Identification of novel chromosomal abnormalities and prognostic cytogenetics markers in intracranial pediatric ependymoma

Available online at www.sciencedirect.com

Cancer Letters 261 (2008) 235–243 www.elsevier.com/locate/canlet

Identification of novel chromosomal abnormalities and prognostic cytogenetics markers in intracranial pediatric ependymoma Annalisa Pezzolo a,*, Valeria Capra b, Alessandro Raso b, Fabio Morandi a, Federica Parodi a, Claudio Gambini c, Paolo Nozza c, Felice Giangaspero e, Armando Cama b, Vito Pistoia a, Maria Luisa Garre` b,d a

Laboratory of Oncology, Department of Laboratory and Experimental Medicine, Giannina Gaslini Institute, Genoa, Italy b Neurosurgery Unit, Department of Neurosurgery, Giannina Gaslini Institute, Genoa, Italy c Department of Pathology, Giannina Gaslini Institute, Genoa, Italy d Neuro-Oncology Unit, Department of Hematology-Oncology, Giannina Gaslini Institute, Genoa, Italy e Department of Experimental Medicine and Pathology, Universita` La Sapienza, Rome, Italy Received 25 July 2007; received in revised form 13 November 2007; accepted 14 November 2007

Abstract Aim of this study was to search for novel chromosomal imbalances and potential prognostic markers in pediatric ependymoma. Tumor DNA, obtained from 20 children with intracranial ependymoma (World Health Organization WHO grades II and III), was analyzed using metaphase-based comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH). The novel copy number aberrations (CNAs) here identified are (i) 4q33-qter loss, (ii) 10q25.2–q26.3 gain, (iii) 3q23-qter losses, (iv) 18q22.2 loss, and (v) 19p13.1–p13.3 gain. The combined presence of 6p22-pter and 13q14.3-qter losses predicted significantly reduced survival. Larger studies are warranted to validate these findings.  2007 Elsevier Ireland Ltd. All rights reserved. Keywords: CGH; Chromosomal imbalances; Ependymoma; FISH; Infant brain tumors

1. Introduction *

Corresponding author. Address: Laboratory of Oncology, Department of Laboratory and Experimental Medicine, Istituto Giannina Gaslini, Largo G. Gaslini, 5, 16147 Genova-Quarto, Italy. Tel.: +39 010 5636342; fax: +39 010 3779820. E-mail address: [email protected] (A. Pezzolo).

Ependymoma is the third most frequent pediatric CNS tumor, and one-third of cases affects children younger than 3 years [1]. In contrast to adult ependymoma that shows predominantly a spinal location, nearly 90% of pediatric ependymoma cases

0304-3835/$ - see front matter  2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2007.11.021

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develop intracranially, with most of them arising in infratentorial location [1]. The World Health Organization (WHO) classification distinguishes three major types of ependymal tumors: myxopapillary ependymoma and sub-ependymoma (WHO grade I), classic ependymoma (WHO grade II), and anaplastic ependymoma (WHO grade III) [2]. While WHO grade I tumors follow a benign course, grade II and III ependymoma show high propensity to recur [1–3]. Prognostic factors with an adverse impact on clinical outcome in pediatric ependymoma include young age at diagnosis [4–6] and inability to completely resect the primary tumor [7,8]. Younger patients (<3 years of age) with intracranial disease have a poorer outcome than older ones [5–7]. Although ependymoma displays numerous copy number aberrations (CNAs) as assessed by comparative genomic hybridization (CGH) [9–21], about 40% of pediatric ependymoma cases show balanced profiles [10,11,15] that have been significantly associated with young age [15]. Quite recently, combination of array CGH and gene expression profiling allowed to detect novel gains or losses in pediatric ependymoma, that correlated with modulation of the expression of genes involved in tumor development [21]. Since the genetic mechanism underlying pediatric ependymoma pathogenesis are still largely unknown, we have here investigated CNAs in primary ependymal tumors from 20 children homogeneously diagnosed, staged, and treated at a single Institution. These studies have been performed using metaphase-based CGH, a technique providing an unbiased screen of the entire genome through the use of a single in situ hybridization reaction [22]. Metaphase-based CGH, although less sensitive than array CGH [23], is highly informative pointing to chromosomal regions involved in tumor growth, cheaper and more easily applicable in many cytogenetics laboratories. 2. Patients and methods 2.1. Patient characteristics Tumor tissues from 20 patients with intracranial ependymoma were collected at the Giannina Gaslini Children’s Hospital in Genoa (Italy), from 1995 to 2003. These patients were consecutively admitted due to intracranial ependymoma and were homogeneously diagnosed, staged, treated, and followed at our Institution.

