MGMT promoter hypermethylation and its associations with genetic alterations in a series of 350 brain tumors

CLINICAL STUDY

MGMT promoter hypermethylation and its associationswith genetic alterations in a series of 350 brain tumors

Marta Mellai • Oriana Monzeglio • Angela Piazzi • Valentina Caldera •

Laura Annovazzi • Paola Cassoni • Guido Valente • Susanna Cordera •

Cristina Mocellini • Davide Schiffer

Received: 2 August 2011 / Accepted: 26 December 2011

� Springer Science+Business Media, LLC. 2012

Abstract MGMT (O6-methylguanine-DNA methyltrans-

ferase) promoter hypermethylation is a helpful prognostic

marker for chemotherapy of gliomas, although with some

controversy for low-grade tumors. The objective of this

study was to retrospectively investigate MGMT promoter

hypermethylation status for a series of 350 human brain

tumors, including 275 gliomas of different malignancy

grade, 21 glioblastoma multiforme (GBM) cell lines, and

75 non-glial tumors. The analysis was performed by

methylation-specific PCR and capillary electrophoresis.

MGMT expression at the protein level was also evaluated

by both immunohistochemistry (IHC) and western blotting

analysis. Associations of MGMT hypermethylation with

IDH1/IDH2 mutations, EGFR amplification, TP53

mutations, and 1p/19q co-deletion, and the prognostic

significance of these, were investigated for the gliomas.

MGMT promoter hypermethylation was identified in 37.8%

of gliomas, but was not present in non-glial tumors, with

the exception of one primitive neuroectodermal tumor

(PNET). The frequency was similar for all the astrocytic

gliomas, with no correlation with histological grade.

Significantly higher values were obtained for oligoden-

drogliomas. MGMT promoter hypermethylation was sig-

nificantly associated with IDH1/IDH2 mutations (P =

0.0207) in grade II–III tumors, whereas it had a borderline

association with 1p deletion (P = 0.0538) in oligoden-

drogliomas. No other association was found. Significant

correlation of MGMT hypermethylation with MGMT

protein expression was identified by IHC in GBMs and

oligodendrogliomas (P = 0.0001), but not by western

blotting. A positive correlation between MGMT protein

expression, as detected by either IHC or western blotting,

was also observed. The latter was consistent with MGMT

promoter hypermethylation status in GBM cell lines. In

low-grade gliomas, MGMT hypermethylation, but not

MGMT protein expression, was associated with a trend,

only, toward better survival, in contrast with GBMs, for

which it had favorable prognostic significance.

Keywords MGMT promoter hypermethylation �Genetics � Immunohistochemistry � Brain tumors

Introduction

MGMT (O6-methylguanine-DNA methyltransferase) is a

DNA repair enzyme involved in the mechanism of resis-

tance of human cancers to alkylating agents. MGMT spe-

cifically removes mutagenic, carcinogenic, and cytotoxic

M. Mellai � O. Monzeglio � V. Caldera � L. Annovazzi �D. Schiffer (&)

Neuro-bio-oncology Center, Policlinico di Monza Foundation,

Via Pietro Micca, 29–13100, Vercelli, Italy

e-mail: [email protected]

A. Piazzi

Department of Medical Sciences, University of Piemonte

Orientale, Novara, Italy

P. Cassoni

Department of Biomedical Sciences and Human Oncology,

University of Turin, Turin, Italy

G. Valente

Department of Clinical and Experimental Medicine,

University of Piemonte Orientale, Novara, Italy

S. Cordera

Department of Neurology, Ospedale Regionale, Aosta, Italy

C. Mocellini

Department of Neurology, Azienda Ospedaliera Santa Croce e

Carle, Cuneo, Italy

123

J Neurooncol

DOI 10.1007/s11060-011-0787-y

O6-alkylguanine DNA adducts induced by radiotherapy or

alkylating agents such as temozolomide (TMZ) or nitro-

sourea derivatives. MGMT-mediated resistance to alkyl-

ating drugs correlates with MGMT expression level [1].

During malignant transformation, the MGMT gene may be

epigenetically silenced by hypermethylation of its pro-

moter regions, leading to an increased sensitivity to

alkylating chemotherapy in a variety of tumors [2].

In glioblastoma multiforme (GBM), MGMT promoter

hypermethylation is detected in approximately 32–72% of

cases [3–7] and in 36–50% of gliosarcomas [8]. In long-

term survivors, the values are higher (74–83.3%) [9]. For

GBMs it is an important prognostic and predictive factor in

chemotherapy with TMZ [3, 4, 10].

In low-grade gliomas (LGG), also, hypermethylation of

the MGMT promoter is a frequent event. It occurs in

43–75% of diffuse astrocytomas [6, 11–14]; most tumors

with MGMT hypermethylation contain TP53 mutations,

particularly G:C?A:T transitions [11, 14, 15], and p53

protein accumulation is observed [12]. MGMT hyperme-

thylation and promoter hypermethylation of the p14 gene

are mutually exclusive [14]. In anaplastic astrocytomas, the

frequency is 38–64% [16, 17].

In grade II and III oligodendrogliomas, the frequency of

MGMT promoter hypermethylation is higher (47–92.9 and

70–94.4%, respectively) whereas it is lower in grade II and

III oligoastrocytomas (27–40 and 30–62.5%, respectively)

[13, 18, 19]. In both oligodendrogliomas and oligoastro-

cytomas, it has recently been found to be associated with

1p/19q co-deletion [18–20], although with some exceptions

[11, 13].

Data regarding MGMT hypermethylation status and its

correlation with LGG patients’ prognosis and treatment

response are conflicting. MGMT hypermethylation has

been demonstrated to be associated with longer overall

survival (OS) [21, 22] and progression-free survival (PFS)

[13, 22], with some exceptions [19]. Its significance in

relation to TMZ chemotherapy is still under investigation

in phase III trials. Whereas in grade III astrocytomas

MGMT promoter hypermethylation is associated with

longer PFS in patients treated either by radio–chemother-

apy or radiation alone [23, 24], in grade II gliomas there are

contrasting results. MGMT promoter hypermethylation was

found to be predictive with TMZ treatment [13, 22], but not

for patients with grade II astrocytomas [12] and oligoas-

trocytomas [11] that did not receive alkylating chemo-

therapy. There is evidence of TMZ efficacy at standard

doses [20, 25] with a PFS increase when a protracted daily

TMZ regimen is used [22]; however, this was also

observed in MGMT unmethylated patients [22]. The pro-

longed TMZ regimen could potentially overcome the

MGMT-mediated resistance by progressive depletion of

MGMT activity and by improving sensitivity to TMZ [22].

