# IDH1/IDH2 but Not TP53 Mutations Predict Prognosis in Bulgarian Glioblastoma Patients

Authors: Gergana Stancheva; Teodora Goranova; Maria Laleva; Margarita Kamenova; Atanaska Mitkova; Nikolay Velinov; George Poptodorov; Vanio Mitev; Radka Kaneva; Nikolay Gabrovsky
Journal: BioMed Research International
 (2014)

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## Abstract

Mutations in genes encoding isocitrate dehydrogenase isoforms 1 (IDH1) and 2 (IDH2) have been associated with good prognosis for patients with brain neoplasias and have been commonly found together with mutated TP53 gene. To determine the prevalence of IDH1, IDH2, and TP53 mutations and their impact on overall survival 106 glioblastoma patients were analysed. IDH1 mutations were detected in 13 and IDH2 mutation in one patient. Two homozygous samples with R132H mutation in IDH1 gene and a novel aberration K129R in IDH2 gene were found. Sixty-four percent of IDH1/IDH2 mutated tumours harboured also a mutation in TP53 gene. Genetic aberrations in TP53 were present in 37 patients. Statistical analysis of the impact of the studied factors on the overall survival showed that the mutations in IDH1/IDH2, but not the ones in TP53, were associated with longer survival. Also, the impact of age on prognosis was confirmed. This is the first comprehensive study on glioblastomas in Bulgaria. Our results suggest that IDH1/IDH2 but not TP53 mutations together with other prognostic factors such as age might be applied in clinical practice for prediction of outcome in patients with glioblastomas.


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## Body

## 1. Introduction

The most common primary brain tumours are gliomas. The worldwide incidence of the most malignant form, glioblastoma multiforme (GBM, WHO grade IV glioma), is 2-3/100000 people per year [1] with a median survival of 6–12 months [2, 3]. In spite of the existing classification, glioma subgroups are not homogeneous in terms of survival [1]. Several prognostic factors have been proposed: age, extent of resection, and KPS, and also some molecular markers [4–6].A study has revealed somatic mutations in a gene encoding isocitrate dehydrogenase 1 (IDH1) in 12% of GBMs [7]. Isocitrate dehydrogenases (IDHs) belong to a family of enzymes catalyzing oxidative decarboxylation of isocitrate to α-ketoglutarate. The human genesIDH1,IDH2,IDH3A,IDH3B, andIDH3G encode three IDH-enzymes: NADP+-dependent IDH1 localized within cytoplasm and peroxisomes, mitochondrial NADP+-dependent IDH2, and NAD+-dependent IDH3, respectively [8–10]. Mutations have been found not only inIDH1 andIDH2, predominantly in gliomas, but also in leukemia samples and very rarely in other cancers [11, 12]. Three amino acid residues, Arg132, Arg109, and Arg100, participate in the formation of the catalytic centre involved in isocitrate binding. Almost all published mutations inIDH1 affect Arg132. Genetic aberrations inIDH1 have been reported in 50–80% of gliomas WHO grade II to IV [13–17]. Only primary GBM shows low frequency ofIDH1 mutations: 5% [17]. Approximately 90% of the R132 mutations are of the R132H type, followed by R132C changes in 4% and R132S and R132G in about 1.5% each, and very rarely the R132L mutation [10, 16, 17]. TheIDH2 aberration in Arg172 residue is an analogue of the one in Arg132 ofIDH1. Mutations in theIDH2 gene have been detected in up to 3% of glial tumours WHO grades II and III but not in GBM [17, 18]. Aberrations in bothIDH1 andIDH2 genes have been associated with better prognosis in glioma patients of various grades [19, 20].In a recent large scale study over 80% of the gliomas withIDH1aberrations carriedTP53 mutations and/or 1p/19q loss (mainly in oligodendrogliomas) [17]. The tumour-suppressor geneTP53 encodes a protein p53 implicated in the pathogenesis of many cancers.TP53mutations have been reported not only in about 30% of gliomas [21, 22], mainly low-grade ones, but also in secondary glioblastomas (65% versus 28% in primary GBM) [23]. In secondary GBM over 50% of all mutations inTP53 have been observed in codons 248 and 273 whereas in primary glioblastomas only 17% occur there. While mostTP53 aberrations resulted in decreased apoptosis in response to DNA damage, thus enabling tumour growth and influencing negatively patient’s overall survival, those in codons 248 and 273 have been associated with better survival for patients [7, 23–26].The objective of the present study was to investigate the frequency ofIDH1,IDH2, andTP53mutations in Bulgarian patients with glioblastomas and their association with survival time and various clinical features in a search for prognostic factors.


