Clinicopathological Features and Molecular Analysis of Primary Glioblastomas in Moroccan Patients

Clinicopathological Features and Molecular Analysis of Primary Glioblastomas in Moroccan Patients

Said Hilmani_&Omar Abidi_&Houda Benrahma_&

Mehdi Karkouri_&Souha Sahraoui_&

Abdessamad El Azhari&Abdelhamid Barakat

Received: 3 July 2012 / Accepted: 25 July 2012 / Published online: 4 August 2012

#Springer Science+Business Media, LLC 2012

Abstract

Glioblastoma is the most frequent and most ag- gressive primary brain tumor. Primary and secondary glio- blastomas develop through different genetic pathways. The aim of this study was to determinate the genetic and clinical features of primary glioblastoma in Moroccan patients. The blood and tumor samples were obtained from a group of 34 Moroccan patients affected with primary glioblastoma. The tumors were investigated for

TP53

,

IDH1

, and

IDH2

muta- tions using PCR sequencing analysis. Clinicopathological data showed that the mean age at diagnosis of patients was 50.06 years, the sex ratio was 11 F/23 M, and the median of Karnofsky performance score was 60. About 18 % of patients were initially treated by total tumor resection, 41 % by subtotal, and 38 % by partial resection, but biopsy was performed for a single patient (3 %). Twenty-five patients (74 %) received radiotherapy. In addition, the

median survival of the all patients was 13 months following diagnosis. There was a significant impact of higher Karnof- sky performance score (KPS) (≥80) on overall survival,

p-

log-rank test

0

0.0002, whereas other parameters did not show any significant differences. The molecular analysis revealed

TP53

mutations in 3/34 (8.82 %) cases; R273H, R306X, and Q136X. However, none of the analyzed sam- ples contained the R132-

IDH1

or R172-

IDH2

mutations.

These results showed the absence of

IDH1

mutation in primary glioblastoma, confirming that this mutation is a hallmark of secondary glioblastoma. It can be used to dis- tinguish primary from secondary glioblastomas. We found also that higher KPS was a significantly favorable factor in patients with primary glioblastoma.

Keywords

Primary glioblastoma .

TP53

.

IDH1

and

IDH2

. Clinical features . Prognosis impact . Morocco

Introduction

Glioblastoma (GBM) is the most common and most aggres- sive primary brain tumor that, despite current therapies, the median survival continues to be approximately 12 months following diagnosis (Fine 1994). GBM may be primary (de novo), in 95 % of cases, when develop rapidly after a short clinical history and without evidence of a less malignant precursor or secondary, in 5 % of cases, when develop slowly through progression from low-grade diffuse or ana- plastic astrocytoma (Ohgaki et al. 2004; Ohgaki and Kleihues 2005, 2007, 2009). Primary (pGBM) and secondary (sGBM) glioblastomas develop through different genetic pathways but histologically are largely indistinguishable, showing the mor- phological criteria of anaplasia, astrocytic tumor cells, prom- inent microvascular proliferation, and necrosis (Kleihues and Ohgaki 1997; Louis et al. 2007; Ohgaki and Kleihues 2009).

Said Hilmani and Omar Abidi contributed equally to this work.

O. Abidi (*)

:

H. Benrahma

:

A. Barakat

Laboratoire de Génétique Moléculaire Humaine, Département de la Recherche Scientifique, Institut Pasteur du Maroc,

1 Place Louis Pasteur, 20360, Casablanca, Morocco e-mail: omar.abidi@pasteur.ma S. Hilmani

:

A. El Azhari Service de Neurochirurgie,

Centre Hospitalier Universitaire Ibn Rochd, Casablanca, Morocco

M. Karkouri

Laboratoire d’Anatomopathologie, Centre Hospitalier Universitaire Ibn Rochd, Casablanca, Morocco

S. Sahraoui

Centre d’Oncologie et Radiothérapie, Centre Hospitalier Universitaire Ibn Rochd, Casablanca, Morocco

At the molecular level, these tumors are the subject of intense investigation. Several genes, including

TP53, IDH1, and IDH2

, are altered in GBM (Bleeker et al. 2009; Nobusawa et al. 2009; Sonoda et al. 2009b; Yan et al. 2009).