The histological diagnosis of ependymoma was formulated according to the criteria of the 2000 WHO classification [2]. Before inclusion in the study all ependymoma samples were centrally reviewed and diagnoses were confirmed by Dr. Felice Giangaspero, University La Sapienza, Rome, Italy. Tumors were classified as classic (WHO grade II) or anaplastic (WHO grade III) ependymoma. Patients were treated according to the Italian Association of Pediatric Emato-Oncology (AIEOP) protocol [1]. Their clinical–pathologic features are summarized in Table 1. 2.2. DNA extraction Tumor tissues and DNA were stored at 80 C in the brain tumor tissue bank at the Neurosurgery Laboratory. Specimens were treated according to the recommendations of the European Society of Human Genetics [24]. Genomic DNA was extracted from frozen tissues by using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma Chemical Co., St. Louis, USA) according to the manufacturer’s instructions. Tumor sections always contained higher than 80% tumor cells. 2.3. Comparative genomic hybridization (CGH) CGH was performed using directly fluorochromeconjugated DNA, as described previously [25]. Tumor and normal DNA were labeled by nick translation using Spectrum Green-dUTP (2 0 -deoxyuridine 5 0 -triphosphate) and Spectrum Red-dUTP (Vysis Inc., Downers Grove, IL, USA), respectively. Labeled test and reference DNA, together with Cot1 DNA (a fraction of DNA which contains a large number of highly repetitive sequences) (Roche, Mannheim, Germany), were prepared as previously described [25]. CGH preparations were analyzed with a Nikon Eclipse E1000 epifluorescence microscope (Nikon Corp., Tokyo, Japan) equipped with filter sets appropriate for DAPI (4 0 -6 0 -diamidino-2phenylindole), FITC (fluorescein isothiocyanate), and TRITC (tetramethyl rhodamine isothiocyanate). Three single-color images corresponding to counterstain, test DNA, and reference DNA were acquired through a high-sensitivity monochrome charge-coupled device (CCD) camera. Evaluation of CGH peak profiles was performed by Genikon software (Nikon Corp.). The standard thresholds for gains (1.20) and losses (0.80) were used [22]. Normal male vs. female hybridization was used as negative control. The MPE-600 breast cancer cell line with well-characterized chromosomal aberrations was used as positive control. 2.4. Fluorescent in situ hybridization (FISH) analysis Dual color interphase FISH was performed on cryosectioned tumor tissues according to the protocol previously described [25]. All experiments were performed on

Table 1 Clinical–pathologic characteristics of 20 intracranial ependymoma patients and chromosomal aberrations detected by CGH ID

Sex

Age (months)

Primary site/ recurrence

Surgery

Grade

Survival (months)

Outcome

Losses

Gains

129

ANED

9p22pter, 18p11.2pter, 20p12pter

1p32pter

m

24

IF/N

Complete

II

2

m

26

IF/local

Complete

II

28.5

DOC

-

1q32qter, 2p23pter, 2q36qter, 8q23.2qter, 10q24qter, 15, 17, 18, 21q22qter

3

m

35

IF/local + dissemin

Partial

II

16

DOD

6q12qter

1p34.3pter, 1q42.3, 4p16pter, 5q35.1, 6p11.2pter, 7q35, 8p23.1, 8q21.3, 9q33qter, 11p15.3pter, 11q23.3, 13q33, 22

4

m

33

IF/N

Complete

II

89

ANED

–

3q28, 4q35.1, 7q36.1, 8p23pter, 8q24.1qter, 9, 10q25qter, 11q22.2q23.1, 15, 16p12pter, 16q22qter