## 2. Materials and Methods

### 2.1. Samples

Tumour tissues from 106 glioblastoma patients have been collected since 2005 in the Department of Neurosurgery, University Multiprofile Hospital for Active Treatment and Emergency Medicine (UMHATEM) “N.I.Pirogov,” Sofia, Bulgaria. All samples were diagnosed and independently histopathologically confirmed in the Department of Pathology, UMHATEM, by experienced neuropathologist according to the 2007 WHO classification system of CNS tumours [27]. GBMs were classified as primary when there was no clinical history of prior lower grade tumour and as secondary when patients had initially lower grade gliomas that progressed to GBM. The patients included 53 males and 53 females with a median age of 56 years old. This age was used to divide the cohort in two groups: young (less than 56 years old) versus old (more than 56 years old). In order to compare the gliomas that developed in one patient, we included tumour samples from two consecutive surgeries of 3 GBM patients and those from 3 surgical extractions in one patient initially diagnosed with astrocytoma WHO grade II, followed by GBM WHO grade IV. The study was approved by the Ethical Committee of Medical University of Sofia and informed consent was obtained from every patient.


### 2.2. DNA Extraction and PCR Amplification

DNA was extracted from 64 fresh-frozen and 47 FFPE tumour tissues using the QIAamp DNA Mini kit (Qiagen, Valencia, CA), according to the manufacturer’s recommendations. The quantity of the DNA was assessed using NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE). For the detection of mutations, primers were designed to amplify exon 4 of theIDH1, exon 4 of theIDH2, and exons 5-8 of theTP53 gene using Primer3 software (http://frodo.wi.mit.edu/primer3/) (Supplementary Table ST1 available online at http://dx.doi.org/10.1155/2014/654727). Polymerase chain reaction amplification was performed in a total volume of 10 μL including 30 ng DNA, 0.5 μM each primer, and 1xRedTaq PCR Master Mix (Sigma-Aldrich Co. LLC, St. Louis, MO, USA). The reaction mixture was subjected to initial denaturation at 95°C for 10 minutes, followed by 40–45 cycles consisting of denaturation at 95°C for 30 seconds, annealing at required temperature (Supplementary Table ST1) for 30 seconds, extension at 72°C for 40 seconds, and a final extension at 72°C for 10 minutes.


### 2.3. Direct Sequencing

A total of 1.5μL of each amplified product was subjected to ExoSap treatment followed by direct sequencing in both directions with the primers listed above using the BigDye Terminator v3.1 Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The sequencing reaction was run on ABI 3130xl Genetic Analyzer (Applied Biosystems) according to the supplier’s protocol and as described elsewhere [28]. The SeqScape Software v.2.5 (Applied Biosystems) was used for the interpretation of the sequence electropherograms.


### 2.4. Loss of Heterozygosity (LOH) Analysis

LOH was studied by fragment analysis of the microsatellite markers D15S996, D15S116, D15S202, and D15S127 in tumour and paired normal glial cells. Primer sequences for each marker were retrieved from NCBI’s UniSTS database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=unists) and each forward primer was labeled on the 5′ end with one of the two fluorescent dyes 6-FAM or HEX. PCR amplification was performed in one multiplex reaction with only 30 cycles of denaturing, annealing, and extension at 95°C for 30 s, 55°C for 30 s, and 72°C for 40 s, respectively. PCR products were separated on a 3130xl DNA Analyzer (Applied Biosystems). The GeneMapper Software v.4.0 (Applied Biosystems) was used for visualization of microsatellite markers.


