doi:10.

1093/brain/awm204

Brain (2007), 130, 2596 ^2606

Long-term survival with glioblastoma multiforme

Dietmar Krex,1 Barbara Klink,2 Christian Hartmann,3 Andreas von Deimling,4 Torsten Pietsch,5
Matthias Simon,6 Michael Sabel,7 Joachim P. Steinbach,8 Oliver Heese,9 Guido Reifenberger,2
Michael Weller8 and Gabriele Schackert1 for the German Glioma Network
1

Department of Neurosurgery, Carl Gustav Carus University Hospital, University of Technology, Dresden, 2Department of
Neuropathology, Heinrich -Heine University, Dusseldorf, 3Department of Neuropathology, Charite-University Medicine
Berlin, 4Department of Neuropathology, Ruprecht-Karls-University, Heidelberg, 5Department of Neuropathology, and
6
Department of Neurosurgery, University of Bonn Medical Centre, Bonn, 7Department of Neurosurgery, Heinrich-Heine
University Dusseldorf, 8Department of General Neurology, Hertie Institute for Clinical Brain Research, Tubingen and
9
Department of Neurological Surgery, University Medical Centre Hamburg-Eppendorf, Germany
Correspondence to: Dietmar Krex, MD, Department of Neurosurgery, Carl Gustav Carus University Hospital,
University of Technology, Dresden, Fetscherstrasse 74, D- 01307 Dresden, Germany
E-mail: dietmar.krex@uniklinikum-dresden.de
The median survival of glioblastoma patients is 12 months. However, 3^5% of the patients survives for more
than 3 years and are referred to as long-term survivors. The clinical and molecular factors that contribute to
long-term survival are still unknown.To identify specific parameters that might be associated with this phenomenon, we performed a detailed clinical and molecular analysis of 55 primary glioblastoma long-term survivors
recruited at the six clinical centres of the German Glioma Network and one associated centre. An evaluation
form was developed and used to document demographic, clinical and treatment-associated parameters. In addition, environmental risk factors, associated diseases and occupational risks were assessed. These patients were
characterized by young age at diagnosis and a good initial Karnofsky performance score (KPS). None of the
evaluated socioeconomic, environmental and occupational factors were associated with long-term survival.
Molecular analyses revealed MGMT hypermethylation in 28 of 36 tumours (74%) investigated. TP53 mutations
were found in 9 of 31 tumours (29%) and EGFR amplification in 10 of 38 tumours (26%). Only 2 of 32 tumours (6%)
carried combined 1p and 19q deletions. Comparison of these data with results from an independent series of
141 consecutive unselected glioblastoma patients registered in the German Glioma Network revealed significantly more frequent MGMT hypermethylation in the long-term survivor group. Taken together, our findings
underline the association of glioblastoma long-term survival with prognostically favourable clinical factors,
in particular young age and good initial performance score, as well as MGMT promoter hypermethylation.
Keywords: EGFR; glioblastoma; long-term survival; MGMT, TP53
Abbreviations: LOH = loss of heterozygosity; WHO = World Health Organization
Received February 16, 2007. Revised July 23, 2007. Accepted July 31, 2007. Advance Access publication September 4, 2007

Introduction
Glioblastoma multiforme is the most common and most
malignant primary tumour of the brain and associated with
one of the worst 5-year survival rates among all human
cancers. Despite multimodal aggressive treatment, comprising surgical resection, local radiotherapy and systemic
chemotherapy, the median survival time after diagnosis is
still in the range of just 12 months (Smith and Jenkins,
2000), with population-based studies indicating even
shorter median survival (Ohgaki et al., 2004). Nevertheless,
a small fraction of glioblastoma patients survive for more
than 36 months. These patients are referred to as long-term
survivors.

With the exception of rare instances of glioblastoma in

patients with a hereditary tumour syndrome, e.g. Turcots
syndrome (Hamilton et al., 1995) or LiFraumeni syndrome (Kleihues et al., 1997), most tumours originate in
a sporadic fashion without any known genetic predisposition. Several studies have evaluated the impact of various
exogenous factors, such as smoking (Zheng et al., 2001),
diet (Huncharek et al., 2003; Lee et al., 1997), ionizing
radiation (Brustle et al., 1992; Neglia et al., 1991), cellular
phones (Inskip et al. 2001), electromagnetic fields (Savitz
et al., 1998; Theriault et al., 1994), socioeconomic status
and education level (Schlehofer et al., 2005), as well as
medical risk factors, such as allergy (Wiemels et al., 2002),

