CLINICAL STUDY

Study of chromosome 9q gain, Notch pathway regulatorsand Tenascin-C in ependymomas

Rakesh Kumar Gupta • Mehar C. Sharma •

Vaishali Suri • Aanchal Kakkar • Manmohan Singh •

Chitra Sarkar

Received: 9 July 2013 / Accepted: 21 October 2013 / Published online: 1 November 2013

� Springer Science+Business Media New York 2013

Abstract Ependymomas are relatively uncommon

tumours of the central nervous system which arise from the

ependymal lining of the ventricles and spinal canal. The

molecular changes leading to ependymal oncogenesis are

not completely understood. We examined chromosome

9q33-34 locus for gain, potential oncogenes at this locus

(Notch-1 and Tenascin-C) and Notch pathway target genes

(Hes-1, Hey-2 & C-myc) in ependymomas by fluorescent

in situ hybridization (FISH) and immunohistochemistry

(IHC), respectively, to assess if they have any correlation

with clinical characteristics. We analyzed 50 cases of

ependymomas by FISH for 9q gain and by IHC for Notch-1

and its target gene proteins (Hes-1, Hey-2 and C-myc)

expression. We also performed IHC for Tenascin-C to rule

out any correlation with aggressiveness/grade of tumour.

FISH study revealed significant chromosome 9q gain in

ependymomas of adult onset (age [ 18 years) and spinal

cord origin. Notch-1 showed significantly more frequent

immunohistochemical expression in supratentorial and

anaplastic ependymomas. Tenascin-C (TN-C) expression

was significant in intracranial, childhood (age B 18 years)

and anaplastic ependymomas. Of the three Notch pathway

target gene proteins (Hes-1, Hey-2 and C-myc), Hes-1 and

C-myc expression showed significant correlation with ana-

plastic and adult onset ependymomas, respectively. Genetic

alterations are independent prognostic markers in

ependymomas. A clinicopathological correlation with vari-

ous molecular signatures may be helpful in the development

of new therapeutic targets.

Keywords Ependymoma � FISH � IHC �Chromosome 9q � Notch-1

Introduction

Ependymomas are glial tumours which originate from the

ependymal lining of the ventricles and central canal of the

spinal cord [1]. They are the third most common paediatric

central nervous system tumours accounting for 6–12 % of all

intracranial tumours in children [2–4]. The prognosis of

childhood ependymomas is relatively poor, with 5-year

overall survival (OS) and progression-free survival (PFS)

rates ranging between 50–60 and 30–50 %, respectively

[2, 4]. A large meta-analysis on 2,408 ependymoma patients

identified WHO grade as an independent prognostic factor

[5], whereas some studies suggested grading is a matter of

subjectivity [4, 6–9]. More than 50 % of cases occur in

children under 5 years of age, which is a potential hurdle for

the use of radiotherapy because of its long term complica-

tions, and the beneficial role of chemotherapy is controver-

sial [2, 6, 10–13]. Patient outcome prediction is difficult

because of heterogeneity in clinical behavior and molecular

changes, and the results of existing studies on prognostic

markers are contradictory [14, 15]. Therefore, there is need

to improve the understanding of the biology of ependymo-

mas in order to develop new therapeutic targets and agents.

Previous studies have demonstrated the role of Notch

pathway in the pathogenesis of central nervous system

tumours including glioblastoma multiforme and medullo-

blastoma, and in several non-neural tumours [16–19].

R. K. Gupta � M. C. Sharma (&) � V. Suri � A. Kakkar �C. Sarkar

Department of Pathology, All India Institute of Medical

Sciences, New Delhi 110029, India

e-mail: sharmamehar@yahoo.co.in

M. Singh

Department of Neurosurgery, All India Institute of Medical

Sciences, New Delhi 110029, India

123

J Neurooncol (2014) 116:267–274

DOI 10.1007/s11060-013-1287-z

However, not much literature is available on the involve-

ment of this pathway in ependymomas. Therefore, we

undertook this study to analyze the expression of Notch

and its target genes in ependymomas.

