Long-term Survival With Glioblastoma Multiforme

Long-term survival with glioblastoma multiformeDietmar 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

1Department of Neurosurgery, Carl Gustav Carus University Hospital, University of Technology, Dresden, 2Department ofNeuropathology, Heinrich -Heine University, Du sseldorf, 3Department of Neuropathology, Charite -University MedicineBerlin, 4Department of Neuropathology, Ruprecht-Karls-University, Heidelberg, 5Department of Neuropathology, and6Department of Neurosurgery, University of Bonn Medical Centre, Bonn, 7Department of Neurosurgery, Heinrich-HeineUniversity Du sseldorf, 8Department of General Neurology, Hertie Institute for Clinical Brain Research, Tu bingen and9Department 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,GermanyE-mail: [email protected]

The median survival of glioblastoma patients is 12 months. However, 3^5% of the patients survives for morethan 3 years and are referred to as long-term survivors. The clinical and molecular factors that contribute tolong-term survival are still unknown.To identify specific parameters thatmight be associated with this phenom-enon, we performed a detailed clinical and molecular analysis of 55 primary glioblastoma long-term survivorsrecruited at the six clinical centres of the German Glioma Network and one associated centre. An evaluationformwas developed and used to document demographic, clinical and treatment-associated parameters. In addi-tion, environmental risk factors, associated diseases and occupational risks were assessed.These patients werecharacterized by young age at diagnosis and a good initial Karnofsky performance score (KPS). None of theevaluated socioeconomic, environmental and occupational factors were associated with long-term survival.Molecular analyses revealed MGMT hypermethylation in 28 of 36 tumours (74%) investigated. TP53 mutationswere found in 9 of 31 tumours (29%) and EGFR amplification in10 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 of141 consecutive unselected glioblastoma patients registered in the German Glioma Network revealed signifi-cantly more frequent MGMT hypermethylation in the long-term survivor group. Taken together, our findingsunderline the association of glioblastoma long-term survival with prognostically favourable clinical factors,in particular young age and good initial performance score, as well asMGMT 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

IntroductionGlioblastoma multiforme is the most common and mostmalignant primary tumour of the brain and associated withone of the worst 5-year survival rates among all humancancers. Despite multimodal aggressive treatment, compris-ing surgical resection, local radiotherapy and systemicchemotherapy, the median survival time after diagnosis isstill in the range of just 12 months (Smith and Jenkins,2000), with population-based studies indicating evenshorter median survival (Ohgaki et al., 2004). Nevertheless,a small fraction of glioblastoma patients survive for morethan 36 months. These patients are referred to as long-termsurvivors.

With the exception of rare instances of glioblastoma inpatients with a hereditary tumour syndrome, e.g. Turcotssyndrome (Hamilton et al., 1995) or LiFraumeni syn-drome (Kleihues et al., 1997), most tumours originate ina sporadic fashion without any known genetic predisposi-tion. Several studies have evaluated the impact of variousexogenous factors, such as smoking (Zheng et al., 2001),diet (Huncharek et al., 2003; Lee et al., 1997), ionizingradiation (Brustle et al., 1992; Neglia et al., 1991), cellularphones (Inskip et al. 2001), electromagnetic fields (Savitzet al., 1998; Theriault et al., 1994), socioeconomic statusand education level (Schlehofer et al., 2005), as well asmedical risk factors, such as allergy (Wiemels et al., 2002),

doi:10.1093/brain/awm204 Brain (2007), 130, 2596^2606

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immunological status (Schlehofer et al., 1999) and viralinfections (Vilchez et al., 2003). However, no unequivocalevidence linking specific risk-factors to glioblastoma hasemerged from these studies, except for an association withthe exposure to ionizing radiation (for review see (Cavenee,2000)). In addition, younger age and a good Karnofskyperformance score (KPS) at the time of diagnosis areestablished clinical parameters associated with longersurvival (Curran Jr et al., 1993).