This effect has also been demonstrated in grade II gliomas

and radiologically verified [26].

MGMT hypermethylation is rarely observed for non-

glial tumors, for example meningiomas, ependymomas,

medulloblastomas, and primitive neuroectodermal tumors

(PNETs) [27–31].

The objective of this study was to investigate MGMT

promoter hypermethylation status and MGMT expression

at the protein level for a series of 311 neuroepithelial

tumors, of which 275 were gliomas and 39 meningiomas.

For gliomas, associations of MGMT hypermethylation with

IDH1/IDH2 mutations, EGFR amplification, TP53 muta-

tions, 1p/19q co-deletion and OS were also studied.

Materials and methods

Patients

Formalin-fixed paraffin-embedded (FFPE) brain tumor

samples were collected from a total of 350 patients

(Table 1) after approval by the relevant Ethics Commit-

tees. Tumors were surgically removed at the Neurosurgery

Table 1 Patients’ demographics

Tumor type WHO

grade

Patients

(n)

Gender

(M/F)

Mean age

(years) and

range

Glial tumors (n = 275)

Pilocytic

astrocytoma

I 20 11/9 44 (19–68)

Diffuse and

gemistocytic

astrocytoma

II 13 5/8 42 (23–68)

Anaplastic

astrocytoma

III 4 3/1 55 (38–75)

Primary GBM IV 161 101/60 61 (23–83)

Secondary GBM IV 2 2/0 46 (42–50)

Gliosarcoma IV 9 6/3 56 (47–73)

Oligoastrocytoma II 3 2/1 38 (28–53)

Anaplastic

oligoastrocytoma

III 1 1/0 58

Oligodendroglioma II 34 20/14 50 (26–79)

Anaplastic

oligodendroglioma

III 28 14/14 54 (31–80)

Non-glial tumors (n = 75)

Meningioma I 30 8/22 63 (23–87)

II 4 2/2 64 (45–73)

III 5 3/2 62 (48–80)

Schwannoma I 15 9/6 58 (25–82)

Ependymoma III 12 8/4 56 (30–84)

Medulloblastoma IV 4 3/1 31 (20–39)

PNET IV 5 3/2 43 (22–59)

J Neurooncol

123

Unit, Department of Neuroscience, University of Turin

(Turin, Italy), Azienda Ospedaliero-Universitaria ‘‘Maggi-

ore della Carita’’ (Novara, Italy), Azienda Ospedaliera

Santa Croce e Carle (Cuneo, Italy), and Clinica Eporediese

(Ivrea, Italy). Histological diagnosis was performed in

accordance with World Health Organization (WHO)

guidelines [32]. All patients underwent either partial or total

resection; their demographics are listed in Table 1. After

informed consent, their tumor and blood samples were pro-

vided for genetic analysis and other research purposes.

One-hundred and four GBMs of the series had already been

investigated for MGMT promoter hypermethylation status [5].

GBMs were regarded as primary (pGBM) or secondary

(sGBM) according to the absence or presence, respectively,

of a previous histologically verified low-grade glioma.

For 21 pGBMs (CV1–21), stabilized cell lines from

primary cultures were also studied.

Glioma patient stratification

Of 172 patients with GBM, 99 received postoperative

standard radiotherapy (RT) (60 Gy total dose in 27–30

fractions by LINAC) and 20 received RT doses of\40 Gy

or died within 1 month after surgery. For 53 patients no

clinical information was available or they were lost to

follow-up. Of the 99 irradiated patients, 55 received stan-

dard TMZ therapy, 75 mg/m2/daily for 6 weeks, followed

by adjuvant TMZ: 200 mg/m2 from day 1 to day 5 every

4 weeks for 6–12 cycles. Treatment and follow-up of

sGBMs were not available.

Of the 3 patients with anaplastic astrocytomas, 1 received

RT and 2 were lost to follow-up. Of the 8 patients with

diffuse astrocytomas and the 7 patients with gemistocytic

astrocytomas, 4 received RT and TMZ, 2 received RT only,

and the others did not receive any treatment. The patients

with pilocytic astrocytomas did not receive any treatment

with the exception of 2 who underwent RT. Of 4 patients

with oligoastrocytomas, 1 received RT only, 2 are still alive

and scheduled for RT, and 1 was lost to follow-up. Of the 34

patients with grade II oligodendrogliomas, 12 received RT

of which 8 also received chemotherapy by PCV, 5 received

PCV only, and 17 did not receive any treatment. Of 28

patients with grade III oligodendrogliomas, 12 received RT

of which 10 also received PCV, 2 did not receive any

treatment, and no information is available for 14.

Molecular genetics

Genomic DNA (gDNA) was extracted from FFPE tumor

samples by use of a standard phenol–chloroform proce-

dure. Before DNA extraction from each sample, only

tumor areas previously identified as proliferating by

hematoxylin and eosin (H&E) staining and microscopic

examination were selected. gDNA from cell lines and

peripheral blood was isolated by use of the QIAmp

DNAMini Kit (Qiagen, Hamburg, Germany) and a salting-

out procedure, respectively.

MGMT promoter hypermethylation status

MGMT promoter hypermethylation status (GenBank

sequence NM_002412) was assessed by methylation-spe-

cific polymerase chain reaction (MS-PCR) followed by

capillary electrophoresis (CE) as reported elsewhere [5].

Sodium bisulfite modification was performed with the

MethylEasyTM

Exceed Rapid DNA Bisulfite Modification

Kit (Human Genetic Signatures, Macquarie Park, Sydney,

Australia) [5]. CpGenomeTM

Universal Methylated DNA

(Chemicon International, Temecula, CA, USA) and normal

lymphocyte DNA were used as methylated and unmethy-

lated controls, respectively. The primer sequences for MS-

PCR and the amplification conditions have already been

reported [2]. After electrophoresis on an ABI� 3130 Genetic

Analyzer (Applied Biosystems), data were collected for

fragment analysis by use of GeneMapper v4.0 software

(Applied Biosystems). A peak height ratio[0.1 was scored

as evidence of the methylated status of the MGMT gene

(mean from two independent experiments) [5].

Because our series contained 2 sGBMs only, for this

analysis 10 supplementary sGBMs were studied, kindly

supplied by Dr Bianca Pollo (Fondazione I.R.C.C.S. Isti-

tuto Neurologico C. Besta, Milan, Italy).