The

TP53

gene, the most commonly mutated gene in human cancers, is a tumor suppressor gene located on the short arm of human chromosome 17 (17p13) (Nayak et al.

2004). The normal

TP53

gene encodes a transcription factor that responds to several forms of cellular stress such as DNA damage, hypoxia, heat shock, metabolic changes, certain cytokines, and multiple antiproliferative functions (Vogelstein et al. 2000; Birner et al. 2002). More than 65 % of secondary glioblastomas display

TP53

mutations, while the rate of these mutations was significantly less frequent in primary glioblas- tomas (<28 %) (Ohgaki 2005; Ohgaki and Kleihues 2009).

On the other hand, mutations of isocitrate dehydrogenase 1 gene (IDH1) were preferentially found in tumors harbor- ing

TP53

mutations (Nobusawa et al. 2009). The

IDH1

gene, located on chromosomal arm 2q33.3, encodes cyto- solic NADP+

−specific IDH and catalyzes the cytosolic

oxidative decarboxylation of isocitrate to

α-ketoglutarate,

resulting in the production of reduced form of NADP + (NADPH) (Narahara et al. 1985). The somatic mutations of

IDH1

gene were identified in approximately 12 % of glioblastomas (Parsons et al. 2008). These mutations are frequent in secondary GBM (>80 %), but absent or rarely detected in primary GBM (<10 %) (Balss et al. 2008;

Hartmann et al. 2009; Ichimura et al. 2009; Nobusawa et al. 2009; Sanson et al. 2009; Watanabe et al. 2009; Yan et al.

2009). They always affect arginine in position 132 in the amino acid sequence, which belongs to an evolutionarily highly conserved region located at the binding site for iso- citrate (Balss et al. 2008; Hartmann et al. 2009; Ichimura et al. 2009; Sanson et al. 2009; Yan et al. 2009). These muta- tions were found to be associated with reduced NADP +

−dependent IDH activity in GBM (Bleeker et al.

2010).

Moreover, it was reported that GBM without

IDH1

muta- tions often have mutations affecting

IDH2

gene (Sonoda et al. 2009a; Yan et al. 2009). This gene, localized in the mitochondria, plays a key role in the regulation of the tricarboxylic acid cycle and, like

IDH1, has been shown to

have a protective role against insults such as oxidative stress (Lee et al. 2004; Reitman and Yan 2010).

IDH2

gene has been reported to be mutated at codon R172, an exact ana- logue of the R132 residue in

IDH1, in 4 to 8 % of low-grade

and anaplastic astrocytic and oligodendroglial gliomas, but in none of the 138 primary or 13 secondary glioblastomas analyzed (Xu et al. 2004; Yan et al. 2009).

In this study, we screened 34 glioblastoma samples look- ing for

TP53

,

IDH1

, and

IDH2

genes and present the epi- demiologic and clinical findings. We investigate also the relationship between overall survival and clinicopathological characteristics.

Materials and Methods

Patients

Thirty-four Moroccan patients with GBM were recruited be- tween January 2009 and December 2011 at the Department of Neurosurgery, Ibn Rochd University Hospital Center of Casa- blanca. All cases were newly diagnosed and the tumors were considered as primary glioblastoma according to the WHO classification of brain tumors (Louis et al. 2007). The Karnof- sky performance score (KPS) was calculated at the time of diagnosis. The patients underwent surgical removal of the tumor followed by radiotherapy, but none of the patients had received chemotherapy. The resection rate was determined by postoperative MRI. Survival times were calculated from the day of surgery of glioblastoma to the day of death, in months.