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Long-term survival with glioblastoma

immunological status (Schlehofer et al., 1999) and viral
infections (Vilchez et al., 2003). However, no unequivocal
evidence linking specific risk-factors to glioblastoma has
emerged from these studies, except for an association with
the exposure to ionizing radiation (for review see (Cavenee,
2000)). In addition, younger age and a good Karnofsky
performance score (KPS) at the time of diagnosis are
established clinical parameters associated with longer
survival (Curran Jr et al., 1993).
Despite the enormous progress in the understanding
of the genetic alterations in glioblastomas, clinically useful
molecular markers that help to predict response to therapy
and prognosis are still rare. To date, only the methylation
status of the O6-methylguanine methyltransferase (MGMT)
gene has become a molecular marker of clinical significance. MGMT encodes a DNA repair protein that causes
resistance to DNA alkylating agents, such as nitrosoureas
and temozolomide. Transcriptional silencing of the
MGMT gene by promoter hypermethylation is seen in

50% of glioblastomas and has been linked to prolonged
progression-free and overall survival in glioblastoma
patients treated with alkylating agents (Esteller et al.,
2000b; Hegi et al., 2005). Combined deletions of the
chromosomal arms 1p and 19q have been shown to be
associated with a favourable prognosis in oligodendroglial
tumours (Cairncross et al., 2006; van den Bent et al., 2006).
In glioblastoma, however, this aberration is rare and
its prognostic significance is less clear (Ino et al., 2000;
Schmidt et al., 2002; Brat et al., 2004; Houillier et al., 2006).
The investigation of glioblastoma long-term survivors
could help to identify yet unknown clinical, environmental
and/or molecular factors that are associated with favourable
prognosis. Here, we report on a retrospective analysis of
55 glioblastoma long-term survivors recruited within the
German Glioma Network. In addition to basic clinical data,
we evaluated the prevalence of environmental, occupational
and socioeconomic risk factors and screened for gliomaassociated genetic aberration, i.e. TP53 mutation, EGFR
amplification and 1p/19q deletion, as well as MGMT
hypermethylation.

Patients and Methods

Patient recruitment and data acquisition
Primary glioblastoma patients with survival longer than
36 months after diagnosis were retrospectively identified by
the six clinical centres of the German Glioma Network (www.
gliomnetzwerk.de). In addition, 13 patients from an associated
centre (Heinrich-Heine University Hospital Dusseldorf) were
included in the study. Five of these patients were included in
a previous report on glioblastoma patients surviving for more
than 5 years (Steinbach et al., 2006). The study was approved by
the local ethics committees at the contributing clinical centres.
A histological diagnosis of glioblastoma according to the World
Health Organization (WHO) classification of brain tumours was
confirmed by central pathology review at the Brain Tumour
Reference Centre of the German Society of Neuropathology and

Brain (2007), 130, 2596 ^2606

2597

Neuroanatomy (T. Pietsch). For this purpose, immunohistochemical characterization with antibodies against lineage-associated
antigens including glial fibrillary acidic protein was performed in
addition to conventional haematoxylin/eosin stainings. Possible
sarcomatous areas were illustrated by silver staining of reticulin
fibres. The clinical data were evaluated by carefully scrutinizing
the patients clinical charts and by interviews with the patients.
Relatives were contacted in case that the patient has already
deceased. A standardized basic evaluation form was used for
demographic, clinical and treatment-associated parameters.
In addition, a special long-term survivor form was completed
in which more details like environmental risk factors, associated
diseases and occupational risks were assessed. Both forms are
available at www.gliomnetzwerk.de. Data were collected at
the Department of Neurosurgery, University Hospital Dresden.
As a control population for the molecular analysis, we investigated
an independent series of primary glioblastomas (137 classic
glioblastomas, one gliosarcoma and three giant cell glioblastoma)
from 141 unselected patients operated at the clinical centres of the
German Glioma Network between October 2004 and December
2005. These tumours were derived from 83 male and 58 female
patients with a median age at operation of 60.9 years.

DNA extraction
Tumour DNA useful for molecular analysis of one or more of the
selected genes could be extracted from tumours of 38 glioblastoma
long-term survivors. In 13 of these tumours, as well as in the 141
glioblastomas of the independent control series, high-molecular
weight DNA was extracted from unfixed frozen tissue samples
as described before (van den Bent et al. 2003). From the remaining 28 long-term survivor tumours, DNA was extracted from
formalin-fixed paraffin-embedded tumour samples as reported
(Reifenberger et al. 1996). A tumour cell content of at least 80%
was histological assured for each specimen used for nucleic
acid extraction.

MGMT promoter methylation analysis

MGMT promoter methylation was analysed by methylationspecific PCR as reported before (Mollemann et al., 2005). The
primer sequences used to detect methylated MGMT promoter
sequences were 50 -gtttttagaacgttttgcgtttcgac-30 and 50 -caccgtcccga
aaaaaaactccg-30 (corresponding to nucleotides 46912-47033,
GenBank accession no. AL355531; fragment size: 122 bp). The
primer sequences used to detect unmethylated MGMT promoter
sequences were 50 -tgtgtttttagaatgttttgtgttttgat-30 and 50 -ctaccacca
tcccaaaaaaaaactcca-30 (corresponding to nucleotides 4690947037;
fragment size: 129 bp). As positive control sample, we used the
A172 glioma cell line, which has a completely methylated MGMT
promoter. Genomic DNA from non-neoplastic brain tissue served
as an unmethylated control sample.