EGFR amplification status

EGFR amplification status was assessed by PCR co-

amplification of both the 110-bp DNA fragment of the

EGFR gene (GenBank sequence NM_005228) and the

85-bp DNA fragment of the INF-c gene (GenBank

sequence NM_000619). INF-c was used as reference

housekeeping gene. The primer sequences and the PCR

conditions have already been reported [33]. After CE, data

were collected by use of GeneMapper v4.0 software for

fragment analysis (Applied Biosystems). The amplification

status of the EGFR gene was determined by measuring the

EGFR/INF-c ratio. A ratio[2.09 was regarded as evidence

of more than two copies of the EGFR gene (mean from two

independent experiments).

IDH1 and IDH2 mutation analysis

Two primer pairs designed on genomic DNA were used to

amplify, by PCR, the IDH1 exon 4 (GenBank sequence

NM_005896), the IDH2 exon 4 (GenBank sequence

NM_002168), and the intron/exon boundaries (including at

least 80 bp of the flanking intronic sequences). The two

J Neurooncol

123

fragments contain, respectively, the arginine residue at IDH1

codon 132 (R132) and the homologous residue at IDH2

codon 172 (R172). The primer sequences are available on

request. PCR conditions have been reported elsewhere [34].

TP53 mutation analysis

Exons 4–8 of the TP53 gene (GenBank sequence

NM_000546) encoding for the highly conserved DNA

binding domain were searched for sequence variations by

direct sequencing. The TP53 gene was amplified from

genomic DNA as six fragments covering the 5 exons and

the intron/exon boundaries (including at least 80 bp of each

flanking intronic sequence). The primer sequences are

available on request. The PCR conditions and thermal

cycling procedure have been described elsewhere [34].

Direct sequencing

All the amplicons for the IDH1, IDH2, and TP53 genes

were analyzed by direct sequencing on an ABI� 3130

Genetic Analyzer by using the BigDye� Terminator v1.1

Cycle Sequencing Kit (Applied Biosystems). Data were

collected by the Sequencing Analysis v.5.3.1 software

(Applied Biosystems). All the identified sequence varia-

tions were confirmed with at least two independent PCR

and sequencing experiments. Mutation nomenclature is in

agreement with http://hgvs.org/mutnomen/recs-prot

(HUGO) recommendations. The reported nucleotide and

amino acid numbering is relative to the transcription start

site (?1) corresponding to the A of the ATG on the cor-

responding GenBank reference sequences.

To establish whether each putative sequence variation

was somatic, i.e. tumor-specific, the corresponding

patient’s constitutional DNA was analyzed when available.

Bioinformatic analysis

Putative functional effects of the identified TP53 missense

mutations were predicted in silico by use of PMUT (http://

mmb.pcb.ub.es/PMut/), PolyPhen (http://genetics.bwh.

harvard.edu/pph/) and SNAP (http://cubic.bioc.columbia.

edu/services/SNAP/) software.

The effect of missense, synonymous, and intronic vari-

ants on splicing was evaluated by use of NNSplice (http://

biologyhelp.awardspace.com/desc7.php?id=14&type=

biotech) and SpliceView (http://bioinfo2.itb.cnr.it/sun/

webgene) software.

Chromosomal status of the 1p and 19q regions

Multiplex ligation-dependent probe amplification (MLPA)

was used to assess allelic losses on the 1p and 19q

chromosomes, because no constitutive DNA was available

for all the archived tumor samples. Analysis was performed

by use of the SALSA-MLPA Kit P088 (lot number 0608)

(MRC-Holland, The Netherlands) in accordance with the

manufacturer’s instructions. The kit includes a total of 15

probes covering 1p, 8 probes covering 19q, and 15 control

probes located on other chromosomes. Additional probes

were included to verify DNA quantity, quality, and the

denaturation and hybridization steps. MLPA products were

analyzed by CE on an ABI� 3130 Genetic Analyzer

(Applied Biosystems) and data were collected by use of

GeneMapper v4.0 software (Applied Biosystems). In each

run, at least four reference samples were included for

normalization. Data were analyzed by use of Coffalyser

v9.4 software (MRC-Holland).

Chromosomal regions were regarded as deleted if a ratio

\0.75 was observed for two or more consecutive probes on

1p or 19q, whereas a gain of function was defined for ratios

[1.40 (mean from two independent experiments) [35].

Combined loss of 1p/19q was defined as either partial or

complete deletion of both chromosome arms 1p and 19q

[35].

We validated the MLPA Kit P088 for a series of 45

tumor samples by parallel LOH (loss of heterozygosity)

analysis with 7 microsatellite markers for 1p (D1S508,

D1S1612, D1S496, D1S2724, D1S457, D1S534, and

D1S2696 from 1p36.23 to 1p11.1, respectively) and 4 for

19q (D19S908, D19S219, D19S412, and D19S902 from

19q13.3 to 19q13.34, respectively).

MGMT immunohistochemistry (IHC)

Analysis of MGMT protein expression was performed on

5 lm-thick sections by a labeled streptavidin–biotin pro-

cedure after heat-induced epitope retrieval (HIER) as

reported elsewhere [5]. Incubation was with the anti-human

MGMT mouse monoclonal antibody (MAB16200, clone

MT3.1, 1:100; Chemicon International, Temecula, CA,

USA). Diaminobenzidine (DAB; Roche Diagnostics,

Penzberg, Germany) was used for detection. Nuclei were

counterstained with Mayer’s hematoxylin. A negative

control was performed by omission of the primary anti-

body. Nuclear expression in endothelial cells and lym-

phocytes provided positive internal controls for binding of

the primary antibody.

The evaluation was performed by use of a semi-quan-

titative score system considering staining intensity (?, ??,

???), percentage of positive cells (\or[20 and[50%),

and diffuse or focal distribution. Further details of the score

system are given in Table 2. Only nuclear staining was

considered for the evaluation. Infiltrating lymphocytes,

microglial cells, and endothelial cells were not included in

the counts.

J Neurooncol

123

Infiltrating lymphocytes, microglial cells, endothelial

cells, and macrophages were not considered in the counts,

being excluded on the basis of CD68 staining. On parallel

sections, IHC with the anti-human CD68 mouse mono-

clonal antibody (790-2931, clone KP1, prediluted; Ventana

Medical Systems, Tucson, AZ, USA) was performed on a

Ventana Full BenchMark� automatic immunostainer

(Ventana) with the UltraViewTM

Universal DAB Detection

Kit as detection system. HIER was performed in Tris–

EDTA, pH 8 (Ventana).