Specimens and Preparation

Blood samples and fresh–frozen tissues were obtained from all patients with microsurgical tumor resection, and clinical follow-up information was collected, in accordance with the Declaration of Helsinki. Clinical details, including the patient’s age at the time of diagnosis, gender, preoperative KPS score, extent of resection, size of tumor, clinical history, and the recorded date of death were noted. This study was approved by the local ethics committee. Informed consent for use of their samples was obtained from all study subjects.

Molecular Analysis

DNA was extracted from frozen tumor tissues and blood samples according to standard phenol/chloroform protocol (Sambrook et al. 1989). The screening of

TP53

gene was carried out by sequencing using a set of previously pub- lished primers for exons 4 to10 (Petitjean et al. 2007). PCR was done in a total volume of 15

μL, consisting of 25–50 ng

of genomic DNA, 3 pmol of each primer, and 7.5

μL of

master mix 2× (AmpliTaq Gold 360 Master Mix; Applied Biosystems, Foster City, CA). PCR conditions were as follows: 94 °C for 5 min; 35 cycles at 94 °C for 40 s, 58 °C for 45 s, 72 °C for 50 s, and 72 °C for 7 min. PCR products were purified using Exonuclease/Shrimp Phosphatase Alka- line method (ExoSAP-IT PCR Purification Kit, GE Health- care, UK) and sequenced using the Big Dye V3.1 ready reaction kit (Applied Biosystems, Foster City, CA, USA).

The capillary electrophoresis was performed on an ABI 3130 Genetic Analyzer model.

The analysis of the

IDH1

and

IDH2

genes was also

performed by automated sequencing. We screened for the

R132 and R172 mutations in the

IDH1

and

IDH2

genes,

respectively, by sequencing exon 4 (encoding the catalytic

domain) of these genes. PCR reactions were performed

using the following primers: forward 5′-AATGAGCTCTA TATGCCATCACTG-3′ and reverse 5′-TTCATACCTTGCT TAATGGGTGT-3

′

for

IDH1

(Bleeker et al. 2009); forward 5

′

TGCACTCTAGACTCTACTGCC-3′ and reverse 5′

ACAAAGTCTGTGGCCTTGTAC-3′ for

IDH2

(Murugan et al. 2010). PCR conditions for

IDH1

and

IDH2

genes were as follows: 95 °C for 5 min; 35 cycles of 94 °C for 35 s, 58 °C for 40 s, and 72 °C for 50 s; followed by 72 °C for 5 min and 14 °C thereafter. The sequences were analyzed by comparison to the consensus data of

TP53, IDH1, and IDH2

genes

using the GenBank accession numbers NM_000546.4, NM_005896.2, and NM_002168.2, respectively.

Statistical Analysis

Kaplan

–

Meier analysis was used to estimate survival distri- butions, and log-rank test was used to compare survival between patient subsets. All statistical analyses were per- formed using MedCalc Software, Version 12.2.1, with sta- tistical significance of

p<0.05.

Table 1 Clinicopathological features of 34 Moroccan patients with primary glioblastomas