5

m

34

IF/local

Partial

II

31

DOD

1q42qter, 2p14p23.2, 4p14p15.2, 5q34q35.1, 9p24

4q33q35.1, 14q32.1, 20p11.23p12.3

6

m

33

IF/N

Complete

II

114

ANED

4p12p15.3, 4q12qter, 9p24

3p25, 5q34, 8q21.3, 9q21.2qter, 10q25.2q26.3

7

f

27

IF/local

Complete

II

163

ANED

1p21p31.2, 1q21qter, 2q33q37, 4q32.2qter, 9p13pter, 12p11.2p13.1

X

8

f

28

IF/N

Complete

II

80

ANED

1q31.2qter, 5, 6, 9p12pter, 15, 22

–

9

m

4

ST/local + dissemin

Complete

III

18

DOD

1p22p31.1, 3p21.3pter, 4, 5p14pter, 9p12pter, 11q14.2qter, 12q15q22, 13q13q22

2p23pter, 7p21.2pter, 17q24qter, 19, 22

10

m

31

IF/local

Complete

III

5

DOD

3, 6p23pter, 8q21.2q22, 13q14.3q21.2, 19q13.3qter, 22

–

11

m

48

IF/local + dissemin

Partial

III

79

DOD

6p22.2pter, 8p23pter, 10p11.2pter, 13, 17, 18q21.3qter, 21

–

12

m

75

IF/local

Complete

III

20

DOD

3q23qter, 6p22pter, 6q23qter, 9p21pter, 10q11.2qter, 13q22qter

1q21qter, 19

13

m

87

IF/local+ dissemin

Partial

II

71

DOD

1p13p34.3, 2, 3, 6, 10p12.2pter, 11, 12p11.2pter, 12q21, 13, 15q22qter, 18q11.2qter, 21, X

7

14

f

83

IF/N

Complete

II

158

ANED

2, 3, 7, 8, 10, 17, 21, 22

5, 12, 13, 20, X

15

m

117

IF/local

Complete

II

78

DOD

2, 6, 12q13.3q22, 13q22qter

19p13.1pter

16

f

131

ST/N

Complete

II

128

ANED

4q33qter, 6p22.2pter, 8p11.2pter, 11q23qter, 18q22.2, 21q22qter

–

17

m

50

IF/N

Complete

II

99

ANED

3p25pter, 6p22pter, 9p12pter, 12, 13q31qter, 18q21.3qter, 21, Xq26qter

–

18

m

47

IF/local

Complete

II

158

ANED

1p31pter, 1q24qter, 2, 3, 4, 5, 6q12qter, 7, 8p11.2pter, 11p13pter, 11q14qter, 13q21qter, 21

18p11.2, 19p13.1pter 237

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1

Abbreviations: m, male; f, female; IF, infratentorial location; ST, supratentorial location; N, none; ANED, alive no evidence of disease; DOD, dead of disease; DOC, dead of complications.

1p34.2pter, 19 1p13p34.1, 1q21qter, 2q21qter, 3, 4, 5, 6q15qter, 9p12pter, 11p11.2p15.1, 11q13q23.3, 12p12pter, 12q21q22, 13q14.1qter, 18 ANED II m 20

55

ST/N

Complete

116

1, 4, 7q11.2q22, 8, 11, 19, 20 3, 6 ANED 141 II Complete m 19

129

IF/N

Grade Age (months) Sex ID

Table 1 (continued)

Primary site/ recurrence

Surgery

Survival (months)