Detection of EGFR gene amplification

EGFR gene dosage was determined by real-time PCR analysis
using the ABI PRISM 5700 (Applied Biosystems, Darmstadt,
Germany) sequence detection system. Continuous quantitative
measurement of the PCR product was achieved by labelling of
double-stranded DNA with SYBR Green fluorescent dye (Applied
Biosystems). The following EGFR-specific primers were used:
50 -cactgcctcatctctcaccatc-30 (sense) and 50 -gactcaccgtagctccagac-30
(antisense). The anonymous marker D2S1743 at chromosome

2598

Brain (2007), 130, 2596 ^2606

2q21.2 was used as reference locus employing the primers

50 -catgactgcgagcccaagatg-30 (sense) and 50 -caggtggtgtcatcagaat
cag-30 (antisense). As constitutional references, we used DNA
extracted from peripheral leukocytes as well as non-neoplastic
brain tissue samples from different patients. DNA from a glioblastoma with known EGFR amplification served as a positive
control. Only tumours showing a normalized increase in EGFR
gene dosage of more than 3-fold relative to constitutional DNA
were considered as showing EGFR amplification.

Multiplex ligation-dependent probe

amplification (MLPA)
MLPA was employed for the determination of allelic losses at
1p and 19q in the long-term survivor group because constitutional
DNA was not available from the majority of patients. Analysis
was performed using the SALSA MLPA KIT P088 lots 0305 and
0705 (MRC Holland, Amsterdam, the Netherlands). MLPA was
performed according to the protocol of Jeuken et al. (Jeuken et al.,
2006). PCR was performed with 35 cycles (95 C, 30 s; 60 C, 30 s;
72 C, 1 min; final extension 20 min, 72 C). Fragments were
separated and quantified on an ABI 3730XL DNA analyzer
(Applied Biosystems, Foster City, CA) and Genemapper 3.7
software utilizing the AFLP algorithm (Applied Biosystems).
In each run at least four reference samples were included.
The allelic status for each sample was determined as follows:
Peak areas of 19 control probes not from chromosomal arms
1p and 19q, which are included in the kit P088 were calculated.
An average peak area for the 19 control genes was determined.
Then peak areas from each individual probe on 1p and 19q were
divided by the value for average peak area. We determined this
average ratio on DNA samples from seven non-neoplastic tissues
for each probe on 1p and 19q and repeated this experiment four
times. Standard deviations of this ratio for each probe were
determined for the data of the multiple rounds of analyses on
these seven control tissues. A ratio deviating for >2 SDs from that
established for each of the probes in control tissues was scored
as reduced gene dosage. Because SDs for all data from controls
were within narrow limits, data from tumours with a ratio smaller
than 0.7 fulfilled these criteria for reduced gene dosage. DNA from
tumours was scored as deleted if two or more probes on 1p or
19q adjacent to each other exhibited a ratio smaller than 0.7.

Loss of heterozygosity (LOH) analysis at

microsatellite markers on 1p and 19q
Allelic losses in the control series were determined by
microsatellite-based LOH analysis as reported (Felsberg et al.,
2004; Hartmann et al., 2005). The following microsatellite loci
were evaluated: D1S211, D1S489 and D1S469 (all located on 1p),
as well as D19S572, D19S1182 and D19S596 (all located on 19q).
Electrophoresis on denaturing 10% polyacrylamide gels, silver
staining and assessment for LOH was carried out as described
elsewhere (Felsberg et al., 2004; Hartmann et al., 2005).

Single strand conformation polymorphism

(SSCP) analysis and direct sequencing
Exons 49 of the TP53 gene were screened for mutations using
SSCP analysis as reported before (Wellenreuther et al., 1995;
von Deimling et al. 2000). Aberrant SSCP bands were excised and
the DNA was extracted as described (Wellenreuther et al., 1995).

D. Krex et al.
Table 1 Patient characteristics
Total population of primary
glioblastoma
Male
Female
Median age at diagnosis
Median KPS
Median survival
Tumour localization
Right hemisphere
Left hemisphere
Both hemispheres
Frontal
Temporal
Parietal
Occipital
Treatment of glioblastoma
Tumour resectionb
Radiotherapy
Any chemotherapy

n = 55
n = 28
n = 27
51 (21^72) years
80 (30 ^100)
4.6 years (3.0 ^15.3 years)
n (%)
31 (56.4)
23 (41.8)
1 (1.8)
24a (43.6)
17 (30.9)
16 (29.1)
11 (20.0)
55 (100.0)
55 (100.0)
37 (67.3)

KPS = Karnofsky performance score, partly retrospectively

evaluated.
a
13 Tumours involved more than one lobe.
b
All patients had gross total resection or partial resection,
a biopsy was performed only once for recurrent tumour.
Following reamplification with the same set of primers the
PCR products were sequenced on a semiautomated sequencer
(Applied Biosystems, model 377, Foster City, CA) using the
Taq cycle sequencing kit (Applied Biosystems, Foster City, CA).
Each amplicon was sequenced bidirectionally.