CD68 immunopositive cells were counted in parallel

sections in areas corresponding to those counted for

MGMT. The number of microglial cells and macrophages

was subtracted from the number of MGMT-positive cells.

Protein extraction and western blotting analysis

Samples drawn from paraffin blocks were deparaffinized

and homogenized in a lysis buffer (50 mM Tris–HCl,

pH 7.4, 150 mM NaCl, 1% v/v Igepal, 2% sodium dodecyl

sulfate (SDS), 0.5% sodium deoxycholate, 10 mM EDTA)

supplemented with a protease inhibitor cocktail (Sigma–

Aldrich, St Louis, MO, USA), 2 mM sodium orthovana-

date, and 10 mM sodium fluoride. Whole protein extracts

were quantified by use of the BCATM

protein assay kit

(Pierce Biotechnology, Rockford, IL, USA) and subjected

to SDS-PAGE (12%). This was followed by immunoblot-

ting analysis as described elsewhere [5] and by probing the

blots with a mouse monoclonal anti-MGMT antibody (#

MS-470-P0, 1:400; NeoMarkers, Fremont, CA, USA).

Immobilon Western Chemiluminescent Substrate (Milli-

pore, Billerica, MA, USA) was used as detection system.

Band intensity was measured and quantified with NIH

Image J software (RSB; NIMH, Bethesda, MD, USA).

Data obtained by densitometric evaluation of MGMT were

expressed relative (arbitrary units) to the signal of a rabbit

polyclonal anti-a-tubulin antibody (# LF-PA0146, 1:5000;

AbFrontier, Seoul, Korea) used for loading and transfer

control.

In-vitro cultures

Tumor surgical tissue was processed as described else-

where [36]. Culture conditions were: Dulbecco’s modified

Eagle’s medium (DMEM)/F-12 with 10 ng/ml bFGF (basic

fibroblast growth factor) and 20 ng/ml EGF (epidermal

growth factor) for neurospheres (NS), and DMEM with

10% fetal bovine serum (FBS) for adherent cells (AC).

Both cultures were maintained in 5% O2/CO2. Human

malignant glioma U87-MG and 010627 cell lines (kindly

supplied by Dr Rossella Galli, DIBIT San Raffaele, Milan,

Italy) were used as reference for both NS and AC.

Statistical methods

Associations between categorical variables were evaluated

by use of 2 9 2 contingency tables and the chi-squared (v2)

or two-tailed Fisher’s exact test, as appropriate.

The correlations between western blotting and immu-

nohistochemistry data were analyzed by use of the two-

sided Pearson’s correlation coefficient test.

OS was defined as the time between diagnosis and death

or last follow-up of the patient. Survival curves were

estimated by use of the Kaplan–Meier method, and the log-

rank test (Mantel–Cox) was performed to compare survival

curves for different groups of individuals. Analysis was

performed by use of SPSS v17.0 software (SPSS, Chicago,

IL, USA).

Results

MGMT methylation status and clinical variables

MS-PCR was performed on 350 brain tumors and MGMT

methylation status was successfully determined in 344

cases (98.3%). MGMT promoter hypermethylation was

detected in 104 of the 275 glial tumors (37.8%). Its fre-

quency in gliomas is reported in Table 3. It was not asso-

ciated with sex, patient age (B50 or [50 years), or tumor

location either for the whole series of gliomas or among

tumor subtypes (P [ 0.05 for all categories).

Among the different types, the frequency of MGMT

hypermethylation was highest for oligodendrogliomas,

with 36 of 62 cases (58.1%) (Table 4). The percentage was

lower for both oligoastrocytic (2 of 4 cases, 50%) and

astrocytic (66 of 207 cases, 31.9%) tumors, with statistical

significance (P = 0.0003) (Table 4).

In oligodendrogliomas, the MGMT gene was hyperme-

thylated in 18 of 34 grade II tumors (52.9%) and in 18 of

28 grade III tumors (64.3%) (Table 3). In astrocytomas, the

frequency of MGMT hypermethylation was as follows:

30% in pilocytic astrocytomas, 38.4% in diffuse and grade

Table 2 Score system for evaluation of MGMT immunostaining

Score Category

Distribution Intensity Percentage

of positive cells

0 No staining - -

1 Heterogeneous ? \20

2 Heterogeneous ??/??? [20

3 Homogeneous ? \20

4 Homogeneous ??/??? [20

5 Homogeneous ??/??? [50

J Neurooncol

123

II gemistocytic astroctyomas, 25% in anaplastic astrocy-

tomas, and 31.5% in pGBMs (Table 3). The frequency of

MGMT hypermethylation in sGBMs was significantly

higher (9 of 12 cases, 75%) (P = 0.0001). In gliosarcomas

the percentage was lower (2 of 9 cases, 22.2%).

In NS, MGMT was hypermethylated in 6 of 9 (66.7%)

cases. Hypermethylated NS originated from hypermethy-

lated GBM primary tumors with the exception of 2 cases.

Four of 6 NS (66.7%) were completely hypermethylated

whereas both hypermethylated and unmethylated MGMT

was observed for 2 cases (CV7 and CV17).

In contrast with NS, in MS-PCR, evidence of a signal

for unmethylated DNA, as a consequence of contamination

of the tumor sample by unmethylated normal and non-

tumor cells, was observed for each hypermethylated and

matched primary tumor. AC were never found to be

hypermethylated.

MGMT promoter hypermethylation was not observed

for non-glial tumors (meningiomas, schwannomas,

ependymomas, medulloblastomas and PNETs), with the

exception of one PNET.

EGFR amplification

EGFR amplification was identified in 57 of 161 GBMs

(35.4%), in one grade III astrocytoma (33.3%), and in one

pilocytic astrocytoma (\1%), but not in grade II astrocy-

tomas (Table 5).

In oligodendrogliomas EGFR amplification was detec-

ted in 2 of 31 (64.6%) grade II and in 8 of 23 (34.8%) grade

III tumors. No gene amplification was identified in

oligoastrocytomas.

IDH1 and IDH2 mutations

Somatic point mutations at IDH1 R132 and IDH2 R172

codons were identified in 53 of 282 gliomas (18.8%). Their

frequency in the different tumor types is depicted in

Table 5. It was higher in oligodendrocytic than in astro-

cytic tumors.