Despite the enormous progress in the understandingof the genetic alterations in glioblastomas, clinically usefulmolecular markers that help to predict response to therapyand prognosis are still rare. To date, only the methylationstatus of the O6-methylguanine methyltransferase (MGMT)gene has become a molecular marker of clinical signifi-cance. MGMT encodes a DNA repair protein that causesresistance to DNA alkylating agents, such as nitrosoureasand temozolomide. Transcriptional silencing of theMGMT gene by promoter hypermethylation is seen in50% of glioblastomas and has been linked to prolongedprogression-free and overall survival in glioblastomapatients treated with alkylating agents (Esteller et al.,2000b; Hegi et al., 2005). Combined deletions of thechromosomal arms 1p and 19q have been shown to beassociated with a favourable prognosis in oligodendroglialtumours (Cairncross et al., 2006; van den Bent et al., 2006).In glioblastoma, however, this aberration is rare andits 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 survivorscould help to identify yet unknown clinical, environmentaland/or molecular factors that are associated with favourableprognosis. Here, we report on a retrospective analysis of55 glioblastoma long-term survivors recruited within theGerman Glioma Network. In addition to basic clinical data,we evaluated the prevalence of environmental, occupationaland socioeconomic risk factors and screened for glioma-associated genetic aberration, i.e. TP53 mutation, EGFRamplification and 1p/19q deletion, as well as MGMThypermethylation.

Patients and MethodsPatient recruitment and data acquisitionPrimary glioblastoma patients with survival longer than36 months after diagnosis were retrospectively identified bythe six clinical centres of the German Glioma Network (www.gliomnetzwerk.de). In addition, 13 patients from an associatedcentre (Heinrich-Heine University Hospital Dusseldorf) wereincluded in the study. Five of these patients were included ina previous report on glioblastoma patients surviving for morethan 5 years (Steinbach et al., 2006). The study was approved bythe local ethics committees at the contributing clinical centres.A histological diagnosis of glioblastoma according to the WorldHealth Organization (WHO) classification of brain tumours wasconfirmed by central pathology review at the Brain TumourReference Centre of the German Society of Neuropathology and

Neuroanatomy (T. Pietsch). For this purpose, immunohistochem-ical characterization with antibodies against lineage-associatedantigens including glial fibrillary acidic protein was performed inaddition to conventional haematoxylin/eosin stainings. Possiblesarcomatous areas were illustrated by silver staining of reticulinfibres. The clinical data were evaluated by carefully scrutinizingthe patients clinical charts and by interviews with the patients.Relatives were contacted in case that the patient has alreadydeceased. A standardized basic evaluation form was used fordemographic, clinical and treatment-associated parameters.In addition, a special long-term survivor form was completedin which more details like environmental risk factors, associateddiseases and occupational risks were assessed. Both forms areavailable at www.gliomnetzwerk.de. Data were collected atthe Department of Neurosurgery, University Hospital Dresden.As a control population for the molecular analysis, we investigatedan independent series of primary glioblastomas (137 classicglioblastomas, one gliosarcoma and three giant cell glioblastoma)from 141 unselected patients operated at the clinical centres of theGerman Glioma Network between October 2004 and December2005. These tumours were derived from 83 male and 58 femalepatients with a median age at operation of 60.9 years.

DNA extractionTumour DNA useful for molecular analysis of one or more of theselected genes could be extracted from tumours of 38 glioblastomalong-term survivors. In 13 of these tumours, as well as in the 141glioblastomas of the independent control series, high-molecularweight DNA was extracted from unfixed frozen tissue samplesas described before (van den Bent et al. 2003). From the remain-ing 28 long-term survivor tumours, DNA was extracted fromformalin-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 nucleicacid extraction.

MGMT promoter methylation analysisMGMT promoter methylation was analysed by methylation-specific PCR as reported before (Mollemann et al., 2005). Theprimer sequences used to detect methylated MGMT promotersequences were 50-gtttttagaacgttttgcgtttcgac-30 and 50-caccgtcccgaaaaaaaactccg-30 (corresponding to nucleotides 46912-47033,GenBank accession no. AL355531; fragment size: 122 bp). Theprimer sequences used to detect unmethylated MGMT promotersequences were 50-tgtgtttttagaatgttttgtgttttgat-30 and 50-ctaccaccatcccaaaaaaaaactcca-30 (corresponding to nucleotides 4690947037;fragment size: 129 bp). As positive control sample, we used theA172 glioma cell line, which has a completely methylated MGMTpromoter. Genomic DNA from non-neoplastic brain tissue servedas an unmethylated control sample.