Materials and methods

We retrieved 50 paraffin embedded tissue blocks of cases

diagnosed as ependymoma in the Department of Pathology

during a period of 10 years (2002–2011). Diagnoses were

confirmed by histopathological assessment of H & E

stained slides by three neuropathologists (RKG, MCS, CS),

according to the 2007 WHO classification of CNS tumours

[1]. Cases with sufficient material in tissue blocks and

diagnostic concurrence were included in the study.

Tumor tissue had been received in neutral buffered

formalin and was routinely processed and paraffin

embedded. Five micron thick sections were cut for hema-

toxylin & eosin (H&E) staining and for immunohisto-

chemistry. Immunohistochemical studies were performed

using antibodies directed against Notch-1(dilution, 1:200,

AntiNotch-1, EP1238Y, Abcam, USA), Tenascin-C (dilu-

tion, 1:200, Anti Tenascin-C, MAB19101, Millipore,

USA), Hes-1(dilution, 1:200, Anti Hes-1, ab71559, Ab-

cam, USA), Hey-2 (dilution, 1:25, Anti Hey-2,

HPA030205, Sigma, USA) and C-myc (dilution, 1:100,

Anti C-myc, 9E10, Santa Cruz, USA). Labeled streptavidin

biotin kit Universal (Dako, Denmark) was used as a

detection system. Antigen retrieval was performed in a

microwave oven using citrate buffer at pH 6.0 for all

antibodies except Tenascin-C, for which Trypsin digestion

(TA-125-TR, Thermo Fisher Scientific, UK) was done. For

each batch, appropriate positive and negative controls were

taken. Immunohistochemistry with all antibodies was

interpreted as follows: 0 % - grade 0; 1–25 % - grade 1;

26–50 % - grade 2; [50 % - grade 3.

Fluorescent in situ hybridization analysis

FISH study was performed on 50 paraffin embedded tissue

samples. Chromosome 9q was studied using a centromeric

probe (CEP9) and dual colour, dual fusion bcr/abl trans-

location probe for 9q33-34 locus (M/S Vysis, Inc., Abbott

Laboratories SA, Downers Grove, IL, USA). Sections were

deparaffinized by immersing in xylene thrice for 10-min

each, followed by two 3-min immersions in 100 % ethanol.

Following rinsing in water, target retrieval was achieved

using citrate buffer, pH 6.0 and boiling in a microwave for

20 min. Slides were exposed to 0.04 % pepsin (P-7000;

Sigma- Aldrich, St. Louis, MO) digestion for 30 min at

37 �C, fixed and dehydrated. Probe mixture (10 ll per

slide) was applied on each section. Simultaneous probe/

specimen denaturation at 73 �C for 5 min with subsequent

overnight incubation at 37 �C was performed in Ther-

mobrite TM hybridization chamber (Vysis Inc). The sec-

tions were washed the next day in 2X SSC (2 min at 73 �C)

followed by 0.5X SSC (2 min at room temperature) and

counterstained with 4,6-diamidino-2-phenylindole (Vysis,

Inc) and visualized under a fluorescent microscope. Signals

were scored at least in 200 non-overlapping, intact nuclei

and the number of test (orange) and control (aqua) signals

was noted. Nuclei with two test and two control signals

were regarded as normal. Cases with [10 % nuclei hav-

ing [3 test signals in comparison to the control signals

were considered as demonstrating gain of 9q33-34. This

cut-off value of 10 % was decided based on study in nor-

mal brain samples from intractable epilepsy cases.

Statistical analysis

Relationships among variables were assessed using stan-

dard statistical techniques: Fisher’s exact test and Chi

square test. We considered a relationship significant at a

p value less than 0.05.