All mutations affected codon R132 with the exception of

one oligodendroglioma with mutation at codon R172.

Details are available elsewhere [34].

TP53 mutations

The TP53 mutation status was investigated in 222 cases of

glioma, and mutations were identified in 57 of these

(25.7%). The incidence of TP53 mutation among tumor

types is reported in Table 5. The range of TP53 mutations

in low and high-grade tumors is illustrated in Fig. 1.

In GBMs, the hotspot codons were Arg158 and Cys176

(exon 5), Tyr220 (exon 6), Cys275, and Pro278 (exon 8); in

low-grade gliomas, the hotspot codons were Ile195 (exon

5), Tyr220 (exon 6), and Arg273 (exon 8). Interestingly, 14

of 222 (6.3%) cases (8 among low-grade gliomas and 6

among GBMs) contained double-nucleotide (doublet) or

multiplet mutations.

Thirty-two of 37 (86.5%) missense mutations are

responsible for either significant or partial loss of p53

protein activity. c.G375A (p.T125T) mutation leads to

aberrant splicing.

Analysis of all point mutations showed that 10 of 46

(21.7%) were G:C?A:T transitions, 8 of which (80%)

affected a CpG site. Of the 10 G:C?A:T transitions, 5 were

identified in methylated tumors and 5 in unmethylated tumors.

Chromosomal status of 1p and 19q regions

This was assessed in 167 astrocytic and 30 oligodendro-

cytic tumors. The frequency of 1p/19q co-deletion in the

different tumor types is reported in Table 5. In

Table 3 Frequency of MGMT promoter hypermethylation according

to WHO grading

Tumor type Patients

(n)

MGMT

hypermethylation

Grade I

Pilocytic astrocytoma 20 6 (30.0%)

Grade II

Diffuse and gemistocytic

astrocytoma

13 5 (38.4%)

Oligodendroglioma 34 18 (52.9%)

Oligoastrocytoma 3 2 (66.7%)

Total 50 25 (49.0%)

Grade III

Anaplastic astrocytoma 4 1 (25%)

Anaplastic oligoastrocytoma 1 0 (0%)

Anaplastic oligodendroglioma 28 18 (64.3%)

Total 33 19 (57.6%)

Grade IV

pGBMs 168 52 (31.0%)

sGBMs 12 9 (75%)

Table 4 Frequency of MGMT promoter hypermethylation among

glioma subtypes

Tumor subtype Patients

(n)

MGMT

hypermethylation

P value

Astrocytic tumors

(Grades I–IV)

207 66 (31.9%) 0.0003

Oligodendrocytic tumors

(Grades II–III)

62 36 (58.1%)

Oligoastrocytic tumors

(Grades II–III)

4 2 (50%) Ns

J Neurooncol

123

oligodendrogliomas, 1p/19q partial or complete co-deletion

was found in 20 of 30 cases (66.7%), and more often in

grade II (80%) than in grade III (53.3%) tumors. Partial

deletion of 1p without 19q loss, no 1p deletion alone, and

no 19q deletion alone were detected in 3, 5, and 2 cases,

respectively. Partial 1p deletions (on 1p36), with or without

19q loss, were detected in 7 of 30 (23.3%) oligodendro-

gliomas, exclusively grade III tumors.

Table 5 Frequency of the genetic alterations investigated in relation to MGMT methylation status

Tumor

type

MGMT

hypermethylation

status

IDH1/IDH2

mutations*

EGFR

amplification

1p

deletion

19q

deletion

1p/19 co-

deletion

TP53

mutations

n n % n % n % n % n % n %

PA Methylated 6 0/6 (0) 1/4 (25) 2/4 (50) 2/4 (50) 2/4 (50) 1/5 (20)

Unmethylated 14 1/14 (7.1) 0/10 (0) 2/11 (18.2) 2/11 (18.2) 2/11 (18.2) 0/14 (0)

DA and GA Methylated 4 3/4 (75) 0/3 (0) 0/1 (0) 1/1 (100) 0/1 (0) 2/2 (100)

Unmethylated 8 0/8 (0) 0/7 (0) 2/4 (50) 1/4 (25) 1/4 (25) 2/4 (50)

AA Methylated 1 1/1 (100) 0/1 (0) 0/1 (0) 0/1 (0) 0/1 (0) –

Unmethylated 3 1/3 (33.3) 1/3 (33.3) 0/1 (0) 0/1 (0) 0/1 (0) 1/3 (33.3)

pGBM Methylated 52 0/52 (0) 22/50 (44) 6/44 (13.6) 2/44 (4.5) 1/44 (2.3) 14/45 (31.1)

Unmethylated 116 1/108 (\1) 35/107(32.7) 10/90 (11.1) 8/90 (8.9) 6/90 (6.7) 19/82 (23.2)

sGBM Methylated 9 7/9 (77.8) 0/2 (0) 1/1 (100) 0/1 (0) 0/1 (0) 1/1 (100)

Unmethylated 3 3/3 (100) – – – – –

OA Methylated 2 2/2 (100) 0/1 (0) 2/2 (100) 1/2 (50) 1/2 (50) 1/2 (50)

Unmethylated 1 1/1 (100) 0/1 (0) 1/1 (100) 1/1 (100) 1/1 (100) 1/1 (100)

AOA Methylated 0 – – – – – –

Unmethylated 1 1/1 (100) 1/1 (100) 1/1 (100) 0/1 (0) 0/1 (0) 1/1 (100)

O Methylated 18 14/18 (77.8) 2/15 (13.3) 7/7 (100) 7/7 (100) 7/7 (100) 3/14 (21.4)

Unmethylated 16 9/16 (56.2) 0/16 (0) 4/7 (57.1) 5/7 (71.4) 4/7 (57.1) 4/14 (28.6)

AO Methylated 18 8/17 (47.1) 5/16 (31.3) 7/9 (77.8) 5/9 (55.6) 4/9 (44.4) 4/16 (25)

Unmethylated 10 2/10 (20) 3/9 (33.3) 4/6 (66.7) 4/6 (66.7) 4/6 (66.7) 1/7 (14.3)

PA pilocytic astrocytoma, DA diffuse astrocytoma, GA gemistocytic astrocytoma, AA anaplastic astrocytoma, GBM glioblastoma multiforme,

O oligodendroglioma, AO anaplastic oligodendroglioma, OA oligoastrocytoma, AOA anaplastic oligoastrocytoma