Detection of EGFR gene amplificationEGFR gene dosage was determined by real-time PCR analysisusing the ABI PRISM 5700 (Applied Biosystems, Darmstadt,Germany) sequence detection system. Continuous quantitativemeasurement of the PCR product was achieved by labelling ofdouble-stranded DNA with SYBR Green fluorescent dye (AppliedBiosystems). The following EGFR-specific primers were used:50-cactgcctcatctctcaccatc-30 (sense) and 50-gactcaccgtagctccagac-30

(antisense). The anonymous marker D2S1743 at chromosome

Long-term survival with glioblastoma Brain (2007), 130, 2596^2606 2597

2q21.2 was used as reference locus employing the primers50-catgactgcgagcccaagatg-30 (sense) and 50-caggtggtgtcatcagaatcag-30 (antisense). As constitutional references, we used DNAextracted from peripheral leukocytes as well as non-neoplasticbrain tissue samples from different patients. DNA from a glio-blastoma with known EGFR amplification served as a positivecontrol. Only tumours showing a normalized increase in EGFRgene dosage of more than 3-fold relative to constitutional DNAwere considered as showing EGFR amplification.

Multiplex ligation-dependent probeamplification (MLPA)MLPA was employed for the determination of allelic losses at1p and 19q in the long-term survivor group because constitutionalDNA was not available from the majority of patients. Analysiswas performed using the SALSA MLPA KIT P088 lots 0305 and0705 (MRC Holland, Amsterdam, the Netherlands). MLPA wasperformed according to the protocol of Jeuken et al. (Jeuken et al.,2006). PCR was performed with 35 cycles (95C, 30 s; 60C, 30 s;72C, 1 min; final extension 20 min, 72C). Fragments wereseparated and quantified on an ABI 3730XL DNA analyzer(Applied Biosystems, Foster City, CA) and Genemapper 3.7software 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 arms1p 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 weredivided by the value for average peak area. We determined thisaverage ratio on DNA samples from seven non-neoplastic tissuesfor each probe on 1p and 19q and repeated this experiment fourtimes. Standard deviations of this ratio for each probe weredetermined for the data of the multiple rounds of analyses onthese seven control tissues. A ratio deviating for >2 SDs from thatestablished for each of the probes in control tissues was scoredas reduced gene dosage. Because SDs for all data from controlswere within narrow limits, data from tumours with a ratio smallerthan 0.7 fulfilled these criteria for reduced gene dosage. DNA fromtumours was scored as deleted if two or more probes on 1p or19q adjacent to each other exhibited a ratio smaller than 0.7.

Loss of heterozygosity (LOH) analysis atmicrosatellite markers on 1p and 19qAllelic losses in the control series were determined bymicrosatellite-based LOH analysis as reported (Felsberg et al.,2004; Hartmann et al., 2005). The following microsatellite lociwere 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, silverstaining and assessment for LOH was carried out as describedelsewhere (Felsberg et al., 2004; Hartmann et al., 2005).

Single strand conformation polymorphism(SSCP) analysis and direct sequencingExons 49 of the TP53 gene were screened for mutations usingSSCP analysis as reported before (Wellenreuther et al., 1995;von Deimling et al. 2000). Aberrant SSCP bands were excised andthe DNA was extracted as described (Wellenreuther et al., 1995).

Following reamplification with the same set of primers thePCR products were sequenced on a semiautomated sequencer(Applied Biosystems, model 377, Foster City, CA) using theTaq cycle sequencing kit (Applied Biosystems, Foster City, CA).Each amplicon was sequenced bidirectionally.

Statistical analysisThe Fishers exact test or the 2 tests were used to compare thefrequencies of molecular aberrations in the long-term survivorgroup versus the control group.

ResultsGeneral demographicsThe study population of glioblastoma long-term survivorsconsisted of 55 patients (male 28; female 27) with a medianage at diagnosis of 51 years (range 2172 years).In comparison, the cohort of 141 unselected glioblastomashowed a significantly higher age (median 60.9 years,P50.001). The median follow-up time in the long-termsurvivor group was 7 years and the median survival timewas 4.6 years (range 3.015.3 years). Median KPS atdiagnosis for the entire population of 55 long-termsurvivors was 80 (range 30100), with five patients havingscores of570 (Table 1).