Results

Clinicopathologic parameters (Table 1)

The 50 tumour samples were comprised of 44 % (22/50)

children and 56 % (28/50) adults. Male to female ratio

was 2:1. In the pediatric group (B18 years), the age

ranged from 2 to 18 years with mean age of 9.1 years,

while in the adult group ([18 years) the age ranged from

19 to 62 years with mean age of 35.9 years. Out of 50

cases, 12 % (6 cases), 52 % (26 cases) and 36 % (18

cases) were of grade I, grade II and grade III, respec-

tively. Site distribution of the cases were 30 % (15 cases)

supratentorial, 32 % (16 cases) infratentorial and 38 %

(19 cases) intraspinal.

Chromosomal 9q33-34 gain in ependymomas

Chromosome 9q gain (Fig. 1) was identified in 48 %

(24/50) of patients, of which 37.5 % (9/24) were intracra-

nial and 62.5 % (15/24) were intraspinal (Fig. 2). This

difference was statistically significant (p = 0.005). Of the

24 patients who showed 9q33-34 gain, 29.1 % (7 cases)

were children and 70.8 % (17 cases) were adults, and this

difference was also statistically significant (p = 0.03).

Although correlation between chromosome 9q gain and

tumour grade was not statistically significant, it showed a

decreasing trend with increase in tumour grade.

268 J Neurooncol (2014) 116:267–274

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Table 1 Clinicopathological features of the cases with their FISH status

Sample Age (years) Sex Site Surgery Grade (WHO) OS (months) Status FISH 9q gain

1 46 F IS GTR I 30 – Yes

5 41 M IS SR I 4 – Yes

30 55 F IS SR I 22.2 ADF Yes

38 21 M IS SR I 12 ADF Yes

49 9 M IS GTR I – – No

50 17 M ST SR I 6 ADF No

4 60 F PF GTR II 2 DOD Yes

7 4 M PF SR II – – No

8 19 M PF SR II 8.2 – No

10 33 F PF SR II 21.2 ADF No

11 35 M IS GTR II 33.5 ADF Yes

12 52 M IS SR II 27.3 ADF Yes

13 2 M PF GTR II 3 – No

15 44 M IS GTR II – – No

16 45 F IS GTR II 15.8 ADF No

17 22 M IS GTR II 24 ADF No

20 5 M PF SR II 3.5 – No

21 9 M PF SR II 35 ADF No

22 23 M IS GTR II – – No

23 18 M PF GTR II 4 – Yes

24 10 M ST GTR II 9.4 ADF Yes

25 25 F IS GTR II 14 ADF Yes

28 35 M IS GTR II – – Yes

31 12 M PF GTR II 3 ADF Yes

32 33 F IS GTR II 2.7 ADF Yes

34 22 M IS GTR II – – Yes

35 30 M IS GTR II 15 ADF Yes

37 30 M IS GTR II 16.5 ADF Yes

39 34 M IS GTR II – – Yes

42 9 M PF GTR II 22.5 ADF No

44 7 F ST SR II 10.6 ADF No

46 13 M ST SR II 20.8 ADF No

2 60 M ST GTR III – – No

3 35 M PF GTR III – – Yes

6 22 F ST GTR III – – No

9 30 M ST GTR III 23.6 ADF No

14 8 F ST GTR III – – No

18 28 M ST SR III – – No

19 1 F PF GTR III 15.5 ADF No

26 10 F ST GTR III 13.3 ADF Yes

27 10 F IS GTR III 23.4 ADF Yes

29 4 M PF SR III 22.4 ADF Yes

33 62 F PF GTR III 17.3 ADF Yes

36 12 M ST GTR III 18 ADF Yes

40 2 M PF SR III 2 DOD No

41 24 M PF SR III 8.5 – No

43 40 M ST SR III 13.4 ADF No

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Immunohistochemical analysis of candidate oncogenes

and target genes (Table 2; Fig. 3)