Case ID patient Age Sex History^a KPS Site Surgery Size RT OS

1 GB01 43 M 0.67 40 F Subtotal 36.09 No 3

2 GB02 46 F 2 60 F Subtotal 61.60 Yes <1

3 GB03 68 F 1.5 60 T Total 23.43 Yes 12

4 GB04 64 M 6 70 F Partial 36.09 Yes 18

5 GB06 52 F 1.5 100 PO Partial 75.33 Yes 30

6 GB07 48 M 1.5 40 TF Subtotal 47.71 Yes 6

7 GB08 33 F 1 90 F Subtotal 113.1 Yes 30

8 GB09 26 F 1 40 T Subtotal 154.68 Yes 11

9 GB11 70 F 3 40 F Total 40.48 No 6

10 GB13 60 M 1.5 40 TP Subtotal 78.84 No 8

11 GB14 62 M 1 60 P Subtotal 69.46 Yes 24

12 GB15 55 F 1 80 F Partial 87.11 Yes 24

13 GB16 40 M 5 90 TP Biopsy 23.23 Yes 46

14 GB17 55 M 1 40 TP Partial 87.11 No <1

15 GB18 48 F 1 50 FT Subtotal 33.51 Yes 22

16 GB19 65 F 1 50 TP Subtotal 103.22 Yes 15

17 GB20 50 M 3 90 PO Total 24.43 Yes 18

18 GB21 55 F 5 60 PO Subtotal NA Yes 6

19 GB22 52 M 18 90 TP Partial 51.63 Yes 18

20 GB24 52 M 3 40 F Partial 56.47 No <1

21 GB25 78 M 4 40 TP Subtotal 38.79 No <1

22 GB26 54 M 5 90 O Total 22.84 Yes 13

23 GB27 54 M 1 100 TF Subtotal NA Yes 18

24 GB28 66 M 2 90 O Partial 33.51 Yes 13

25 GB29 16 M 0.7 90 P Partial 83.37 Yes 13

26 GB32 45 M 3 60 F Partial 14.14 Yes 13

27 GB33 40 F 2 40 T Partial 38.79 Yes 2

28 GB34 24 M 5 100 F Total 31.54 Yes 2

29 GB35 65 M 1.5 50 F Partial 50.97 Yes 2

30 GB36 43 M 1 100 F Subtotal 45.21 Yes 7

31 GB37 11 M 6 100 TP Total 57.91 No 22

32 GB38 65 M 24 60 P Partial NA No 11

33 GB39 47 M 7 100 F Subtotal 51.63 Yes 13

34 GB40 50 M 6 20 TP Partial 143.79 No 22

Mmale,Ffemale,OSoverall survival,Ssurgery,RTradiotherapy,NAnon available,Ffrontal,Ttemporal,Pparietal,Ooccipital,POparieto– occipital,TPtemporo–parietal,TFtemporo–frontal,OSoverall survival,KPSKarnofsky performance score,RTradiotherapy

aTime (in months) between onset of the first clinical symptoms and diagnosis

Results

Clinical and Epidemiological Features of Patients

We studied a series of 34 primary GBM, and the clinical and epidemiological data are summarized in Table 1. Histolog- ically, all the tumors demonstrated features of glioblastoma with pleomorphic, astrocytic tumor cells, prominent micro- vascular proliferation, and necroses. Twenty-three patients were males and 11 were females, reflecting the male pre- dominance. The patients’ ages ranged from 11 to 78 years, with a mean age of 50.06±5.28 years (females 50.72, males 49.74). About 67.65 % (23/34) of patients presented with clinical symptoms less than 3 months. The preoperative median KPS was 60 (95 % CI, 50–90) with six cases scored 100, seven cases scored 90, one case scored 80, one case scored 70, six cases scored 60, six cases scored 50, nine cases scored 40, and one case scored 20. The tumor size ranged between 14.14 and 154.68 cm

³

with a mean of 58.5±

12.71 cm

³

. The tumor locations affected the frontal lobe in 13 cases, temporal lobe in 3 cases, parietal lobe in 3 cases, occipital lobe in 2 cases, temporo

–

parietal lobes in 8 cases, parieto–occipital lobes in 3 cases, and temporo–frontal lobes in 2 cases. The patients were initially treated with tumor resection, 6 (18 %) by total resection, 14 (41 %) by subtotal resection, and 13 (38 %) by partial resection, but biopsy was performed for a single patient (3 %). Among 34 patients, 74 % (25/34) had received radiotherapy, but 9 were (26 %) not treated with radiotherapy. The median overall survival (OS) for all patients averaged 13 months (95 % CI, 6.83–18) with medians of 12 for women and 13 for men. The prognosis relevance of the clinical parameters is summarized in Table 2.

It shows a significant impact of KPS at diagnosis on OS.

Indeed, patients with KPS

≥

80 have longer OS compared with those with KPS<80; 18 vs. 7 months, respectively,

p-log-rank

test00.0002. However, there were no significant differences in OS according to age, gender, clinical history, extent of resection, or therapy parameters. Kaplan–Meier estimates for OS by these parameters are shown in Fig. 1.