### 2.5. Statistical Analysis

Statistical processing of the data was performed using Statistical Package for Social Sciences (SPSS) ver.17.0 (SPSS Inc., Chicago, IL, USA). Overall survival (OS) was calculated from the date of diagnosis until death or end of follow-up. Kaplan-Meier survival curves were plotted and the log-rank test was used to compare survival between groups. Also, univariate and multivariate Cox regression analyses were done to test the association of various factors with survival time. Six patients were excluded from the analysis because of insufficient clinical data.


## 2.1. Samples

Tumour tissues from 106 glioblastoma patients have been collected since 2005 in the Department of Neurosurgery, University Multiprofile Hospital for Active Treatment and Emergency Medicine (UMHATEM) “N.I.Pirogov,” Sofia, Bulgaria. All samples were diagnosed and independently histopathologically confirmed in the Department of Pathology, UMHATEM, by experienced neuropathologist according to the 2007 WHO classification system of CNS tumours [27]. GBMs were classified as primary when there was no clinical history of prior lower grade tumour and as secondary when patients had initially lower grade gliomas that progressed to GBM. The patients included 53 males and 53 females with a median age of 56 years old. This age was used to divide the cohort in two groups: young (less than 56 years old) versus old (more than 56 years old). In order to compare the gliomas that developed in one patient, we included tumour samples from two consecutive surgeries of 3 GBM patients and those from 3 surgical extractions in one patient initially diagnosed with astrocytoma WHO grade II, followed by GBM WHO grade IV. The study was approved by the Ethical Committee of Medical University of Sofia and informed consent was obtained from every patient.


## 2.2. DNA Extraction and PCR Amplification

DNA was extracted from 64 fresh-frozen and 47 FFPE tumour tissues using the QIAamp DNA Mini kit (Qiagen, Valencia, CA), according to the manufacturer’s recommendations. The quantity of the DNA was assessed using NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE). For the detection of mutations, primers were designed to amplify exon 4 of theIDH1, exon 4 of theIDH2, and exons 5-8 of theTP53 gene using Primer3 software (http://frodo.wi.mit.edu/primer3/) (Supplementary Table ST1 available online at http://dx.doi.org/10.1155/2014/654727). Polymerase chain reaction amplification was performed in a total volume of 10 μL including 30 ng DNA, 0.5 μM each primer, and 1xRedTaq PCR Master Mix (Sigma-Aldrich Co. LLC, St. Louis, MO, USA). The reaction mixture was subjected to initial denaturation at 95°C for 10 minutes, followed by 40–45 cycles consisting of denaturation at 95°C for 30 seconds, annealing at required temperature (Supplementary Table ST1) for 30 seconds, extension at 72°C for 40 seconds, and a final extension at 72°C for 10 minutes.


## 2.3. Direct Sequencing

A total of 1.5μL of each amplified product was subjected to ExoSap treatment followed by direct sequencing in both directions with the primers listed above using the BigDye Terminator v3.1 Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The sequencing reaction was run on ABI 3130xl Genetic Analyzer (Applied Biosystems) according to the supplier’s protocol and as described elsewhere [28]. The SeqScape Software v.2.5 (Applied Biosystems) was used for the interpretation of the sequence electropherograms.


## 2.4. Loss of Heterozygosity (LOH) Analysis

LOH was studied by fragment analysis of the microsatellite markers D15S996, D15S116, D15S202, and D15S127 in tumour and paired normal glial cells. Primer sequences for each marker were retrieved from NCBI’s UniSTS database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=unists) and each forward primer was labeled on the 5′ end with one of the two fluorescent dyes 6-FAM or HEX. PCR amplification was performed in one multiplex reaction with only 30 cycles of denaturing, annealing, and extension at 95°C for 30 s, 55°C for 30 s, and 72°C for 40 s, respectively. PCR products were separated on a 3130xl DNA Analyzer (Applied Biosystems). The GeneMapper Software v.4.0 (Applied Biosystems) was used for visualization of microsatellite markers.