Gains Losses

A. Pezzolo et al. / Cancer Letters 261 (2008) 235–243

Outcome

238

preparations containing 80% tumor cells and 20% normal cells, as assessed by morphology and immunohistochemistry. The following BAC clones, selected on the basis of available physical maps (USC Human Genome Working Draft: http://genome.ucsc.edu; Ensemble http://www.ensemble.org) RP11-676K14 (6p25.3) and RP11-484I6 (13q33.1) were used as probes. BAC probe DNAs (kindly provided by Prof. M. Rocchi, University of Bari, Italy) were labeled with Spectrum Green deoxyuridine triphosphate (Vysis, Downers Grove, IL) by use of a Nick-Translation Kit (Vysis) according to the manufacturer’s instructions. In addition alphoid DNA specific for chromosome 6 and LSI (RB1) 13q14 probe direct-labeled with Spectrum Orange deoxyuridine triphosphate (Vysis) were used. A minimum of 100 nuclei were counted. The FISH probes were validated on normal control metaphase spreads to verify chromosomal localization. 2.5. Statistical analysis Data were analyzed using Fisher’s exact test (P values <0.05 were considered significant). Progression-free survival was measured from the date of diagnosis to the date of disease relapse. Observations were censored at the date of most recent control. Survival curve was constructed using the Kaplan–Meier method and P value was calculated with the use of the logrank test. 3. Results 3.1. Patient samples The cohort under study (Table 1) consisted of 20 patients all included in the AIEOP Italian protocol for ependymoma treatment, whose frozen primary tumor tissue was available. The frequency of genders (4/20 females = 20%) differed from the disease characteristics (approximately 50%), disease onset was age 3 years or younger in 10 of 20 patients, and tumor location was infratentorial in 17 of 20 patients. 3.2. CGH analysis of intracranical pediatric ependymomas Intracranial ependymomas were analyzed for CNAs by metaphase-based CGH, a standard method for genomewide screening [12,25–28]. The cumulative losses and gains detected by CGH in the 20 tumor samples investigated are summarized in Fig. 1A. All tumors exhibited multiple CNAs, and chromosomal abnormalities were identified in all chromosomes but the Y chromosome. In general, the total number of underrepresented regions observed in individual tumors exceeded that of overrepresented regions. The most common regions of overlap of detected genomic losses were

A. Pezzolo et al. / Cancer Letters 261 (2008) 235–243

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Fig. 1. CGH ideograms. Vertical red lines to the left of each ideogram indicate regions of DNA loss; vertical green lines to the right represent regions of DNA gain. Each line represents a chromosomal imbalance found in the individual tumors. The thick lines reflect the number of tumor, shown above the lines, with the same aberrations. (A) Summary of total chromosomal imbalances detected in 20 intracranial ependymoma samples. The most common regions of overlap of chromosomal imbalances involving chromosome arms were losses on 6p22.2-pter (9 cases), 9p24 (9 cases), 13q14.3-qter (9 cases), 3p25-pter (8 cases), 6q23-qter (8 cases), 3q23-qter (7 cases); gains on 19p13.1–p13.3 (6 cases), 1q42.3 (4 cases) and 1q32-qter (3 cases). (B) Summary of chromosomal imbalances in 10 intracranial ependymoma samples from patients younger than 3 years at diagnosis. The most frequent regions of overlap of chromosomal imbalances were losses on 9p24 (6 cases), 1q42-qter (3 cases), 4q32.2-qter (3 cases); gains on 9q33-qter (3 cases), and 10q25.2–q26.3 (3 cases). (C) Summary of chromosomal imbalances in 10 intracranial ependymoma samples from patients older than 3 years at diagnosis. The most frequent regions of overlap of altered chromosomes were losses on 6p22.2-pter (7 cases), 13q31-qter (7 cases), 3p25-pter (6 cases), 3q23qter (6 cases), 6q23-qter (6 cases), 21q22-qter (6 cases), 2q21-qter (5 cases), 18q 22.2 (5 cases), and gain on 19p13.1–p13.3 (5 cases).

found on chromosome 6p22.2-pter (9 cases), 9p24 (9 cases), 13q14.3-qter (9 cases), 3p25-pter (8 cases), 6q23qter (8 cases), 3q23-qter (7 cases), 2q21-qter (6 cases), 4q33-qter (6 cases), and 21q22-qter (6 cases). The most frequent regions of overlap of gains involved chromosome arms 19p13.1–p13.3 (6 cases), 1q42.3 (4 cases), and 1q32qter (3 cases). Interphase FISH analysis was performed on 6 tumor samples (cases no. 10, 11, 12, 13, 15, and 17) carrying the combined presence of 6p22-pter and 13q14.3-qter losses using specific BAC probes. In all experiments a minor fraction 20% of the analyzed cells exhibited a nor-

mal pattern of hybridization signals. These experiments confirmed the heterozygous deletions of 6p and 13q chromosomal regions detected by CGH (Fig. 2A). 3.3. Correlation between CGH results and clinical parameters We next stratified the 20 ependymoma patients according to the presence or absence of CNAs. Only those lesions detected in at least six patients (30%) were correlated to clinical outcome and patient age at diagnosis (Table 1).