*Significantly associated with MGMT hypermethylation in grade II–III A (P = 0.0357) and in grade II–III A ? O (P = 0.0207)

Fig. 1 Genomic structure of human TP53 gene. The hatched boxcorresponds to untranslated exon 1. All the identified mutations in low

and high-grade gliomas are reported. Each mutation was verified to be

previously described in gliomas or glioma cell lines in the UMD_TP53

mutation database (R1 release, July 2010, http://p53.free.fr/), in the

Catalogue of Somatic Mutations in Cancer (COSMIC database, R15

release, November 2010, http://www.sanger.ac.uk/genetics/CGP/

cosmic/) and by the International Agency for Research on Cancer

(IARC TP53 database, R15 release, November 2010, http://www.p53.

iarc.fr/). Mutations identified de novo in gliomas in this study are

indicated by asterisks. Mutations responsible for aberrant splicing are

reported in gray. Hotspot codons are in bold

J Neurooncol

123

In the two cases of oligoastrocytomas, one had 1p/19q

co-deletion and the other had 1p deletion only.

MGMT hypermethylation status and molecular markers

After stratification of patients for tumor subtypes and

grades, MGMT promoter hypermethylation was signifi-

cantly associated with IDH1/IDH2 mutations in grade II–

III astrocytomas (P = 0.0357), even more when consid-

ering them together with grade II–III oligodendrocytic

tumors (P = 0.0207), for which the incidence of IDH1/

IDH2 mutation was greater (Table 5).

In grade II–III oligodendrogliomas, no association was

found between MGMT promoter hypermethylation and 1p/

19 co-deletion. However, for 11 of 16 (68.8%) MGMT

hypermethylated tumors 1p/19q co-deletion was observed,

in contrast with 8 of 13 (61.5%) of unmethylated tumors. A

borderline association was identified with 1p deletion only

(P = 0.0538).

In GBMs, neither EGFR amplification nor TP53 muta-

tions was associated with MGMT promoter hypermethyla-

tion (P [ 0.05 for both categories). EGFR amplification was

detected in 22 of 50 (44%) and in 34 of 109 (31.2%) meth-

ylated and unmethylated tumors, respectively. TP53 muta-

tions were identified in 25 of 86 (29.1%) and in 28 of 121

(23.1%) methylated and unmethylated cases, respectively.

MGMT immunohistochemistry

A total of 102 grade I–III gliomas, 172 GBMs, and 96 non-

glial brain tumors were studied. Nuclear staining only was

considered. The evaluation according to the score system is

reported in Table 6. Positive endothelial cells, infiltrating

lymphocytes, microglial cells, and macrophages were

excluded from the counts.

In gliomas, MGMT expression decreases from low to

high-grade tumors without statistical significance. The

percentage of positive tumor cells was highest for pilocytic

astrocytomas (Fig. 2a, b). This percentage was intermedi-

ate and variable for grade II astrocytomas (Fig. 2c, d),

anaplastic astrocytomas, grade II–III oligodendrogliomas

(Fig. 2e, f), and GBMs (Fig. 3a, b). For gemistocytes and

minigemistocytes weak cytoplasmic positivity was

observed. In gliosarcomas, both glial and sarcomatousus

components were positive, with staining intensity slightly

weaker for the latter.

Contamination by macrophages, lymphocytes, and

endothelial cells was regionally heterogeneous (Fig. 3c–e).

After correction for contamination, MGMT protein

expression correlated significantly with MGMT methyla-

tion status in GBMs (P = 0.0001) but not in low-grade

astrocytic tumors (P = 0.2861). It must be emphasized that

most astrocytic tumor samples with MGMT expression had

low MGMT hypermethylation values. In contrast, for oli-

godendrocytic tumors the correlation was statistically sig-

nificant, with 3 of 36 methylated tumors positively stained

and 8 of 23 unmethylated ones without MGMT expression

(P = 0.0001).

Absence of MGMT hypermethylation in meningiomas,

schwannomas, ependymomas, medulloblastomas and

PNETs corresponded to their positive protein expression,

with the exception of one PNET (Fig. 3f).

Western blotting

By western blotting analysis tumors were scored as positive

for MGMT protein expression on the basis of a clearly

visible band of molecular size 25 kDa (Fig. 4a). The dis-

tribution is shown in Table 7. A positive linear correlation

with statistical significance was found between MGMT

expression levels detected by IHC (taking into account the

percentage of positive cells, the staining intensity, and type

of distribution) and western blotting analysis, for both the

oligodendrocytic (Pearson’s correlation coefficient

r = 0.328; P = 0.0212) and astrocytic series (Pearson’s

correlation coefficient r = 0.637; P = 0.0011) (Table 7).

Table 6 MGMT immunopositivity in gliomas according to the score system

Score PA

(n = 20)

DA and GA

(n = 13)

AA

(n = 4)

O (n = 34) AO

(n = 27)

OA and AOA

(n = 4)

pGBM

(n = 160)

sGBM

(n = 12)

0 5 (25%) 10 (76.9%) 1 (25%) 19 (55.9%) 16 (59.3%) 2 (50%) 36 (22.6%) 7 (58.5%)

1 2 (10%) 0 (0%) 0 (0%) 5 (14.7%) 4 (14.8%) 1 (25%) 29 (18.1%) 1 (8.3%)

2 3 (15%) 2 (15.4%) 3 (75%) 4 (11.8%) 0 (0%) 1 (25%) 30 (18.8%) 0 (0%)

3 0 (0%) 0 (0%) 0 (0%) 2 (5.9%) 2 (7.4%) 0 15 (9.4%) 2 (16.7%)

4 8 (40%) 0 (0%) 0 (0%) 3 (8.8%) 3 (11.1%) 0 30 (18.8%) 0 (0%)

5 2 (10%) 1 (7.8%) 0 (0%) 1 (2.9%) 2 (7.4%) 0 20 (12.5%) 1 (8.3%)

PA pilocytic astrocytoma, DA diffuse astrocytoma, GA gemistocytic astrocytoma, AA anaplastic astrocytoma, GBM glioblastoma multiforme,

O oligodendroglioma, AO anaplastic oligodendroglioma, OA oligoastrocytoma, AOA anaplastic oligoastrocytoma

J Neurooncol

123

No correlation was found between MGMT hyperme-

thylation status, as detected by MS-PCR, and MGMT

expression by western blotting analysis in primary tumors.