## 2.5. Statistical Analysis

Statistical processing of the data was performed using Statistical Package for Social Sciences (SPSS) ver.17.0 (SPSS Inc., Chicago, IL, USA). Overall survival (OS) was calculated from the date of diagnosis until death or end of follow-up. Kaplan-Meier survival curves were plotted and the log-rank test was used to compare survival between groups. Also, univariate and multivariate Cox regression analyses were done to test the association of various factors with survival time. Six patients were excluded from the analysis because of insufficient clinical data.


## 3. Results

### 3.1. Genetic Aberrations inIDH1,IDH2, andTP53 Genes

A total of 111 glioma samples from 106 patients were analyzed forIDH1, IDH2, andTP53 mutations. Genetic aberrations in at least one of the genes were found in 46 samples from 42 patients (39.6%) (Supplementary Table ST2). Exon 4 ofIDH1 gene was mutated in 13 patients (12.3%). AllIDH1 aberrations were missense mutations at codon 132 leading to the substitution arginine to histidine (c.395G>A, R132H) (Table 1).Table 1
MutationsinIDH1,IDH2, and TP53 genes that were detected in 106 brain tumour patients.
Gene
Exon
Nucleotide change (amino acid change)
Number of patients with mutations (which are homozygous)
% mutations among all mutations in the gene
IDH1
Exon 4
c.395G>A
(R132H)
13 (1)
100 (7.7)
IDH2
Exon 4
c.386A>G
(K129R)
1 (1)
100
TP53
Exon 5
c.427G>A
(V143M)
1
2.4
c.455C>T
(P152L)
1
2.4
c.473G>A
(R158H)
1 (1)
2.4 (2.4)
c.495G>C
(Q165H)
1
2.4
c.523C>A
(R175S)
1
2.4
c.524G>A
(R175H)
3
7.3
c.535C>A
(H179N)
1
2.4
Exon 6
c.584T>C
(I195T)
1
2.4
c.632C>T
(T211I)
1
2.4
c.643A>G
(S215G)
1
2.4
c.659A>G
(Y220C)
1
2.4
Exon 7
c.725G>A
(C242Y)
1
2.4
c.733G>A
(G245S)
1
2.4
c.742C>T
(R248W)
2
4.9
c.773A>T
(E258V)
5
12.2
c.775G>C
(D259H)
1
2.4
del 716_21
(NA)
1
2.4
del 759_61
(NA)
1
2.4
Exon 8
c.799C>T
(R267W)
1
2.4
c.806G>T
(S269I)
1
2.4
c.817C>T
(R273C)
5 (3)
12.2 (7.3)
c.821T>C
(V274A)
1
2.4
c.841G>C
(D281H)
3
7.3
c.844C>T
(R282W)
1
2.4
c.847C>T
(R283C)
1
2.4
c.850A>T
(T284S)
1
2.4
c.853G>A
(E285K)
1
2.4
c.857A>T
(E286V)
1
2.4A sample with a homozygous R132H mutation inIDH1 was found (Supplementary Figure SF1). Deletion of the secondIDH1 allele was suspected but could not be confirmed due to lack of normal tissue from the patient. Another novel aberration in homozygous state was detected inIDH2, c.386A>G, affecting codon 129. This mutation led to lysine-to-arginine replacement. Deletion of the secondIDH2 allele was confirmed by LOH analysis for the microsatellite markers D15S996, D15S116, D15S202, and D15S127 (Supplementary Figure SF2). LOH was observed in tumour DNA for three of the markers (D15S116, D15S202, and D15S127). D15S996 and D15S116 are located centromerically, while D15S202 and D15S127 are telomeric to IDH2, confirming LOH of at least 1.38 Mb including the entireIDH2 gene. Further, three algorithms were employed to predict the impact of this substitution on protein activity. It was classified as “low confidence” with a score rate of 3.46 through SIFT (http://sift.jcvi.org/), as “benign” with a score rate of 0.270 using PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), and as “not very reliable” using the SNPs&GO (http://snps-and-go.biocomp.unibo.it/snps-and-go/).All samples were analyzed for mutations in hot-spot exons 5 to 8 ofTP53, encoding its DNA binding domain.TP53 mutations were detected in 37 patients (34.9%). Four patients carried two simultaneous mutations inTP53 gene. Of the 41 aberrations found, 9 (21.9%) were in exon 5, 4 (9.8%) in exon 6, 12 (29.3%) in exon 7, and 16 (39%) in exon 8. The most frequentTP53 mutations were c.773A>T (E258V) in exon 7 and c.817C>T (R273C) in exon 8, each detected in 5 patients (Table 1). Nine of the patients withIDH1/IDH2 mutations (64%) carried also mutatedTP53 (Supplementary Table ST2).Comparing the samples from two consecutive operations of 3 patients we found the same mutation status: two patients carried the sameTP53 mutation and one patient had no mutations. However, there was a difference in the patient with samples from three surgeries. The first operation was in 2005 with diagnosis astrocytoma WHO grade II (sample 16, Supplementary Table ST2). A mutation inIDH1 (R132H) and in exon 7 ofTP53 (del 759_61) was found. The sample from the second surgery in 2009 (sample 52) revealed the same two aberrations although the tumour had developed in glioblastoma. In 2010 the third operation was performed but the tumour (sample 41) did not carry the deletion in exon 7 ofTP53. As all the resections were subtotal, it might be speculated that during the second surgery the part of the tumour carryingTP53 mutated clone was removed, but the cells withIDH1 mutation were not and they formed the third glioma.Except for the above mutations one synonymous change inIDH1 and 2 aberrations in intron 6 ofTP53 were found. The SNP inIDH1, c. 315G>T, G105G (rs11554137) was located in exon 4 in the same region where the R132 mutation was found. The SNP was detected in 9 patients, one of them harbouring also the mutation R132H. The aberrations found in intron 6 ofTP53 were rs17884607 (g.7578129T>C) and rs34949160 (g.7578146C>T).