Statistical analysis
The Fishers exact test or the 2 tests were used to compare the
frequencies of molecular aberrations in the long-term survivor
group versus the control group.

Results
General demographics
The study population of glioblastoma long-term survivors
consisted of 55 patients (male 28; female 27) with a median
age at diagnosis of 51 years (range 2172 years).
In comparison, the cohort of 141 unselected glioblastoma
showed a significantly higher age (median 60.9 years,
P50.001). The median follow-up time in the long-term
survivor group was 7 years and the median survival time
was 4.6 years (range 3.015.3 years). Median KPS at
diagnosis for the entire population of 55 long-term
survivors was 80 (range 30100), with five patients having
scores of 570 (Table 1).
Patients were married (49%), single (18%) or divorced
(2%); data on marital status were not available for
17 patients (31%). The average number of children was
two. There were nine (16%) current smokers among
the entire study population, consuming 345 cigarettes
per day [no data in 22 cases (40%)]. Occasional alcohol
consumption was admitted by 10 (18%) patients,

Long-term survival with glioblastoma

while there were no data available for 23 patients (42%).
No other drug consumption was recorded.

History
Inborn defects or inherited diseases were recorded in one
case each; one patient had an anomaly of the ear and
one had thalassaemia minor. Regarding childhood diseases
and vaccination status, only data from 10 (18%) and
7 (13%) patients were available. Within these groups the
prevalence of measles, small-pox, scarlet fever, whoopingcough, mumps and German measles was identical
with those found in the general population. Similarly,
7/7 patients had vaccination for tetanus, 6/7 for diphtheria,
and 4/7 for whooping-cough, which is comparable with
the status in the general population (Reiter, 2004).

Associated risk factors

There was no preponderance of any occupation among
the study population. Twenty-four different occupations
were documented, in addition to retired patients, homemakers and patients without any occupation. One profession (book-keeper) was represented four times, another one
(farmer) three times and six jobs were represented twice.
Coexisting tumour within the family was found in
nine (16%) patients. The recorded tumour entities were:
hepatocellular carcinoma, stomach carcinoma, breast carcinoma (twice), renal carcinoma, lung carcinoma, colon
carcinoma and abdominal wall carcinoma.
An accident in the history was found in 12 (22%)
patients resulting in fractures in nine (16%), tendon
rupture in two (4%), and in incision injury in one patient.
There was only one patient with a significant head injury
in history (skull fracture).
Any other operation not for repair of accident related
injuries was reported in 27 (49%) patients. There were
13 different procedures including cholecystectomy (three
times), endoscopic knee joint exploration (four times) and
appendectomy (twice). No patient had undergone prior
surgery for an intracranial tumour.
Sixteen different diagnoses in 22 patients were registered
as co-morbidities. Diabetes mellitus was found in four and
depression in two patients. Gall bladder disease and arterial
hypertension were found in two patients each. Among all
co-morbidities there was no preponderance of any organ
system. In order to evaluate disorders associated with
intracranial location, only the two with depression were
identified.

Tumour localization and histological features

Tumour localization was in the left cerebral hemisphere in
23 (42%), right cerebral hemisphere in 31 (56%) patients
and in both frontal lobes in one patient. Thirteen (23%)
tumours involved more than one lobe. The frontal lobe was

Brain (2007), 130, 2596 ^2606

2599

involved in 24 (44%) patients, followed by temporal (31%),

parietal (29%) and occipital (20%) lobe affliction.
In five of the tumours, the histology corresponded
to giant cell glioblastoma (WHO grade IV). Two additional
cases were classified as glioblastoma with sarcomatous
component (gliosarcoma WHO grade IV). Two cases exhibited a significant oligodendroglial component in addition
to the typical areas of glioblastoma with astrocytic differentiation. These cases were classified as glioblastoma with
oligodendroglial component. All other tumours corresponded histological to classic glioblastoma multiforme
(WHO grade IV).

Surgical treatment
All patients had planned gross total resection during the
first surgery. The extent of surgery was determined on
postoperative MR images. Due to the multicentric retrospective evaluation of data, the time between surgery
and MRI data acquisition was not uniform. However, the
first post-operative MRI data were recorded. Accordingly,
16 (29%) complete resections, i.e. no contrast-enhancing
residual tumour, and 25 (45%) partial resections,
i.e. contrast-enhancing tumour detectable irrespective of
size were documented whereas no data were available in
14 (25%) patients. Twenty-five patients (45%) had a second
operation (seven complete and 10 partial resections, one
biopsy, seven no data), 12 patients (22%) had a third
operation (three complete, six partial resections, three no
data), and four patients (7%) were operated on a fourth
time (two partial resections, two no data).