Notch-1 showed significantly higher immunoexpression in

supratentorial tumours in comparison to infratentorial

(p = 0.001) and intraspinal (p = 0.01) tumours. However,

this difference was not statistically significant between

intracranial (supratentorial & infratentorial) and intraspinal

tumours. Immunoexpression of TN-C was significantly

higher in intracranial tumours in comparison to intraspinal

tumours (p = 0.05). Notch-1, TN-C and Hes-1 showed a

significantly higher expression in grade III tumours in

comparison to grade II tumours with a p value of 0.001,

0.006 and 0.02, respectively. Notch-1 showed increased

expression with tumour grade and the difference between

grade I & grade III tumours was statistically significant

(p = 0.004). TN-C immunoexpression was seen more

commonly in childhood tumours in comparison to adults

(p = 0.003). C-myc immunoexpression was contrary to

Fig. 1 Representative fluorescent in situ hybridization (FISH) show-

ing gain of 9q33-34 (red signals) as compared to control blue signals

(CEP9). Green signals represent chromosome 22 ‘BCR’ region and

are not part of this study

Fig. 2 Flowchart showing

clinicopathological correlation

of the cases with 9q33-34 gain

Table 1 continued

Sample Age (years) Sex Site Surgery Grade (WHO) OS (months) Status FISH 9q gain

45 9 F ST GTR III 17.5 ADF No

47 3 F ST GTR III 17 ADF No

48 12 M ST GTR III – – No

M male, F female, IS intraspinal, PF posterior fossa, ST supratentorial, GTR gross total resection, SR subtotal resection, ADF alive disease free,

DOD died of disease

270 J Neurooncol (2014) 116:267–274

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TN-C expression and more commonly seen in tumours

which occurred in adults as compared to childhood

tumours (p = 0.03). None of the oncogenes (Notch-1 and

TN-C) and target genes (Hes-1, Hey-2 & C-myc) showed

correlation with chromosome 9q gain.

Discussion

Several cytogenetic and molecular studies have been pub-

lished, highlighting the chromosomal and molecular alter-

ations in ependymomas, but have failed to show specific

target genes involved in ependymal oncogenesis, unlike

astrocytic gliomas. This failure of demonstrating specific

molecular changes proved to be a hindrance in developing

specific targeted therapies in ependymomas. These tumours

frequently occur in young children, and the usual chemo-

therapeutic agents and radiotherapy have high toxicity,

resulting in long term complications. Therefore, this

necessitates the urgent unraveling of molecular alterations

in ependymomas to discover newer therapeutic agents.

In the last decade, several authors have studied cyto-

genetic changes in ependymomas either by conventional

comparative genomic hybridization (CGH) on metaphase

chromosomes, karyotyping or array based expression pro-

filing CGH [20–32]. A few studies have shown gain of 9q

in ependymomas [20–24, 26]. Reardon et al. [20] studied

32 cases of ependymomas by CGH and found either gain of

chromosome 1q or 9. Hirose et al. [21] found that gains of

1q and loss of 9 were preferentially associated with grade III

ependymomas. None of their cases showed 9q gain. Jeuken

et al. [24] recorded 9q gain more commonly in posterior

fossa ependymomas and in adult patients. Rousseau et al.

[31] have shown that combination of chromosome 9q gain

in association with other chromosomal gains and losses is

more frequently seen in spinal cord grade II and myxo-

papillary grade I ependymomas. However, the exact sig-

nificance of this gain was not demonstrated in these studies.

To date, only a single study has examined 9q gain along

with expression of oncogenes identified at this locus, and

their downstream targets. Puget et al. performed bacterial

artificial chromosome (BAC) based array-comparative

genomic hybridization(aCGH) on 59 paediatric ependy-

moma samples and found that gain of chromosome 9q is

frequently associated with relapse, age older than 3 years

and posterior fossa location. This gain was further localized

at 9q33-34 region by FISH. Analysis of various genes at

this locus confirmed mutations of TN-C and Notch-1 genes

located in this region [28]. These authors have shown that

Notch pathway activation leads to overexpression of target

genes Hes-1, Hey-2 and C-myc which conferred a growth

advantage on the tumour cells. In our study, we identified

chromosome 9q gain by FISH in a significant proportion of

ependymomas (48 %). We also found a significantly higher

percentage of chromosome 9q33-34 gain in spinal cord

ependymomas, including myxopapillary. Our results are

similar to previous studies, [21, 22, 31, 33] but contrary to

that of Puget et al. [30]. In the present series, chromosome

9q gain was observed more commonly in adult patients

(p = 0.03), as shown in earlier studies [20, 22, 34]. There

was no significant difference in frequency of 9q gain in

different tumour grades, however there was a decreasing

trend in 9q gain with increase in tumour grade, and these

results are in concordance with published studies [32, 35].