Molecular Features

On the other hand, the

TP53

analysis revealed three muta- tions in different tumor samples with frequency of 8.82 % (3/34). Two of these mutations affected exon 8, one missense (c.818 G>A; R273H) in GB12 patient, and one nonsense (c.916 C>T; R306X) in patient GB09. The other nonsense mutation (c.406 C>T; Q136X) affects exon 5 in GB18. These mutations were not detected in blood samples of the corresponding patients. In addition, the

TP53

analysis revealed two previously reported polymorphisms: g.12708A

>G (c.639A>G) and g.12803A>G (c.672+62A>G). Finally, the screening of all 34 glioblastomas for R132-

IDH1

and

R172-IDH2

mutations revealed the absence of these mutations in our patients.

Discussion

The pathogenic mechanisms leading to the development and progression of glioblastoma are still unclear. However, a number of genetic alterations are described as involved in the development of GBMs (Furnari et al. 2007; Ohgaki and Kleihues 2007). Of these, mutations in

TP53, IDH1, and IDH2

genes are more investigated in glioblastoma. No in- formation is available about the molecular and epidemio- logic profiles of glioblastoma in our country. Here, we present the results of molecular analysis of these genes in 34 Moroccan patients with pGBM and we report their clinical and epidemiological characteristics.

It has been reported that

TP53

mutations occurs in 25–

30 % of GBMs and significantly more frequent in secondary glioblastomas (65 %) than in primary glioblastomas (<30 %) (Watanabe et al. 1996, 1997; Ohgaki et al. 2004;

Ohgaki and Kleihues 2005; Benito et al. 2010). In our series, we found a low prevalence of

TP53

mutations with

Table 2 Clinicopathologic parameters in association with overall survival

Parameter Number Median OS (months) p-log-rank

All patients 34 13

Age (years)

<50 14 12 0.8900^a

≥50 20 13

Gender

Male 23 13 0.3696^a

Female 11 12

KPS

<80 20 7 0.0002^b

≥80 14 18

Surgery

Biopsy 1 – 0.7229^a

Partial 13 13

Subtotal 14 9.5

Total 6 12.5

Radiotherapy

With RT 25 13 0.0710^a

Without RT 9 6

Clinical history

<3 months 19 12 0.5159^a

≥3 months 15 13

OSoverall survival,KPSKarnofsky performance score,RTradiotherapy

aNo significant difference

bVery highly significant difference

about 9 % (3 of 34) that corresponds to that found in primary glioblastomas. Additionally, the type and distribu- tion of

TP53

mutations may differ between GBM subtypes.

In secondary GBMs, 57 % of mutations are located in two hotspot codons 248 and 273, whereas in primary GBMs, mutations are more equally distributed through all exons.

Moreover, the G:C to A:T mutations at CpG sites, especially in hotspot codons, appear to be an early event associated with malignant transformation in the pathway to secondary GBMs (Batchelor et al. 2004; Ohgaki et al. 2004). In our

study, one of three mutations was found at the hotspot codon R273. The prognosis significance of p53 mutations in glio- blastoma remains unclear (Shiraishi et al. 2002; Rich et al.

2005; Chen et al. 2006). Indeed, Burton et al. noted higher p53 protein expression in long-term survivor patients, al- though there was no significant difference in the mutation rate of the p53 gene between long-term- and short-term survivor glioblastoma patients (Burton et al. 2002).