Patients were married (49%), single (18%) or divorced(2%); data on marital status were not available for17 patients (31%). The average number of children wastwo. There were nine (16%) current smokers amongthe entire study population, consuming 345 cigarettesper day [no data in 22 cases (40%)]. Occasional alcoholconsumption was admitted by 10 (18%) patients,

Table 1 Patient characteristics

Total population of primaryglioblastoma

n=55

Male n=28Female n=27

Median age at diagnosis 51 (21^72) yearsMedian KPS 80 (30^100)Median survival 4.6 years (3.0^15.3 years)Tumour localization n (%)Right hemisphere 31 (56.4)Left hemisphere 23 (41.8)Both hemispheres 1 (1.8)Frontal 24a (43.6)Temporal 17 (30.9)Parietal 16 (29.1)Occipital 11 (20.0)

Treatment of glioblastomaTumour resectionb 55 (100.0)Radiotherapy 55 (100.0)Any chemotherapy 37 (67.3)

KPS=Karnofsky performance score, partly retrospectivelyevaluated.a13 Tumours involved more than one lobe.bAll patients had gross total resection or partial resection,a biopsy was performed only once for recurrent tumour.

2598 Brain (2007), 130, 2596^2606 D. Krex et al.

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

HistoryInborn defects or inherited diseases were recorded in onecase each; one patient had an anomaly of the ear andone had thalassaemia minor. Regarding childhood diseasesand vaccination status, only data from 10 (18%) and7 (13%) patients were available. Within these groups theprevalence of measles, small-pox, scarlet fever, whooping-cough, mumps and German measles was identicalwith 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 withthe status in the general population (Reiter, 2004).

Associated risk factorsThere was no preponderance of any occupation amongthe study population. Twenty-four different occupationswere documented, in addition to retired patients, home-makers and patients without any occupation. One profes-sion (book-keeper) was represented four times, another one(farmer) three times and six jobs were represented twice.

Coexisting tumour within the family was found innine (16%) patients. The recorded tumour entities were:hepatocellular carcinoma, stomach carcinoma, breast carci-noma (twice), renal carcinoma, lung carcinoma, coloncarcinoma and abdominal wall carcinoma.

An accident in the history was found in 12 (22%)patients resulting in fractures in nine (16%), tendonrupture in two (4%), and in incision injury in one patient.There was only one patient with a significant head injuryin history (skull fracture).

Any other operation not for repair of accident relatedinjuries was reported in 27 (49%) patients. There were13 different procedures including cholecystectomy (threetimes), endoscopic knee joint exploration (four times) andappendectomy (twice). No patient had undergone priorsurgery for an intracranial tumour.

Sixteen different diagnoses in 22 patients were registeredas co-morbidities. Diabetes mellitus was found in four anddepression in two patients. Gall bladder disease and arterialhypertension were found in two patients each. Among allco-morbidities there was no preponderance of any organsystem. In order to evaluate disorders associated withintracranial location, only the two with depression wereidentified.

Tumour localization and histological featuresTumour localization was in the left cerebral hemisphere in23 (42%), right cerebral hemisphere in 31 (56%) patientsand in both frontal lobes in one patient. Thirteen (23%)tumours involved more than one lobe. The frontal lobe was

involved in 24 (44%) patients, followed by temporal (31%),parietal (29%) and occipital (20%) lobe affliction.

In five of the tumours, the histology correspondedto giant cell glioblastoma (WHO grade IV). Two additionalcases were classified as glioblastoma with sarcomatouscomponent (gliosarcoma WHO grade IV). Two cases exhi-bited a significant oligodendroglial component in additionto the typical areas of glioblastoma with astrocytic differ-entiation. These cases were classified as glioblastoma witholigodendroglial component. All other tumours corre-sponded histological to classic glioblastoma multiforme(WHO grade IV).

Surgical treatmentAll patients had planned gross total resection during thefirst surgery. The extent of surgery was determined onpostoperative MR images. Due to the multicentric retro-spective evaluation of data, the time between surgeryand MRI data acquisition was not uniform. However, thefirst post-operative MRI data were recorded. Accordingly,16 (29%) complete resections, i.e. no contrast-enhancingresidual tumour, and 25 (45%) partial resections,i.e. contrast-enhancing tumour detectable irrespective ofsize were documented whereas no data were available in14 (25%) patients. Twenty-five patients (45%) had a secondoperation (seven complete and 10 partial resections, onebiopsy, seven no data), 12 patients (22%) had a thirdoperation (three complete, six partial resections, three nodata), and four patients (7%) were operated on a fourthtime (two partial resections, two no data).