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1)

2)

Cumulative proportion surviving (%)

OS 100

6p/13q losses association Single 6p and 13q loss or no 6p and 13q loss

80 60

P = 0.023

40 20 0 0

12 24 36 48 60 72 84 96 108 120 132 144 156 168 180

Months after surgery

Fig. 2. (A) Confirmation of CGH data by FISH analysis. (1) The tumor nuclei from case no. 17 (see Table 1) showed a heterozygous deletion of 6p represented by three centromere 6 signals (red spots) coupled with two 6p25.3 signals (green spots) using CEP6/BAC clone RP11-676K14 as FISH probes. (2) The tumor nuclei from case no. 17 showed a heterozygous deletion of 13q represented by two 13q14 signals (red spots) and one 13q33.1 signal (green spot) using LSI13 (RB1)/BAC clone RP11-484I6 as FISH probes. The nuclei were stained in blue by DAPI (magnification 100·). (B) Correlation of 6p/13q losses association with clinical outcome. Kaplan–Meier plot of estimated overall survival time distributions showing a significant shorter survival in patients with association of 6p22-pter and 13q14.3-qter loss than in patients with single CNA or no losses 6p and 13q. P value was calculated with the use of the log-rank test.

We further analyzed the relationship between tumor CNAs and patient age at diagnosis since patients younger than three years are characterized by worse clinical course and little information is available on the genetic differences underlying the whole spectrum of pediatric ependymomas. In addition, the issue of the relation between CNAs and disease outcome in the latter tumors has never been addressed. The number (n = 100) of total CNAs in 10 tumors from patients older than 3 years was greater than that (n = 87) in tumors from the 10 patients younger than 3 years. The pattern of chromosomal abnormalities according to patient age at diagnosis (<3/>3 years) allowed the identification of two separate genetic groups. The ideogram illustrating the regions and frequencies of alterations found in tumors from patients younger than 3 years is depicted in Fig. 1B, and the ideogram for tumors from patients older than 3 years is illustrated in Fig. 1C. In the group below three years, 71/87 tumor CNAs detected by CGH (81.6%) involved part of a chromosome or a chromosome arm, consistent with structural chromosomal rearrangements. Conversely, in tumors from children older than 3 years, 52 out of 100 CNAs (52%) were structural chromosomal rearrangements. The difference between the two groups of patients was statistically significant (P < 0.0001). Numerical CNAs represented 16/87 abnormalities (18%) in patients younger than three years and 48/100 (48%) in those older than 3 years (P < 0.0001). In the 10 tumors from children younger than three years, the most frequent regions of chromosomal imbalance overlap were losses on 9p24 (6 cases), 1q42-qter (3 cases), and gains on 9q33-qter (3 cases) and 10q25.2– q26.3 (3 cases) (Fig. 1B).

In the 10 tumors from children older than 3 years, the most frequent regions of chromosomal imbalance overlap were: losses on 6p22.2-pter (7 cases), 13q31-qter (7 cases), 3p25-pter (6 cases), 3q23-qter (6 cases), 6q23-qter (6 cases), 21q22-qter (6 cases), 2q21-qter (5 cases), 18q 22.2 (5 cases), and gain on 19p13.1–p13.3 (5 cases) (Fig. 1C). Thus, losses on chromosome 21q22-qter, 13q31-qter, and 18q22.2 were detected in older, but not in younger, patients (P = 0.0054, 0.0015, and 0.0163, respectively, one-tailed Fisher’s exact test, 90% CI). In contrast, gains on chromosomes 9q33-qter and 10q25.2–q26.3 were found in the younger group only. Loss on chromosome 9p24 occurred more frequently in younger than in older patients. Gain on chromosome 19p13.1–p13.3 was predominant in older patients (Table 2). Associations among CNAs occurring in at least 15% of tumors were absent below 3 years, whereas they were numerous in the older patient group (Table 3).