On the other hand, it has been reported that mutations of

IDH1

gene are absent or rare in primary GBM (<11 %) and

Fig. 1 Kaplan–Meier survival curves showing the relationship between overall survival of glioblastoma patients according to clinicopathological parameters.KPSKarnofsky performance score,RTradiotherapy

Table 3 Frequency ofTP53,IDH1, andIDH2mutations in glioblastomas from various studies

Study No. of

patients

IDH1(%) IDH2(%) TP53(%)

GBM pGBM sGBM GBM pGBM sGBM

(Nobusawa et al.2009)

407 8.8 (36/407) 3.7 (14/377) 73.3 (22/30) 0 (0/367) ND ND ND

(Yan et al.2009) 151 11.26 (17/151) 4.9 (6/123) 85 (11/13) 0 (0/151) 26.47 (36/136) 23 (28/123) 62 (8/13)

(Bleeker et al.2009) 109 20 (22/109) 11.7 (11/94) 73.33 (11/15) ND ND ND ND

(Antonelli et al.2010) 22 0 (0/22) ND ND ND 18.18 (4/22) ND ND

(Parsons et al.2008) 105 11.43 (12/105) 7.07 (7/99) 83.33 (5/6) ND 35 (37/105) ND ND

(Krell et al.2011) 47 12 (6/47) ND ND 0 (0/47) ND ND ND

(Zhou et al.2011) 16 56.25 (9/16) 16.7 (1/6) 80 (8/10) ND ND ND ND

(Weller et al.2009) 291 5.6 (16/286) ND ND ND 15 (43/291) ND ND

(Yan et al.2012) 118 ND 16.1 (19/118) ND ND ND ND ND

(Mellai et al.2011) 186 10.22 (19/186) 1.8 (3/167) 84.2 (16/19) 0 0/186 ND 21.2 (28/132) ND

(Uno et al.2011) 161 11.8 (19/161) 9.7 (15/155) 66.7 (4/6) ND ND ND ND

Present study 34 0 (0/34) 0 (0/34) ND 0 (0/34) ND 8.82 (3/34) ND

GBMglioblastomas,pGBMprimary glioblastomas,sGBMsecondary glioblastomas,NDnot determined

frequent in secondary GBM (>80 %) (Ohgaki et al. 2004;

Ohgaki and Kleihues 2005; Antonelli et al. 2010) (Table 3).

In this study, no

IDH1

or

IDH2

mutations were found in 34 primary glioblastoma samples. This finding coincides with some earlier reports concerning pGBM (Nobusawa et al.

2009; Yan et al. 2009; Antonelli et al. 2010; Krell et al.

2011; Mellai et al. 2011) (Table 3).

IDH1

mutations pre- dominantly occurred in younger patients and considered as strong predictors of more favorable prognosis with a signif- icantly longer survival (Parsons et al. 2008). Furthermore,

IDH1

and

IDH2

mutations were preferentially (80 %) found in tumors harboring

TP53

mutations (Nobusawa et al. 2009;

Yan et al. 2009; Uno et al. 2011).

Finally, we found that a higher preoperative KPS was a favorable factor associated with longer survival (the median OS was 18 months for patients with KPS

≥80 versus

7 months for patients with KPS < 80;

p-log-rank test0

0.0002). This result is consistent with previously described data (Krex et al. 2007; Sonoda et al. 2009b). Although it has been reported that younger age, female gender, clinical history, more extensive tumor resection, and radiotherapy are possible factors contributing to the long-term survival of the few reported patients (Vertosick and Selker 1992; Scott et al. 1999; Stummer et al. 2006; Krex et al. 2007; Sonoda et al. 2009b; Weller et al. 2009), we did not found a significant association between these parameters and OS in our patients. The lack of statistical impact might be attributed to a low number of studied cases, to the follow-up period that is relatively short, and to follow-up documentation that became incomplete in some patients. In addition, it has been reported that two thirds of patients with pGBM have a clinical history of less than 3 months (Ohgaki et al. 2004).

Similarly, we found about 67.65 % (23/34) of our series presented clinical history within 3 months before diagnosis.

In summary, this study established the clinical, epidemi- ological, and molecular profiles of primary glioblastoma in a Moroccan series. This would help to improve the progno- sis and the management of patients. However, supplement and profound molecular and clinical studies should be nec- essary to describe other genetic markers and their impact on diagnosis, survival, and therapy for GBM.

Acknowledgments We sincerely thank the neurosurgeons of CHU Ibn Rochd of Casablanca. We also thank the patients for their partic- ipation in this study.

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