In contrast, in GBM cell lines MGMT protein expression

detected by western blotting was consistent with MGMT

promoter hypermethylation status (Fig. 4b).

Fig. 2 MGMT immunohistochemistry in low-grade gliomas. a Pilo-

cytic astrocytoma (unmethylated MGMT promoter), [75% positive

nuclei. b Pilocytic astrocytoma (slightly methylated MGMT pro-

moter), 20% positive nuclei. c Diffuse astrocytoma (unmethylated

MGMT promoter), [75% positive nuclei. d Diffuse astrocytoma

(highly methylated MGMT promoter), non-positive nuclei. e Anaplas-

tic oligodendroglioma (unmethylated MGMT promoter), [75%

positive nuclei, and f Anaplastic oligodendroglioma (highly methyl-

ated MGMT promoter), non-positive nuclei. All DAB. Scale bar50 lm

J Neurooncol

123

Fig. 3 MGMT immunohistochemistry in high-grade gliomas.

a GBM (unmethylated MGMT promoter), [75% positive nuclei.

b GBM (highly methylated MGMT promoter), non-positive nuclei.

c GBM (unmethylated MGMT promoter), [50% positive nuclei.

d The same area with a high number of CD68 positive cells. e GBM

(highly methylated MGMT promoter), positive perivascular lympho-

cytes, and f PNET (methylated MGMT promoter), non-positive

nuclei. All DAB. Scale bar 50 lm in a, b, e, f; Scale bar 10 lm in c, d

J Neurooncol

123

MGMT methylation status and survival

OS was evaluated for 35 patients with grade II–III astro-

cytomas and oligodendrogliomas and for 121 GBM

patients. For the remaining patients data were not available.

Survival analysis by the Kaplan–Meier method showed

that MGMT promoter hypermethylation as detected by MS-

PCR correlates with a significantly longer OS for GBM

patients (P = 0.030) whereas for grade II–III gliomas a

trend toward positive correlation, only, was found

(P [ 0.05). It must be remarked that our OS data are lar-

gely incomplete for these tumors.

In contrast, MGMT protein expression by either IHC or

western blotting did not seem to be prognostic (P [ 0.05

for both categories).

Discussion

In this study MGMT promoter hypermethylation was

detected in 37.8% of gliomas but not in non-glial brain

tumors. The first general consideration is that our collection

of gliomas is unbalanced between high and low-grade

tumors, with the number of the latter being small enough to

negatively affect the statistical analysis. The same obser-

vation applies to grade III astrocytomas. However, MGMT

promoter hypermethylation was more frequent in low than

in high-grade gliomas (53.0 and 31.9%, respectively) as

previously reported [5–8, 11–14, 18]. In GBMs, it occurred

in secondary more than in primary tumors, in concordance

with the literature [12, 37]. Among low-grade gliomas,

MGMT promoter hypermethylation was more prevalent in

oligodendrocytic (58.1%) than in oligoastrocytic (50%) or

astrocytic (31.9%) tumors, in accordance with previous

data [8, 11–14, 18, 19, 22, 24, 38]. In astrocytic gliomas, no

correlation was found with histological grade, not con-

firming previous observations [6, 11, 13, 18, 19].

The similar frequencies of MGMT promoter hyperme-

thylation found in NS and in the matched primary tumors is

related with DNA hypermethylation as an epigenetic event

during tumor development and transformation as already

shown [36, 39]. This is in agreement with the negative

finding in AC and demonstrates that DNA hypermethyla-

tion belongs to the stemness properties.

We have no explanation for the prevalence of the het-

erogeneity of MGMT protein expression in low-grade

compared with high-grade astrocytic tumors, because in the

latter the contamination is greater. However, the correla-

tion between MGMT protein expression and promoter

Fig. 4 Western blotting analysis and densitometric quantification.

MGMT protein expression in a FFPE tumor samples and b GBM cell

lines. U unmethylated MGMT promoter, M methylated MGMT

promoter, PA pilocytic astrocytoma, GA gemistocytic astrocytoma,

AA anaplastic astrocytoma, GBM glioblastoma multiforme, O oligo-

dendroglioma, AO anaplastic oligodendroglioma

Table 7 Correlations between MGMT protein expression by western

blotting and IHC

Tumor type Total

cases (n)

WB

positive

cases

WB

negative

cases

P value

n % n %

Pilocytic astrocytoma 7 6 85.7 1 14.3 0.0011

Diffuse and

gemystocytic

astrocytoma

11 5 45.5 6 54.6

Anaplastic astrocytoma 5 3 60.0 2 40.0

GBM 86 81 94.2 5 5.8 0.0039

Oligodendroglioma 28 11 39.3 17 60.7 0.0212

Anaplastic

oligodendroglioma

47 16 84.2 3 15.8

J Neurooncol

123

hypermethylation is poorer in the former than in the latter

[40].

No MGMT promoter hypermethylation was identified in

non-glial brain tumors (meningiomas, schwannomas,

ependymomas, medulloblastomas, and PNETs) by MS-

PCR, with the exception of one PNET, in agreement with

previous observations [27–31]. The unmethylated status of

MGMT promoter was confirmed by both IHC and western

blotting analysis.

Overall, there is variability in assessment of MGMT

hypermethylation status among the different series in the

literature. This might be because of the different sensitiv-

ities of the methods used to assess MGMT status. The

different methods currently used can generate inter-labo-

ratory inconsistencies and, consequently, different selec-

tions of patients for treatment [7, 41]. In our and others’

experience, MS-PCR is a reproducible and accurate semi-

quantitative method [5, 42], particularly when followed by

capillary electrophoretic analysis [5], with sensitivity of

0.1% methylated tumor cells in a heterogeneous cell pop-

ulation [43]. As a reference test in clinical practice it cor-

relates with OS [2, 4, 5, 44], but this does not exclude

alternative methods from being reliable in patient selection.

By IHC, there was intratumoral heterogeneity in MGMT

protein expression in all FFPE tumor samples, both

because of the wide heterogeneity of gliomas and the

clonal origin of MGMT hypermethylation. However, hy-

permethylation was found in a previous study analyzing

multiple samples from the same tumor by stereotactic

procedures, intratumoral homogeneity of MGMT [45].

Contamination by non-neoplastic cells (microglial cells,

macrophages, endothelial cells, and infiltrating lympho-

cytes) expressing MGMT is involved [5, 46]. Contamina-

tion is important in phenotypically homogeneous and

heterogeneous tumors. IHC can also be invalidated by the

up-regulation of protein expression by either radio or

chemotherapy or steroid treatment [4, 47]. Its altering

effect on the number of positive cells can be nullified by

identifying contaminating cells with CD68. This obviously

applies to IHC, but not to western blotting.