Table 2 Cytogenetics differences between tumors affecting younger and older patients (<3/>3 years) Chromosomal aberrations

Incidence <3 years %

Incidence >3 years %

P value

Loss 9p24 Gain 9q33-qter Gain 10q25.2–q26.3 Loss 13q31-qter Loss 18q22.2 Loss 21q22-qter Gain 19p13.1–p13.3

60 30 30 – – – 10

30 – – 70 50 60 50

0.1849 0.1053 0.1053 0.0015 0.0163 0.0054 0.0937

Abbreviations: %, percentage. One-tailed Fisher’s exact test was used to calculate P value (confidence interval 90%).

A. Pezzolo et al. / Cancer Letters 261 (2008) 235–243 Table 3 Association among frequent cytogenetics aberrations in 10 intracranial ependymoma that occurred in older patients (>3 years of age at diagnosis) Combination of chromosomal aberrations

Observed cases No.

Loss/loss Loss 6p22.2-pter/loss 13q14.3-qter Loss 3q23-qter/loss 3p25-pter Loss 3q23-qter/loss 6p22.2-pter Loss 3q23-qter/loss 13q31-qter Loss 3q23-qter/loss 6q23-qter Loss 6p22.2-pter/loss 6q23-qter Loss 6p22.2-pter/loss 21q22-qter Loss 6p22.2-pter/loss 18q22.2 Loss 3p25-pter/loss 13q31-qter Loss 3q23-qter/loss 2q21-qter Loss 13q31-qter/loss 18q22.2 Loss 13q31-qter/loss 2q21-qter Loss 3q23-qter/loss 21q22-qter Loss 6p22.2-pter/loss 3p25-pter Loss 3q23-qter/loss 21q22-qter Loss 2q21-qter/loss 18q22.2 Loss 3q23-qter/loss 18q22.2 Loss 6p22.2-pter/loss 2q21-qter

6 5 4 4 4 4 4 4 4 4 4 4 3 3 3 3 2 2

Loss/gain Loss 13q31-qter/gain 19p13.1–p13.3 Loss 3q23-qter/gain 19p13.1–p13.3 Loss 6p22.2-pter/gain 19p13.1–p13.3 Loss 2q21-qter/gain 19p13.1–p13.3

4 4 3 3

Abbreviations: No., absolute number of cases with combination of aberrations pattern represented.

Losses of 9p24, 3p25-pter, 6q23-qter, 3q23-qter, 2q21qter, 21q22-qter, and gain of 19p13.1–p13.3 were equally distributed in patients alive with no evidence of disease or in patients dead of disease (Table 1). Loss of 6p22-pter, detected in 45% of tumors (n = 9) and loss of 13q14.3qter, detected in 45% of tumors (n = 9), were more common in the group of patients with adverse clinical outcome (Table 1). When the presence or absence of 6p22pter or 13q14.3-qter loss was correlated with patient survival, neither CNA showed any statistical significance (Fig. 2B). Nonetheless, analysis of the impact of these CNAs tested in combination demonstrated that the presence of 6p22-pter and 13q14.3-qter losses predicted significantly reduced survival (P = 0.023) (Fig. 2B). Loss of 4q33-qter, detected in 30% of tumors (n = 6), was more common in patient with favorable prognosis (Table 1). Therefore we investigated whether this CNA correlated with survival; indeed, the presence of 4q33-qter loss did not predict a longer survival.