Adjuvant treatment
Following surgery, all patients had involved-field radiotherapy. A total dose between 59 and 60 Gy was applied
to 46 out of 55 patients (84%); in seven patients, a lower
dose (between 46 and 56 Gy) was applied because of side
effects and in two patients higher doses of 70 and 80 Gy
were applied. A combination of involved-field radiotherapy
and stereotactic convergence radiotherapy was implemented
in two patients; one patient had stereotactic convergence
radiotherapy only. Any kind of chemotherapy was applied
to 37 patients (67%). Some patients received more than one
protocol (Table 2). Chemotherapy was applied to 31 (56%)
patients after the first tumour resection and radiotherapy,
to nine patients after the second, to five patients after the
third, and to one patient after the fourth operation.
Nimustine (ACNU) was the most frequently (16 out of
31 patients) used drug in first-line chemotherapy protocols
as monotherapy in one, or in combination either with
teniposide in thirteen, or with cytarabine in three patients.
Temozolomide was applied to 21 patients, including five
patients who received combined radio-chemotherapy.
The PCV (procarbacine, lomustine, vincristine) protocol
was applied to seven patients after radiotherapy. ACNU,
temozolomide and PCV were also used for chemotherapy

2600

Brain (2007), 130, 2596 ^2606

D. Krex et al.

Table 2 Chemotherapy in primary glioblastoma

After First OP
Nimustine
PCV
Temozolomide
More than one chemotherapy
regimen in total n (%)a
No chemotherapy n (%)a
Total number of patients treated n (%)a

After second OP

After third OP

After fourth OP

16
7
12
4 (7.2)

1
3
7
7 (12.7)

2
1
3
13 (23.6)

0
1
0
13 (23.6)

24 (43.6)
31 (56.4)

19 (34.5)
36 (65.5)

18 (32.7)
37 (67.3)

18 (32.7)
37 (67.3)

Percentage of all glioblastoma patients (n = 55).

Table 3 Incidences of molecular aberrations detected in glioblastomas from long-term survivors and from an unselected
control series

TP53 mutation
EGFR amplification
Combined 1p/19q loss
MGMT hypermethylation

Glioblastomas from
long-term survivors

Glioblastomas from
control series

Statistical results P

9/31 (29%)
10/38 (26%)
2/32 (6%)
28/36 (74%)

30/135 (22%)
60/138 (44%)
14/136 (10%)
59/136 (43%)

0.4199 (2 test)

0.0556 (2 test)
0.7394 (Fishers exact test)
0.0002 (2 test)

after the second to fourth operation. A total of 13 patients

received more than one chemotherapy regime (Table 2).

Molecular analyses
DNA suitable for molecular analysis could be extracted
from 38 of 55 long-term survivors. However, due to DNA
degradation and limited amounts of DNA, not all 38
tumours could be investigated for aberrations in each of the
four selected genes. Exons 49 of the TP53 gene were
screened for mutations in 31 tumours. TP53 mutations
were identified in tumours of nine patients (16%). The
detected mutations included nine distinct missense mutations (c.451C>T/p.P151S, c.481G>C/p.A161T, c.568C>A/
p.P190T,
c.733G>A/p.G245S,
c.736A>G/p.M256V,
c.742C>T/p.R248W,
c.817C>T/p.R273C,
c.823T>C/
p.C275R) and one nonsense mutation (c.135G>delG
p.45fs). The EGFR gene dose could be assessed in all
38 tumours and revealed EGFR gene amplification in
10 patients (26%). Combined allelic losses on chromosome
arms 1p and 19q were detected in 2 of 32 tumours
investigated by MLPA (6%). In both cases, histological
evaluation did not reveal an oligodendroglial component.
The methylation status of the MGMT promoter was
determined in 36 patients and revealed MGMT hypermethylation in tumours of 28 patients (74%). In 10
patients, tissue samples from recurrent tumours were
additionally analysed for MGMT methylation. In all
instances, the MGMT status in the primary tumour was
retained in the recurrent tumour (eight methylated,
two unmethylated). There was no correlation of molecular
features with tumour location.

Similar to the long-term survivor group, not all

141 tumours of the control group of unselected consecutive
primary glioblastomas could be studied for each of the
genes. Data on the EGFR copy number were obtained from
138 tumours and revealed EGFR gene amplification in
60 tumours (44%). A total of 136 tumours from this series
were studied for allelic losses on 1p and 19q, which revealed
combined losses on both chromosome arms in 14 tumours
(10%). MGMT hypermethylation was detected in 59 of
136 tumours investigated (43%). Mutational analysis of
135 tumours for TP53 aberrations identified mutations
in 30 tumours (22%). Statistical comparisons revealed that
MGMT hypermethylation was significantly more common
in glioblastomas from the long-term survivor group as
compared to glioblastomas from the unselected patient
group (P = 0.0002, 2 test). In contrast, the frequencies of
TP53 mutation, EGFR amplification and 1p/19q losses
did not significantly differ between both groups, although
there was a trend towards less frequent EGFR amplification
in the long-term survivor group (Table 3).