On the whole, our data are in favour of a correlation

between assessment of MGMT hypermethylation status by

MS-PCR and the MGMT protein expression as detected by

IHC, but not in low-grade astrocytomas. These results are

partially inconsistent with previous observations [40, 44,

46, 48, 49]. However, the lack of a significant association

between the immunoistochemical MGMT protein expres-

sion and patient outcome advises against the use of anti-

MGMT immunohistochemistry as a clinical biomarker for

routine diagnostic purposes [40]. A recent systematic

review and meta-analysis demonstrates that evaluation of

MGMT protein expression by IHC alone fails to reflect

promoter hypermethylation status and to predict patient

survival or glioma chemosensitivity in a way that is

interchangeable with MS-PCR [50]. The two methods

select different groups of patients [51].

A significant correlation was observed between IHC and

western blotting analysis, both in astrocytic and oligo-

dendrocytic tumors [5]. MGMT hypermethylation detected

by MS-PCR does not correlate with western blotting for

primary tumors, but it does for GBM cell lines [36]. On the

whole, most observations favor the hypothesis that MGMT

protein expression, whether by IHC or western blotting, has

poor prognostic significance, as already described [52].

Our MGMT promoter hypermethylation data confirm the

statistically significant association with IDH1/IDH2 muta-

tions for grade II–III astrocytic and oligodendrocytic

tumors [38, 53] but not for GBMs [6, 54].

For oligodendrogliomas and oligoastrocytomas, MGMT

promoter hypermethylation has previously been associated

with 1p/19q co-deletion [18–20, 53], with some exceptions

[6, 11, 13]. In our study, because of the small number of

cases, we did not make a distinction between partial or

complete 1p deletions, even though this might be important

for prognosis. Complete and partial 1p deletions associated

with 19q deletions may [55, 56] or may not [35] have

prognostic significance. Because partial 1p deletions may

have an unfavorable effect, and not only in oligodendro-

gliomas [55], it should be mandatory to distinguish partial

from complete deletions. This can be achieved only by

multiple loci techniques, for example CGH or MLPA [57].

In our experience, MLPA is confirmed by LOH analysis

with 7 microsatellites on 1p and it can resolve the problem

of allelic imbalance (loss vs. gain). In anaplastic oligo-

dendrogliomas with 1p/19q co-deletion, unfavorable

prognostic significance may be associated with a polisomy

of 1q and 19p detected by FISH [58].

In our series, partial and complete 1p/19q co-deletions

did not correlate significantly with MGMT promoter hy-

permethylation; however, 68.8% of 1p/19q co-deleted

tumors with MGMT promoter hypermethylation may

indicate a trend, with all the limitations discussed above.

The association between MGMT promoter hyperme-

thylation and TP53 mutations is controversial. A positive

association was found for diffuse astrocytomas [11, 14,

15], with greater occurrence of G:C?A:T transitions in

MGMT methylated tumors [15], and with p53 protein

accumulation [12]. The p53 immunopositivity of methyl-

ated GBMs is of little significance because GBMs with

negative MGMT immunohistochemical expression had a

significantly higher number of TP53 mutations [59]. In

contrast, no association was found by others with either

TP53 mutations in low and high-grade astrocytic tumors [6,

13, 17] or p53 immunopositivity [53]. In our series, we did

not find any correlation with TP53 mutation status and

EGFR amplification, but there are exceptions.

J Neurooncol

123

Together with IDH1/IDH2 mutations, MGMT promoter

hypermethylation is a frequent and early epigenetic event

during gliomagenesis of both astrocytic and oligodendro-

cytic tumors [17, 60], preceding the differentiation of

precursors. Its prevalence among diffuse astrocytomas and

grade II–III oligodendrogliomas and oligoastrocytomas

suggests that these tumors may originate from a common

glial precursor cell population of NCSCs (neural cancer

stem cells) through two different IDH-dependent or inde-

pendent pathways [61].

Promoter-associated CpG island hypermethylation in

general has widely been reported for human GBMs and

other glioma subtypes [62]. Our observations of MGMT

promoter hypermethylation are in agreement with the

suggestion that a glioma-CpG island methylator phenotype

(G-CIMP) does exist [63]. This phenotype would prevail

among low-grade gliomas and have distinct copy number

alterations that are closely associated with IDH1/IDH2

somatic mutations and improved patient survival [63]. As a

matter of fact, a significant association between IDH1/

IDH2 mutations and MGMT promoter hypermethylation

was found in this series of low-grade gliomas [34], and this

has recently been regarded as useful in the molecular

subclassification of gliomas [64]. Together with 1p/19q co-

deletion and the newly identified G-CIMP phenotype,

MGMT promoter hypermethylation should belong to the

transcriptionally defined proneuronal glioma subclass

which characterizes low rather than high-grade gliomas,

the latter belonging to the mesenchymal glioma subclass

[65]. Therefore, this epigenetic alteration may be used as a

stratification marker to identify subgroups of glioma

patients with a better survival according to their methyla-

tion profile [66].

MGMT hypermethylation as detected by MS-PCR con-

fers a survival benefit on GBM patients [3–5, 10, 24, 38],

as confirmed by us, whereas its prognostic and predictive

significance for low-grade gliomas is still debated. On this

matter, our data are still insufficient for definite ascertain-

ment; however, our preliminary results suggests MGMT

hypermethylation is not prognostic for these tumors.

Conclusions

MGMT hypermethylation has prognostic significance for

high-grade but not low-grade gliomas. The reliability of

IHC for evaluation of MGMT protein expression and

therefore, indirectly, of MGMT hypermethylation status, is

lower than that of MS-PCR, mainly because of contami-

nation. Contamination also affects western blotting

analysis.

MGMT promoter hypermethylation is significantly

associated with IDH1/IDH2 mutations in grade II–III gli-

omas. It has a borderline association with 1p deletion for

oligodendrogliomas. No correlation is found with either

TP53 mutations or EGFR amplification.

Acknowledgments This work was supported by a Grant from

Compagnia di San Paolo, Turin. We are greatly indebted to Dr Bianca

Pollo (Fondazione I.R.C.C.S. Istituto Neurologico C. Besta, Milan,

Italy) for providing sGBMs.

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