Adjuvant treatmentFollowing surgery, all patients had involved-field radio-therapy. A total dose between 59 and 60 Gy was appliedto 46 out of 55 patients (84%); in seven patients, a lowerdose (between 46 and 56 Gy) was applied because of sideeffects and in two patients higher doses of 70 and 80 Gywere applied. A combination of involved-field radiotherapyand stereotactic convergence radiotherapy was implementedin two patients; one patient had stereotactic convergenceradiotherapy only. Any kind of chemotherapy was appliedto 37 patients (67%). Some patients received more than oneprotocol (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 thethird, and to one patient after the fourth operation.Nimustine (ACNU) was the most frequently (16 out of31 patients) used drug in first-line chemotherapy protocolsas monotherapy in one, or in combination either withteniposide in thirteen, or with cytarabine in three patients.Temozolomide was applied to 21 patients, including fivepatients who received combined radio-chemotherapy.The PCV (procarbacine, lomustine, vincristine) protocolwas applied to seven patients after radiotherapy. ACNU,temozolomide and PCV were also used for chemotherapy


after the second to fourth operation. A total of 13 patientsreceived more than one chemotherapy regime (Table 2).

Molecular analysesDNA suitable for molecular analysis could be extractedfrom 38 of 55 long-term survivors. However, due to DNAdegradation and limited amounts of DNA, not all 38tumours could be investigated for aberrations in each of thefour selected genes. Exons 49 of the TP53 gene werescreened for mutations in 31 tumours. TP53 mutationswere identified in tumours of nine patients (16%). Thedetected mutations included nine distinct missense muta-tions (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>delGp.45fs). The EGFR gene dose could be assessed in all38 tumours and revealed EGFR gene amplification in10 patients (26%). Combined allelic losses on chromosomearms 1p and 19q were detected in 2 of 32 tumoursinvestigated by MLPA (6%). In both cases, histologicalevaluation did not reveal an oligodendroglial component.The methylation status of the MGMT promoter wasdetermined in 36 patients and revealed MGMT hyper-methylation in tumours of 28 patients (74%). In 10patients, tissue samples from recurrent tumours wereadditionally analysed for MGMT methylation. In allinstances, the MGMT status in the primary tumour wasretained in the recurrent tumour (eight methylated,two unmethylated). There was no correlation of molecularfeatures with tumour location.

Similar to the long-term survivor group, not all141 tumours of the control group of unselected consecutiveprimary glioblastomas could be studied for each of thegenes. Data on the EGFR copy number were obtained from138 tumours and revealed EGFR gene amplification in60 tumours (44%). A total of 136 tumours from this serieswere studied for allelic losses on 1p and 19q, which revealedcombined losses on both chromosome arms in 14 tumours(10%). MGMT hypermethylation was detected in 59 of136 tumours investigated (43%). Mutational analysis of135 tumours for TP53 aberrations identified mutationsin 30 tumours (22%). Statistical comparisons revealed thatMGMT hypermethylation was significantly more commonin glioblastomas from the long-term survivor group ascompared to glioblastomas from the unselected patientgroup (P= 0.0002, 2 test). In contrast, the frequencies ofTP53 mutation, EGFR amplification and 1p/19q lossesdid not significantly differ between both groups, althoughthere was a trend towards less frequent EGFR amplificationin the long-term survivor group (Table 3).

DiscussionThis is the largest series of glioblastoma long-term survivorsreported to date. We provide a clinical characterization andreport on molecular analyses of 55 primary glioblastomapatients with a survival time of >36 months. There wasno association between socioeconomic, environmental andoccupational factors and long-term survival. Molecularanalyses indicated a higher proportion of MGMT methyla-tion in the long-term survivor group when comparedwith a reference sample of an unselected consecutive seriesof glioblastomas.

Table 2 Chemotherapy in primary glioblastoma

After First OP After second OP After third OP After fourth OP

Nimustine 16 1 2 0PCV 7 3 1 1Temozolomide 12 7 3 0More than one chemotherapyregimen in total n (%)a

4 (7.2) 7 (12.7) 13 (23.6) 13 (23.6)

No chemotherapy n (%)a 24 (43.6) 19 (34.5) 18 (32.7) 18 (32.7)Total number of patients treated n (%)a 31 (56.4) 36 (65.5) 37 (67.3) 37 (67.3)

aPercentage of all glioblastoma patients (n=55).