### 3.2. Association of Mutation Status with Clinical Features

Table2 shows the distribution of mutations inIDH1/IDH2 andTP53 among patients according to their clinical characteristics. Mutations were predominantly found in young patients (age ≤ 56 years) with higher KPS (>70). This trend was more pronounced in the group withIDH1/IDH2 mutations than in the one withTP53 mutations. In confirmation of the previous publicationsIDH1/IDH2 mutations were more frequent in secondary than in primary GBM. Also, median OS was longer in patients harbouring aberrations, 30.9 months forIDH1/IDH2 mutated and 9.1 months forTP53 mutated, compared to 6.2 months for the group without mutations.Table 2
Clinical features of the patients with mutations inIDH1/IDH2 and TP53 genes.
Clinical characteristics
N (n
=
106)
IDH1/IDH2 mutated (n
=
14)
TP53 mutated (n
=
37)
Gender
Male
53
8 (15.1%)
20 (37.7%)
Female
53
6 (11.3%)
17 (32.1%)
Age
<56 years old
51
12 (23.5%)
22 (43.1%)
≥56 years old
55
2 (0.36%)
15 (27.3%)
Karnofsky Scalea
KPS ≤70
56
3 (0.5%)
15 (26.8%)
KPS >70
45
10 (22.2%)
21 (46.6%)
Resectiona
Subtotal or partial
54
10 (18.5%)
21 (38.8%)
Total
50
4 (0.8%)
16 (32%)
Glioblastoma type
Primary
93
10 (10.7%)
29 (31.2%)
Secondary
13
4 (30.7%)
8 (61.5%)
Overall survivala (months)
Median
7.7
30.9
9.1
aMissing data for several patients.
N: number of patients.Clinical data of the patients with homozygousIDH1/IDH2 mutations were analyzed. The patient with homozygousIDH1 mutation was 53 years old and had OS of 34.6 months; the tumour was a primary GBM and surgery was followed by both chemo- and radiotherapy. The clinical features seemed similar to the others carrying heterozygousIDH1 mutations. The patient with the novel mutation inIDH2 gene had long OS (30.3 months), as expected for a secondary GBM, although he was 74 years old. Because of the long OS this case was added to those with mutations inIDH1 for further analysis.Univariate analysis using log-rank test was used to evaluate the prognostic value ofIDH1/IDH2 mutations; the patients carrying mutations had better OS (P
=
0.001) (Figure 1(a)). Although genetic aberrations inIDH1/IDH2 have been correlated with those inTP53 [14], no significant difference in survival was found between mutated and nonmutatedTP53 groups (median survival of 9.1 versus 7.6 months) (Figure 1(b)). Other factors with prognostic value were also examined. The younger patients showed longer OS (P
<
0.001) (Figure 1(c)). Further, the secondary GBM was the favourable type (P
=
0.001) (Figure 1(d)). KPS over 70 predicted longer OS as well (Figure 1(e)). The impact of theIDH1 polymorphism was also analysed, but no association with OS was found (P
=
0.454) (Figure 1(f)).Kaplan-Meier plots of glioma patients showing the association of the following factors with overall survival: (a)IDH1/IDH2 mutations, (b)TP53 mutations, (c) age, (d) primary versus secondary GBM, (e) KPS, and (f)IDH1 rs11554137.
(a)
(b)
(c)
(d)
(e)
(f)Cox proportional hazards model was used to calculate the hazard ratios for each of the examined factors (Table3). Univariate Cox regression demonstrated significant association of OS with the age (per year), type of GBM (primary versus secondary), andIDH1/IDH2 mutation status. In the multivariate analysis only age andIDH1/IDH2 mutation status showed statistical significance as independent prognostic factors. While age was a predictor of poor survival,IDH1/IDH2 mutations were predicting longer OS (Table 3).Table 3
Univariate and multivariate Cox analyses of the association between the features of the patients and overall survival.
Univariate Cox regression
Multivariate Cox regression
HR
95% CI
P
HR
95% CI
P
Age (per year)
1.048
1.029–1.067
<0.001
1.039
1.018–1.061
<0.001
Gender (female versus male)
0.884
0.575–1.361
0.576
*
*
*
Secondary versus primary GBM
0.271
0.123–0.597
0.001
0.487
0.215–1.106
0.086
Resection (total versus others)
0.854
0.559–1.303
0.463
*
*
*
KPS (per 10 points)
0.990
0.979–1.001
0.083
*
*
*
IDH1/IDH2 (mutated versus nonmutated)
0.274
0.124–0.604
0.001
0.237
0.157–0.791
0.011
IDH1 SNP (SNP versus no SNP)
0.745
0.343–1.617
0.456
*
*
*
TP53 (mutated versus nonmutated)
0.764
0.489–1.196
0.240
*
*
*
*Not included in multivariate analysis.
HR: hazard ratio; CI: confidence interval;P: P value; IDH1 SNP refers to rs11554137.We investigated further the impact ofIDH1/IDH2 mutations on survival in groups stratified by the type of GBM (Figure 2). When the patients were divided into primary and secondary GBM the most favourable group included patients with secondary GBM andIDH1/IDH2 mutations (median OS: 68.6 months) while the worst was primary GBM with noIDH1/IDH2 aberrations (median OS: 5.5 months). It was also observed that the group with secondary GBMs without IDH1/IDH2 mutations had a worse overall survival than the group with primary GBM but with mutated IDH1/IDH2 (Figure 2(a)).Kaplan-Meier plots of overall survival in groups divided by two factors:IDH1/IDH2 mutation status and (a) primary versus secondary GBM and (b) age.
(a)
(b)Coevaluation of age andIDH1/IDH2 mutation status showed that both factors contribute to prognosis (Figure 2(b)). Younger patients with mutatedIDH1/IDH2 had the most favourable clinical course while those over 56 and carrying wild-typeIDHgenes were with the worst prognosis (P
<
0.001).