Discussion
This is the largest series of glioblastoma long-term survivors
reported to date. We provide a clinical characterization and
report on molecular analyses of 55 primary glioblastoma
patients with a survival time of >36 months. There was
no association between socioeconomic, environmental and
occupational factors and long-term survival. Molecular
analyses indicated a higher proportion of MGMT methylation in the long-term survivor group when compared
with a reference sample of an unselected consecutive series
of glioblastomas.

Long-term survival with glioblastoma

Brain (2007), 130, 2596 ^2606

2601

Table 4 Review of the literature for patients with glioblastoma who survived longer than 3 years (long-term survivors)

Netsky et al., 1950

Roth and Elvidge, 1960
Ley et al., 1962
Taveras et al., 1962
Elvidge and Barone, 1965
Gullotta and Bettag, 1967
Jelsma and Bucy, 1967
Dara et al., 1980
Johnson 1981
Hatanaka et al., 1984
Bucy et al., 1985
Salford et al., 1988
Akslen et al., 1989
Ishikura et al., 1989
Margetts and Kalyan-Raman, 1989
Imperato et al., 1990
Shibamoto et al., 1990
Rutz et al., 1991
Vertosick, Jr. and Selker, 1992
Hiesiger et al., 1993
Chandler et al., 1993
Phuphanich et al., 1993
Archibald et al., 1994
Wester et al., 1994
Morita et al., 1996
New et al., 1997
Pollak et al., 1997
Klein et al., 1998
Cervoni et al., 1998
Salvati et al., 1998
Puzzilli et al., 1998
Scott et al., 1999
Yoshida et al., 2000
Sabel et al., 2001
Burton et al., 2002ab
Valery et al., 2002
Floeth et al., 2003
Ho et al., 2003
McLendon and Halperin 2003
Schmidinger et al., 2003
Jagannathan et al., 2004
Rainov and Heidecke 2004
Shinojima et al., 2004
Deb et al., 2005
Yamanaka et al., 2006
Steinbach et al., 2006
Total

Number of cases

Mean age

male

female

Mean KPS score

GTR

RT

Chemotherapy

8
20
6
13
2
1
6
3
1
1
1
2
2
1
3
5
1
1
13
10
22
7
7
1
10
1
2
1
1
11
1
15
2
1
41
1
1
6
17
5
4
1
6
6
1
10
281

na
na
na
na
24.5
45
na
na
32
50
30
14.5
41.5
8
41.3
42.6
51
21
40.9
47
39.2
37
37.7
45
39.2
34
20
11
13
39
50
43.5
40
69
39
42
37
na
40.2
47
46.3
27
44.2
27
42
42
36.9

na
na
na
na
1
1
na
na
na
1
1
1
1
na
2
3
1
1
7
na
10
5
2
na
7
na
1
na
na
5
1
9
1
1
16
na
1
na
11
3
2
1
na
3
1
4
104

na
na
na
na
1
na
na
na
1
na
na
1
1
1
1
2
na
na
6
na
12
2
5
1
3
1
1
1
1
6
na
6
1
na
25
1
na
na
6
2
2
na
6
3
na
6
105

na
na
na
na
na
na
na
na
na
100
na
na
na
na
90a
na
na
na
86a
81
80
na
na
na
na
100
na
na
80
80
90
90
na
na
90
na
100
na
na
na
na
90
85
na
90
90
89

na
na
na
na
2
1
6
na
1
0
1
1
2
1
3
5
1
1
na
1
2
na
2
1
10
1
2
1
1
11
0
na
1
1
na
1
1
na
na
na
na
na
4
na
1
9
75

na
na
6
na
1
1
na
na
0
1
1
2
2
0
3
5
1
1
13
10
22
7
7
1
na
1
2
1
1
11
1
na
2
0
na
1
1
na
na
na
4
na
6
na
1
10
126

na
na
na
na
0
0
na
3
0
0
0
0
1
0
3
4
0
0
13
10
18
6
7
0
na
1
0
1
1
11
0
na
2
0
na
1
1
na
na
na
na
na
6
na
na
9
98

Data from a recent review for glioblastoma patiens living longer than 5 years (Shinojima et al., 2004) are included.
Calculated on the basis of data that were provided in the concerning paper.
b
The study population is also published in another paper by Burton et al., 2002b.
na = not available, Data were not provided in the paper.
KPS = Karnofsky Performance score; GTR = gross total resection; RT = radiotherapy.
a

The median age of glioblastoma patients is >60 years

according to population-based studies (www.cbtrus.org)
(Ohgaki and Kleihues, 2005; Chakrabarti et al., 2005).
In line with these data, the median age of our control
group of unselected consecutive glioblastoma patients was
60.9 years. In contrast, the median age of the long-term
survivors in our study was considerably lower, i.e. 51 years