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

Glioblastomas fromlong-term survivors

Glioblastomas fromcontrol series

Statistical results P

TP53 mutation 9/31 (29%) 30/135 (22%) 0.4199 (2 test)EGFR amplification 10/38 (26%) 60/138 (44%) 0.0556 (2 test)Combined 1p/19q loss 2/32 (6%) 14/136 (10%) 0.7394 (Fishers exact test)MGMT hypermethylation 28/36 (74%) 59/136 (43%) 0.0002 (2 test)

2600 Brain (2007), 130, 2596^2606 D. Krex et al.

The median age of glioblastoma patients is >60 yearsaccording 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 controlgroup of unselected consecutive glioblastoma patients was60.9 years. In contrast, the median age of the long-termsurvivors in our study was considerably lower, i.e. 51 years

(P50.001). This is in accordance with numerous clinicalstudies indicating that young age at the time of diagnosisis an important parameter associated with longer survival(Burger and Green 1987; Curran Jr et al., 1993; Devauxet al., 1993; Chang et al., 2005). Taking data from all 281published glioblastoma long-term survivors (Table 4), theirmedian age is 36.9 years, which supports that age is of

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

Number of cases Mean age male female Mean KPS score GTR RT Chemotherapy

Netsky et al., 1950 8 na na na na na na naRoth and Elvidge, 1960 20 na na na na na na naLey et al., 1962 6 na na na na na 6 naTaveras et al., 1962 13 na na na na na na naElvidge and Barone, 1965 2 24.5 1 1 na 2 1 0Gullotta and Bettag, 1967 1 45 1 na na 1 1 0Jelsma and Bucy, 1967 6 na na na na 6 na naDara et al., 1980 3 na na na na na na 3Johnson 1981 1 32 na 1 na 1 0 0Hatanaka et al., 1984 1 50 1 na 100 0 1 0Bucy et al., 1985 1 30 1 na na 1 1 0Salford et al., 1988 2 14.5 1 1 na 1 2 0Akslen et al., 1989 2 41.5 1 1 na 2 2 1Ishikura et al., 1989 1 8 na 1 na 1 0 0Margetts and Kalyan-Raman, 1989 3 41.3 2 1 90a 3 3 3Imperato et al., 1990 5 42.6 3 2 na 5 5 4Shibamoto et al., 1990 1 51 1 na na 1 1 0Rutz et al., 1991 1 21 1 na na 1 1 0Vertosick, Jr. and Selker, 1992 13 40.9 7 6 86a na 13 13Hiesiger et al., 1993 10 47 na na 81 1 10 10Chandler et al., 1993 22 39.2 10 12 80 2 22 18Phuphanich et al., 1993 7 37 5 2 na na 7 6Archibald et al., 1994 7 37.7 2 5 na 2 7 7Wester et al., 1994 1 45 na 1 na 1 1 0Morita et al., 1996 10 39.2 7 3 na 10 na naNew et al., 1997 1 34 na 1 100 1 1 1Pollak et al., 1997 2 20 1 1 na 2 2 0Klein et al., 1998 1 11 na 1 na 1 1 1Cervoni et al., 1998 1 13 na 1 80 1 1 1Salvati et al., 1998 11 39 5 6 80 11 11 11Puzzilli et al., 1998 1 50 1 na 90 0 1 0Scott et al., 1999 15 43.5 9 6 90 na na naYoshida et al., 2000 2 40 1 1 na 1 2 2Sabel et al., 2001 1 69 1 na na 1 0 0Burton et al., 2002ab 41 39 16 25 90 na na naValery et al., 2002 1 42 na 1 na 1 1 1Floeth et al., 2003 1 37 1 na 100 1 1 1Ho et al., 2003 6 na na na na na na naMcLendon and Halperin 2003 17 40.2 11 6 na na na naSchmidinger et al., 2003 5 47 3 2 na na na naJagannathan et al., 2004 4 46.3 2 2 na na 4 naRainov and Heidecke 2004 1 27 1 na 90 na na naShinojima et al., 2004 6 44.2 na 6 85 4 6 6Deb et al., 2005 6 27 3 3 na na na naYamanaka et al., 2006 1 42 1 na 90 1 1 naSteinbach et al., 2006 10 42 4 6 90 9 10 9Total 281 36.9 104 105 89 75 126 98

Data from a recent review for glioblastoma patiens living longer than 5 years (Shinojima et al., 2004) are included.aCalculated on the basis of data that were provided in the concerning paper.bThe 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.


predictive value in glioblastoma. On the other hand, fourof our patients were 65 years or older, indicating that olderage does not exclude long-term survival of glioblastoma.Interestingly, the median age in our study is almost 15 yearsabove to that of all published cases. This might in partbe explained by the fact that older studies are possiblycontaminated with low-grade tumours in young adults thatwere mistaken for glioblastoma, in particular pleomorphicxanthoastrocytomas. In our study population we hadfive 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 oflong-term survivors (9.1% versus 2.1%, P= 0.041) support-ing the hypothesis that this histological variant is associatedwith longer survival (Shinojima et al., 2004). A contamina-tion of glioblastoma patients cohorts by high gradetumours of the oligodendroglial lineage may also occur.However, in our series, only two cases showed a minoroligodendroglial component in an otherwise typicalglioblastoma.

