma formation. J Pathol 2008; 216: 337-344.
9. Grajkowska W, Kotulska K, Matyja E, Larysz-Brysz M, Mandera M, Roszkowski M, Domańska-Pakieła D, Lewik-Kowalik J, Jóźwiak S. Expression of tuberin and hamartin in tuberous sclerosis complex-associated and sporadic cortical dysplasia of Taylor's balloon cell type. Folia Neuropathol 2008; 46: 43-48.
10. Hirose T, Scheithauer BW, Lopes MB, Gerber HA, Altermatt HJ, VandenBerg SR. Ganglioglioma: An ultrastructural and immunohistochemical study. Cancer 1997; 79: 989-1003.
11. Huang J, Grotzer MA, Watanabe T, Hewer T, Pietsch T, Rutkowski S, Ohgaki H. Mutations in the Nijmegen breakage syndrome gene in medulloblastomas. Clin Cancer Res 2008; 14: 4053-4058.
12. Izycka-Swieszewska E, Majewska H, Szurowska E, Mazurkiewicz-Bełdzińska M, Drozyńska E. Papillary glioneuronal tumour of the precentral gyrus. Folia Neuropathol 2008; 46: 158-163.
13. Kobayashi J, Antoccia A, Tauchi H, Matsuura S, Komatsu K. NBS1 and its functional role in the DNA damage response. DNA Repair 2004; 3: 855-861.
14. Luyken C, Blumcke I, Fimmers R, Urbach H, Wiestler OD, Schramm J. Supratentorial gangliogliomas: histopathologic grading and tumor reccurence in 184 patients with a median follow-up of 8 years. Cancer 2004; 101: 146-155.
15. Meyer P, Eberle MM, Probst A, Tolnay M. Ganglioglioma of optic nerve in neurofibromatosis type 1. Case report and review of the literature. Klin Monatsbl Augenheilkd 2000; 217: 55-58.
16. Mosor M, Ziółkowska I, Pernak-Schwarz M, Januszkiewicz-Lewandowska D, Nowak J. Association of the heterozygous germline I171V mutation of the NBS1 gene with childhood acute lymphoblastic leukemia. Leukemia 2006; 20: 1454-1456.
17. Nowak J, Mosor M, Ziółkowska I, Wierzbicka M, Pernak-Schwarz M, Przyborska M, Roznowski K, Pławski A, Słomski R, Januszkiewicz D. Heterozygous carriers of the I171V mutation of the NBS1 gene have a significantly increased risk of solid malignant tumours. Eur J Cancer 2008; 44: 627-630.
18. Osborne AG. Diagnostic neuroradiology. Mosby, St Louis 1994.
19. Petrini JHJ, Stacker TH. The cellular response to DNA double-strand breaks: defining the sensors and mediators. Trends Cell Biol 2003; 13: 458-462.
20. Plisiecka-Hałasa J, Dansonka-Mieszkowska A, Rembiszewska A, Bidziński M, Steffen J, Kupryjańczyk J. Nijmegen breakage syndrome gene (NBS1) alterations and its protein (nibrin) expression in human ovarian tumours. Ann Hum Genet 2002; 66: 353-359.
21. Roznowski K, Januszkiewicz-Lewandowska D, Mosor M, Pernak M, Litwiniuk M, Nowak J. I171V germline mutation in the NBS1 gene significantly increases risk of breast cancer. Breast Cancer Res Treat 2008; 110: 343-348.
22. Seemanova E, Jarolim P, Seemann P, Varon R, Digweed M, Swift M, Sperling K. Cancer risk of heterozygotes with the NBN founder mutation. J Natl Cancer Inst 2007; 99: 1875-1880.
23. Squire JA, Arab S, Marrano P, Bayani J, Karaskova J, Taylor M, Becker L, Rutka J, Zielenska M. Molecular cytogenetic analysis of glial tumors using spectral karyotyping and comparative genomic hybridization. Mol Diagn 2001; 6: 63-108.
24. Steffen J, Varon R, Mosor M, Maneva G, Maurer M, Stumm M, Nowakowska D, Rubach M, Kosakowska E, Ruka W, Nowecki Z, Rutkowski P, Demkow T, Sadowska M, Bidziński M, Gawrychowski K, Sperling K. Increased cancer risk of heterozygotes with NBS1 germline mutations in Poland. Int J Cancer 2004; 111: 67-71.
25. Varon R, Vissinga C, Platzer M, Cerosaletti KM, Chrznowska KH, Saar K, Beckmann G, Seemanova E, Cooper PR, Nowak NJ, Stumm M, Weemaes CM, Gatti RA, Wilson RK, Digweed M, Rosenthal A, Sperling K, Concannon P, Reis A. Nibrin, a nowel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome. Cell 1998; 93: 467-476.
26. von Deimling A, Fimmers R, Schmidt MC, Bender B, Fassbender F, Nagel J, Jahnke R, Kaskel P, Duerr EM, Koopmann J, Maintz D, Steinbeck S, Wick W, Platten M, Müller DJ, Przkora R, Waha A, Blümcke B, Wellenreuther R, Meyer-Puttlitz B, Schmidt O, Mollenhauer J, Poustka A, Stangl AP, Lenartz D, von Ammon K. Comprehensive allelotype and genetic anaysis of 466 human nervous system tumors. J Neuropathol Exp Neurol 2000; 59: 544-558.
27. Watanabe T, Nobusawa S, Lu S, Huang J, Mittelbronn M, Ohgaki H. Mutational inactivation of the Nijmegen Breakage Syndrome gene (NBS1) in glioblastomas is associated with multiple TP53 mutations. J Neuropathol Exp Neurol 2009; 68: 210-215.
28. Yang M, Chiang W, Chou T, Chang S, Chen P, Teng S, Wu K. Increased NBS1 expression is a marker of aggressive head and neck cancer and overexpression of NBS1 contributes to transformation. Clin Cancer Res 2006; 12: 507-515.
29. Ziólkowska I, Mosor M, Wierzbicka M, Rydzanicz M, Pernak-Schwarz M, Nowak J. Increased risk of larynx cancer in heterozygous carriers of the I171V mutation of the NBS1 gene. Cancer Sci 2007; 98: 1701-1705.
30. Zhu XD, Kuster B, Mann M, Petrini JH, de Lange T. Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres. Nat Genet 2000; 25: 347-352.