(P50.001). This is in accordance with numerous clinical

studies indicating that young age at the time of diagnosis
is an important parameter associated with longer survival
(Burger and Green 1987; Curran Jr et al., 1993; Devaux
et al., 1993; Chang et al., 2005). Taking data from all 281
published glioblastoma long-term survivors (Table 4), their
median age is 36.9 years, which supports that age is of

2602

Brain (2007), 130, 2596 ^2606

predictive value in glioblastoma. On the other hand, four

of our patients were 65 years or older, indicating that older
age does not exclude long-term survival of glioblastoma.
Interestingly, the median age in our study is almost 15 years
above to that of all published cases. This might in part
be explained by the fact that older studies are possibly
contaminated with low-grade tumours in young adults that
were mistaken for glioblastoma, in particular pleomorphic
xanthoastrocytomas. In our study population we had
five tumours characterized as giant cell glioblastoma.
In comparison to the control cohort of 141 patients,
which included three cases of giant cell glioblastoma,
this histological variant was overrepresented in the group of
long-term survivors (9.1% versus 2.1%, P = 0.041) supporting the hypothesis that this histological variant is associated
with longer survival (Shinojima et al., 2004). A contamination of glioblastoma patients cohorts by high grade
tumours of the oligodendroglial lineage may also occur.
However, in our series, only two cases showed a minor
oligodendroglial component in an otherwise typical
glioblastoma.
In general, glioblastomas are more frequent in males,
with a male/female ratio between 1.3 and 1.45, corresponding to a proportion of female patients of 43 and 41%,
respectively (Barnholtz-Sloan et al., 2003; Ohgaki and
Kleihues 2005). In line with these data, the male to
female ratio in our control series of 141 unselected
glioblastoma patients was 1.43. The long-term survivors
showed a trend to a higher proportion (50%, 95% CI 38
62%) of female patients, although the proportion reported
in the literature is still within the limits of the confidence
interval. Nevertheless, it seems that glioblastoma long-term
survival is favoured by the combination of two basic clinical
parametersyoung age and female gender. Unfortunately,
the median follow up time in the control group did not
allow validation of these findings. Several environmental
and socioeconomic risk factors have been associated with
the development of malignant glial tumours (Lee et al.,
1997; Inskip et al., 2001; Huncharek et al., 2003; Schlehofer
et al., 2005). One might assume that the absence of
such factors could be associated with a better prognosis in
those tumours. However, in the present study we failed
to identify any of those factors evaluated by our questionnaire to be obviously under- or overrepresented in longterm survivors. Although such data are difficult to obtain in
a retrospective setting, our results support the hypothesis
that socioeconomic, environmental and occupational
factors do not play major roles in determining whether
a glioblastoma patient becomes a long-term survivor or not.
All patients of our series were initially treated by
(gross total) tumour resection. Recent data have confirmed
that the extent of resection is associated with improved
progression-free survival (Stummer et al., 2006). Although
we had no uniform early postoperative MRI scanning
to document the extent of resection in our patients,
the fact that all 55 glioblastoma long-term survivors had

D. Krex et al.
intended gross total resection as initial treatment confirms
that tumour resection enhances the chances for a favourable
outcome.
All patients had adjuvant radiotherapy. Standard treatment for glioblastoma includes postoperative radiotherapy;
hence radiotherapy is unlikely to be a positive selection
factor. Chemotherapy for malignant gliomas has experienced a renaissance since the publication of the EORTC
26981/22981-NCIC CE3 phase III randomized trial, which
demonstrated that concomitant and adjuvant temozolomide chemotherapy has a positive effect on survival of
patients with glioblastoma (Stupp et al., 2005). In our
study, patients were treated between 1986 and 2003.
Therefore, only 21 patients received temozolomide treatment, among these were 12 patients who had temozolomide
for initial adjuvant treatment. A total of 37 patients (67%)
had at least one or up to three different adjuvant chemotherapy regimens, although chemotherapy was not included
in standard of care when those patients were treated.
Therefore, these data are in support of a positive role for
chemotherapy in glioblastoma with respect to long-term
survival. However, the study design does not allow to define
chemotherapy as a prognostic factor. Here, the same
limitations apply as for the number of surgical interventions. Patients who have a less malignant course of disease
also have more options to undergo multiple surgical
and chemotherapeutic interventions. Yet, nitrosoureabased chemotherapy has previously been defined as
a factor favouring long-term survival in the classical
meta-analyses published in 1993 by Fine and colleagues
and in 2002 by the Glioma Meta-analysis Trialists group.
These authors reported that there are consistently more
long-term surviving patients in the experimental trial arms
of adjuvant nitrosourea-based chemotherapy (Fine et al.,
1993; Stewart 2002).
Recent translational studies have reported that hypermethylation of the MGMT promoter is associated with
prolonged progression-free and overall survival in glioblastoma patients treated with alkylating agents (Esteller et al.,
2000a; Hegi et al., 2005). In line with these data, MGMT
promoter methylation was observed in the majority of
glioblastomas from long-term survivors. We found MGMT
hypermethylation to be significantly (almost two-fold)
more common in long-term survivors (74%) as compared
to an unselected consecutive series of glioblastoma patients
treated at the clinical centres of the German Glioma
Network (43%). On the other hand, it is also important to
note that the absence of MGMT promoter methylation is
still compatible with long-term survival in individual
patients.
In addition to MGMT methylation, we investigated
genetic alterations that are characteristic of either primary
(EGFR amplification) or secondary glioblastomas (TP53
mutation) (Kleihues and Ohgaki, 2000), as well as allelic
losses on 1p and 19q, which are an established prognostic marker for anaplastic oligodendroglial tumours