In general, glioblastomas are more frequent in males,with a male/female ratio between 1.3 and 1.45, correspond-ing to a proportion of female patients of 43 and 41%,respectively (Barnholtz-Sloan et al., 2003; Ohgaki andKleihues 2005). In line with these data, the male tofemale ratio in our control series of 141 unselectedglioblastoma patients was 1.43. The long-term survivorsshowed a trend to a higher proportion (50%, 95% CI 3862%) of female patients, although the proportion reportedin the literature is still within the limits of the confidenceinterval. Nevertheless, it seems that glioblastoma long-termsurvival is favoured by the combination of two basic clinicalparametersyoung age and female gender. Unfortunately,the median follow up time in the control group did notallow validation of these findings. Several environmentaland socioeconomic risk factors have been associated withthe development of malignant glial tumours (Lee et al.,1997; Inskip et al., 2001; Huncharek et al., 2003; Schlehoferet al., 2005). One might assume that the absence ofsuch factors could be associated with a better prognosis inthose tumours. However, in the present study we failedto identify any of those factors evaluated by our question-naire to be obviously under- or overrepresented in long-term survivors. Although such data are difficult to obtain ina retrospective setting, our results support the hypothesisthat socioeconomic, environmental and occupationalfactors do not play major roles in determining whethera 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 confirmedthat the extent of resection is associated with improvedprogression-free survival (Stummer et al., 2006). Althoughwe had no uniform early postoperative MRI scanningto document the extent of resection in our patients,the fact that all 55 glioblastoma long-term survivors had

intended gross total resection as initial treatment confirmsthat tumour resection enhances the chances for a favourableoutcome.

All patients had adjuvant radiotherapy. Standard treat-ment for glioblastoma includes postoperative radiotherapy;hence radiotherapy is unlikely to be a positive selectionfactor. Chemotherapy for malignant gliomas has experi-enced a renaissance since the publication of the EORTC26981/22981-NCIC CE3 phase III randomized trial, whichdemonstrated that concomitant and adjuvant temozolo-mide chemotherapy has a positive effect on survival ofpatients with glioblastoma (Stupp et al., 2005). In ourstudy, patients were treated between 1986 and 2003.Therefore, only 21 patients received temozolomide treat-ment, among these were 12 patients who had temozolomidefor initial adjuvant treatment. A total of 37 patients (67%)had at least one or up to three different adjuvant chemo-therapy regimens, although chemotherapy was not includedin standard of care when those patients were treated.Therefore, these data are in support of a positive role forchemotherapy in glioblastoma with respect to long-termsurvival. However, the study design does not allow to definechemotherapy as a prognostic factor. Here, the samelimitations apply as for the number of surgical interven-tions. Patients who have a less malignant course of diseasealso have more options to undergo multiple surgicaland chemotherapeutic interventions. Yet, nitrosourea-based chemotherapy has previously been defined asa factor favouring long-term survival in the classicalmeta-analyses published in 1993 by Fine and colleaguesand in 2002 by the Glioma Meta-analysis Trialists group.These authors reported that there are consistently morelong-term surviving patients in the experimental trial armsof adjuvant nitrosourea-based chemotherapy (Fine et al.,1993; Stewart 2002).

Recent translational studies have reported that hyper-methylation of the MGMT promoter is associated withprolonged progression-free and overall survival in glioblas-toma patients treated with alkylating agents (Esteller et al.,2000a; Hegi et al., 2005). In line with these data, MGMTpromoter methylation was observed in the majority ofglioblastomas from long-term survivors. We found MGMThypermethylation to be significantly (almost two-fold)more common in long-term survivors (74%) as comparedto an unselected consecutive series of glioblastoma patientstreated at the clinical centres of the German GliomaNetwork (43%). On the other hand, it is also important tonote that the absence of MGMT promoter methylation isstill compatible with long-term survival in individualpatients.