4. Discussion Using metaphase-based CGH we have here identified novel CNAs and new prognostic cytogenetics

241

markers in pediatric intracranial ependymoma. These results were obtained from the study of twenty consecutive patients who were homogeneously diagnosed, staged, treated, and followed at a single Institution. The female to male ratio in our study was lower than that reported in historical series [2]. Whether or not this ratio may represent a potential source of bias is unknown. Although more powerful techniques, such as CGH array [21], can identify CNAs that escape conventional CGH detection, this study provides proof of the concept that a relatively easy and cheap technique such as metaphase-based CGH still represents an useful tool in clinical oncology. Remarkable cytogenetics differences in ependymoma occurring in patients younger vs. older than three years were identified. At variance with a previous study that identified a balanced CGH profile in all ependymomas below three years of age analyzed [15], we found that this group of patients was characterized by structural chromosome rearrangements. In contrast, patients older than three years displayed both structural and numerical rearrangements, as well as a higher number of chromosomal imbalances (see Fig. 1B and C). These results suggest that chromosomal instability may be higher in tumors from patients older than three years and account for the higher number of CNAs detected in this group. The novel CNAs here identified are the following: (i) 4q33-qter loss, (ii) 10q25.2–q26.3 gain, (iii) 3q23-qter losses, (iv) 18q22.2 loss, and (v) 19p13.1–p13.3 gain. Among them, 10q25.2–q26.3 gain was detected exclusively in patients younger than three years, whereas 18q22.2 loss was significantly associated with patients over three years. Furthermore, 21q22-qter was an exclusive marker of the latter group. In the attempt to establish the clinical impact of newly discovered and previously published CNAs, those present in at least six out of 20 patients were selected for further studies without taking into consideration age. This decision, dictated by the limited number of cases studied, did not allow to analyze separately the prognostic relevance of CNAs in patients younger and older than three years, respectively. Additional CNAs with prognostic value were found to be 6p22-pter and 13q14.3-qter losses. Recurrent losses of 13q (9–12, 15) and 6p (9–12, 15) have been reported in other studies with similar frequencies. Either 6p22-pter or 13q14.3-qter loss

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tested individually did not display any correlation with patient survival. However, the combined presence of 6p22-pter and 13q14.3-qter losses predicted significantly reduced survival. In this study, 1q gain, whose role in predicting unfavorable clinical outcome of mixed populations of adult and pediatric ependymoma patients has been established [10,15,19], was detected in few cases (4/20) and therefore excluded from prognostic analyses. Likewise, chromosome 22 loss was found only in 3/20 patients. A possible explanation for this latter finding is that chromosome 22 loss occurs usually in adult patients or in pediatric patients with spinal ependymoma [29], that were not included in the study. In conclusion, clinical correlation of the newly discovered CNAs in intracranial ependymoma patients, although promising, warrant further validation in larger series of cases. Acknowledgements This study was supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC, Inter-Regional grant on Pediatric Oncology 2005– 2007) and Associazione per la Ricerca sui Tumori Cerebrali del Bambino. References [1] M. Massimino, L. Gandola, F. Giangaspero, et al., Hyperfractionated radiotherapy and chemotherapy for childhood ependymoma: final results of the first prospective AIEOP (Associazione Italiana di Ematologia-Oncologia Pediatrica) study, Int. J. Radiat. Oncol. Biol. Phys. 58 (2004) 1336–1345. [2] O.D. Wiestler, D. Schiffer, S.W. Coons, et al., Ependymal tumors, in: P. Kleihues, W.K. Cavence (Eds.), World Health Organization (WHO) Classification of Tumors: Pathology and Genetics of Tumors of the Nervous System, IARC Press, Lyon, 2000, pp. 71–82. [3] C. Teo, P. Nakaji, P. Symons, et al., Ependymoma, Childs Nerv. Syst. 19 (2003) 270–285. [4] S.J. Mork, A.C. Loken, Ependymoma: a follow-up study of 101 cases, Cancer 40 (1997) 907–915. [5] T.E. Merchant, Current management of childhood ependymoma, Oncology (Huntingt) 16 (2002) 629–644. [6] I.F. Pollack, P.C. Gerszten, A.J. Martinez, et al., Intracranial ependymomas of childhood: long term outcome and prognostic factors, Neurosurgery 37 (1995) 655–666. [7] A.C. Paulino, Radiotherapeutic management of intracranial ependymoma, Pediatr. Hematol. Oncol. 19 (2002) 295–308. [8] P.L. Robertson, P.M. Zelrzer, J.M. Boyett, et al., Survival and prognostic factors following radiation therapy and chemotherapy for ependymomas in children: a report of the Children’s Cancer Group, J. Neurosurg. 88 (1998) 695– 703.

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