We identified significant upregulation of Notch-1

expression in supratentorial tumours, similar to previous

studies [28–30, 36]. Modena et al. [28] in 2006, showed

overexpression of Notch pathway proteins in supratentorial

ependymomas. TN-C, which is involved in central nervous

system embryogenesis, expression was significantly more

frequent in ependymomas which were located intracrani-

ally, as well as in childhood tumours, and these results

were in concordance to earlier studies [28, 30]. Overex-

pression of TN-C has been shown in many glial tumours,

and this expression has been shown to be associated with

increased vascularity, poor prognosis and relapse [37, 38].

In the present series, higher expression of TN-C, Notch-1,

as well as its target gene Hes-1 was observed in anaplastic

ependymomas (grade III) in comparison to grade I and

grade II. These results were similar to the observation

made by Puget et al. [30]. The discordance between

chromosome 9q gain and Notch-1 & TN-C oncogenes

overexpression at different sites may be due to activating or

inactivating mutations in these genes [30].

Table 2 Correlation of various gene proteins expression with age, sex and grade of the tumour

Markers No. of positive

cases (n = 50)

Age Site Grade

Children Adults ST PF IS I II III

TNC 16 (32 %) 12 (75.0 %) 4 (25.0 %) 5 (31.2 %) 8 (50.0 %) 3 (18.8 %) 3 (18.8 %) 5 (31.2 %) 8 (50.0 %)

Notch1 18 (36.0 %) 7 (38.8 %) 11 (61.1 %) 11 (61.1 %) 2 (11.1 %) 5 (27.7 %) 1 (5.5 %) 5 (27.7 %) 12 (66.6 %)

HES1 20 (40.0 %) 7 (35.0 %) 13 (65.0 %) 3 (15.0 %) 7 (35.0 %) 10 (50.0 %) 3 (15.0 %) 14 (70.0 %) 3 (15.0 %)

HEY2 31 (62.0 %) 12 (38.7 %) 19 (61.2 %) 10 (32.2 %) 10 (32.2 %) 11 (35.4 %) 2 (6.4 %) 16 (51.6 %) 13 (41.9 %)

c-myc 26 (52.0 %) 5 (19.2 %) 21 (80.7 %) 7 (26.9 %) 7 (26.9 %) 12 (46.1 %) 4 (15.3 %) 14 (53.8 %) 8 (30.6 %)

J Neurooncol (2014) 116:267–274 271

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272 J Neurooncol (2014) 116:267–274

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To summarize, we found a significantly higher expres-

sion of Notch-1 and TN-C in supratentorial and intracranial

(both supratentorial & posterior fossa) tumours, respec-

tively, as well as in anaplastic ependymomas. These

markers can help in risk stratification of ependymomas and

may further serve as targets for novel therapeutic agents.

Immunotherapy using radiolabelled anti TN-C antibodies

has shown promising results in hematological malignancies

and brain tumours [39, 40]. c-Secretase (a Notch pathway

enzyme) inhibitors might be an important therapeutic

option in future for supratentorial ependymomas [30].

Ependymomas can be categorized based on their unique

molecular profiles at different site, grade and age groups;

accordingly target oriented therapeutic interventions might

be developed in near future.

Acknowledgments We would like to acknowledge Mr Sujit for

help in performing FISH studies, Mr Pankaj & Mrs Kiran for helping

in immmunohistochemistry. Our special thanks to Mr Guresh for

statistical analysis.

Conflict of interest None.

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