In addition to MGMT methylation, we investigatedgenetic alterations that are characteristic of either primary(EGFR amplification) or secondary glioblastomas (TP53mutation) (Kleihues and Ohgaki, 2000), as well as alleliclosses on 1p and 19q, which are an established prog-nostic marker for anaplastic oligodendroglial tumours

2602 Brain (2007), 130, 2596^2606 D. Krex et al.

(Cairncross et al., 2006; van den Bent et al., 2006).In comparison to our control group of unselected consecu-tive glioblastoma patients, there was a trend towards lesscommon EGFR amplification in glioblastomas from long-term survivors. However, statistical analysis did not confirma significant difference in EGFR amplification frequencybetween the long-term survivor group and the controlgroup (Table 3). In the literature, conflicting data on theprognostic relevance of EGFR amplification in glioblasto-mas have been reported. While EGFR amplification and/oroverexpression has been associated with a poor prognosisin some studies (Hiesiger et al., 1993; Zhu et al., 1996;Etienne et al., 1998; Korshunov et al., 1999), other authorscould not substantiate a prognostic significance (Quanet al., 2005), or even reported an association with betterprognosis (Houillier et al., 2006; Smith et al., 2001). Ourfinding suggests that the likelihood of long-term survival ishigher in glioblastomas without EGFR amplification.However, it has to be considered that EGFR amplificationis more common in glioblastomas of older patients(von Deimling et al., 2000). Therefore, the lower percentageof EGFR amplified tumours among the long-term survivorsmight be due to the significantly lower median age ofthis patient group.

Similar to EGFR amplification, the prognostic signifi-cance of TP53 mutations in glioblastoma is unclear.In glioblastomas, some studies reported a better prognosisof 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 notsignificant when adjusted for age at diagnosis (Ohgaki et al.,2004). In our study, the frequencies of TP53 mutation didnot differ significantly between the long-term survivorgroup and the control group, which does not supportan association between the TP53 mutation status andlong-term survival.

In contrast to anaplastic oligodendrogliomas andoligoastrocytomas (Cairncross et al., 2006; van den Bentet al., 2006), the prognostic significance of 1p/19q deletionin glioblastomas is not clear yet (Ino et al., 2000; Schmidtet al., 2002; Brat et al., 2004; Houillier et al., 2006). Whilethe incidence of combined 1p/19q losses is low in glio-blastomas (von Deimling et al., 2000), some studiesreported that the presence of an oligodendroglial compo-nent is associated with more frequent 1p/19q deletion andbetter prognosis (He et al., 2001; Kraus et al., 2001). Otherauthors did not confirm a better outcome of glioblastomapatients with oligodendroglial component in a multivariateanalysis adjusted for age and gender (Homma et al., 2006).However, these authors found that allelic losses on 1p wereassociated with longer survival in glioblastoma. Our studydid not identify an increased frequency of 1p/19q lossesin glioblastoma long-term survivors when compared toour control series of unselected glioblastoma patients.

The central pathology review confirmed classic glioblastomahistology in the three cases with 1p/19q loss. These datasuggests that long-term survival of classic glioblastomais unrelated to 1p/19q loss. Furthermore, we can excludea major contamination of our series with malignantoligodendroglial neoplasms.

We conclude that although we confirmed certain clinicalfactors and MGMT promoter hypermethylation to be asso-ciated with prolonged survival, our current study does notexplain why the 55 glioblastoma patients reported herebecame long-term survivors. Thus, our results do not allowrefining current treatment and overall counselling strategiesfor patients with glioblastoma. We assume that furthermolecular analyses employing large-scale microarray-basedgenomic and expression profiling approaches will identifymolecular features that are specific to glioblastomas oflong-term survivors. For this purpose, the German GliomaNetwork is prospectively collecting fresh tissue from allglioblastoma patients operated at its participating centresand shall thus be able to perform comparative profilingexperiments on a reasonable number of long-term survivorsfrom this large population within the next few years.

AcknowledgementsThe German Glioma Network is funded by the DeutscheKrebshilfe e.V., grant no. 70-3163-Wi33. We would liketo thank all study nurses of the German Glioma Networkfor their work in CRF documentation and evaluation ofclinical data. The German Glioma Network is sponsoredby the Deutsche Krebshilfe (German Cancer Aid).

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Long-term Survival With Glioblastoma Multiforme