Long-term survival with glioblastoma

(Cairncross et al., 2006; van den Bent et al., 2006).
In comparison to our control group of unselected consecutive glioblastoma patients, there was a trend towards less
common EGFR amplification in glioblastomas from longterm survivors. However, statistical analysis did not confirm
a significant difference in EGFR amplification frequency
between the long-term survivor group and the control
group (Table 3). In the literature, conflicting data on the
prognostic relevance of EGFR amplification in glioblastomas have been reported. While EGFR amplification and/or
overexpression has been associated with a poor prognosis
in some studies (Hiesiger et al., 1993; Zhu et al., 1996;
Etienne et al., 1998; Korshunov et al., 1999), other authors
could not substantiate a prognostic significance (Quan
et al., 2005), or even reported an association with better
prognosis (Houillier et al., 2006; Smith et al., 2001). Our
finding suggests that the likelihood of long-term survival is
higher in glioblastomas without EGFR amplification.
However, it has to be considered that EGFR amplification
is more common in glioblastomas of older patients
(von Deimling et al., 2000). Therefore, the lower percentage
of EGFR amplified tumours among the long-term survivors
might be due to the significantly lower median age of
this patient group.
Similar to EGFR amplification, the prognostic significance of TP53 mutations in glioblastoma is unclear.
In glioblastomas, some studies reported a better prognosis
of patients with TP53-mutant tumours (Chen et al. 2006)
while others did not detect any prognostic significance
(Kraus et al., 2001; Shiraishi et al., 2002; Rich et al., 2005).
In a population-based study of glioblastoma patients,
TP53 mutation was predictive of longer survival but not
significant when adjusted for age at diagnosis (Ohgaki et al.,
2004). In our study, the frequencies of TP53 mutation did
not differ significantly between the long-term survivor
group and the control group, which does not support
an association between the TP53 mutation status and
long-term survival.
In contrast to anaplastic oligodendrogliomas and
oligoastrocytomas (Cairncross et al., 2006; van den Bent
et al., 2006), the prognostic significance of 1p/19q deletion
in glioblastomas is not clear yet (Ino et al., 2000; Schmidt
et al., 2002; Brat et al., 2004; Houillier et al., 2006). While
the incidence of combined 1p/19q losses is low in glioblastomas (von Deimling et al., 2000), some studies
reported that the presence of an oligodendroglial component is associated with more frequent 1p/19q deletion and
better prognosis (He et al., 2001; Kraus et al., 2001). Other
authors did not confirm a better outcome of glioblastoma
patients with oligodendroglial component in a multivariate
analysis adjusted for age and gender (Homma et al., 2006).
However, these authors found that allelic losses on 1p were
associated with longer survival in glioblastoma. Our study
did not identify an increased frequency of 1p/19q losses
in glioblastoma long-term survivors when compared to
our control series of unselected glioblastoma patients.

Brain (2007), 130, 2596 ^2606

2603

The central pathology review confirmed classic glioblastoma

histology in the three cases with 1p/19q loss. These data
suggests that long-term survival of classic glioblastoma
is unrelated to 1p/19q loss. Furthermore, we can exclude
a major contamination of our series with malignant
oligodendroglial neoplasms.
We conclude that although we confirmed certain clinical
factors and MGMT promoter hypermethylation to be associated with prolonged survival, our current study does not
explain why the 55 glioblastoma patients reported here
became long-term survivors. Thus, our results do not allow
refining current treatment and overall counselling strategies
for patients with glioblastoma. We assume that further
molecular analyses employing large-scale microarray-based
genomic and expression profiling approaches will identify
molecular features that are specific to glioblastomas of
long-term survivors. For this purpose, the German Glioma
Network is prospectively collecting fresh tissue from all
glioblastoma patients operated at its participating centres
and shall thus be able to perform comparative profiling
experiments on a reasonable number of long-term survivors
from this large population within the next few years.

Acknowledgements
The German Glioma Network is funded by the Deutsche
Krebshilfe e.V., grant no. 70-3163-Wi33. We would like
to thank all study nurses of the German Glioma Network
for their work in CRF documentation and evaluation of
clinical data. The German Glioma Network is sponsored
by the Deutsche Krebshilfe (German Cancer Aid).
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