## 3.1. Genetic Aberrations inIDH1,IDH2, andTP53 Genes

A total of 111 glioma samples from 106 patients were analyzed forIDH1, IDH2, andTP53 mutations. Genetic aberrations in at least one of the genes were found in 46 samples from 42 patients (39.6%) (Supplementary Table ST2). Exon 4 ofIDH1 gene was mutated in 13 patients (12.3%). AllIDH1 aberrations were missense mutations at codon 132 leading to the substitution arginine to histidine (c.395G>A, R132H) (Table 1).Table 1
MutationsinIDH1,IDH2, and TP53 genes that were detected in 106 brain tumour patients.
Gene
Exon
Nucleotide change (amino acid change)
Number of patients with mutations (which are homozygous)
% mutations among all mutations in the gene
IDH1
Exon 4
c.395G>A
(R132H)
13 (1)
100 (7.7)
IDH2
Exon 4
c.386A>G
(K129R)
1 (1)
100
TP53
Exon 5
c.427G>A
(V143M)
1
2.4
c.455C>T
(P152L)
1
2.4
c.473G>A
(R158H)
1 (1)
2.4 (2.4)
c.495G>C
(Q165H)
1
2.4
c.523C>A
(R175S)
1
2.4
c.524G>A
(R175H)
3
7.3
c.535C>A
(H179N)
1
2.4
Exon 6
c.584T>C
(I195T)
1
2.4
c.632C>T
(T211I)
1
2.4
c.643A>G
(S215G)
1
2.4
c.659A>G
(Y220C)
1
2.4
Exon 7
c.725G>A
(C242Y)
1
2.4
c.733G>A
(G245S)
1
2.4
c.742C>T
(R248W)
2
4.9
c.773A>T
(E258V)
5
12.2
c.775G>C
(D259H)
1
2.4
del 716_21
(NA)
1
2.4
del 759_61
(NA)
1
2.4
Exon 8
c.799C>T
(R267W)
1
2.4
c.806G>T
(S269I)
1
2.4
c.817C>T
(R273C)
5 (3)
12.2 (7.3)
c.821T>C
(V274A)
1
2.4
c.841G>C
(D281H)
3
7.3
c.844C>T
(R282W)
1
2.4
c.847C>T
(R283C)
1
2.4
c.850A>T
(T284S)
1
2.4
c.853G>A
(E285K)
1
2.4
c.857A>T
(E286V)
1
2.4A sample with a homozygous R132H mutation inIDH1 was found (Supplementary Figure SF1). Deletion of the secondIDH1 allele was suspected but could not be confirmed due to lack of normal tissue from the patient. Another novel aberration in homozygous state was detected inIDH2, c.386A>G, affecting codon 129. This mutation led to lysine-to-arginine replacement. Deletion of the secondIDH2 allele was confirmed by LOH analysis for the microsatellite markers D15S996, D15S116, D15S202, and D15S127 (Supplementary Figure SF2). LOH was observed in tumour DNA for three of the markers (D15S116, D15S202, and D15S127). D15S996 and D15S116 are located centromerically, while D15S202 and D15S127 are telomeric to IDH2, confirming LOH of at least 1.38 Mb including the entireIDH2 gene. Further, three algorithms were employed to predict the impact of this substitution on protein activity. It was classified as “low confidence” with a score rate of 3.46 through SIFT (http://sift.jcvi.org/), as “benign” with a score rate of 0.270 using PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), and as “not very reliable” using the SNPs&GO (http://snps-and-go.biocomp.unibo.it/snps-and-go/).All samples were analyzed for mutations in hot-spot exons 5 to 8 ofTP53, encoding its DNA binding domain.TP53 mutations were detected in 37 patients (34.9%). Four patients carried two simultaneous mutations inTP53 gene. Of the 41 aberrations found, 9 (21.9%) were in exon 5, 4 (9.8%) in exon 6, 12 (29.3%) in exon 7, and 16 (39%) in exon 8. The most frequentTP53 mutations were c.773A>T (E258V) in exon 7 and c.817C>T (R273C) in exon 8, each detected in 5 patients (Table 1). Nine of the patients withIDH1/IDH2 mutations (64%) carried also mutatedTP53 (Supplementary Table ST2).Comparing the samples from two consecutive operations of 3 patients we found the same mutation status: two patients carried the sameTP53 mutation and one patient had no mutations. However, there was a difference in the patient with samples from three surgeries. The first operation was in 2005 with diagnosis astrocytoma WHO grade II (sample 16, Supplementary Table ST2). A mutation inIDH1 (R132H) and in exon 7 ofTP53 (del 759_61) was found. The sample from the second surgery in 2009 (sample 52) revealed the same two aberrations although the tumour had developed in glioblastoma. In 2010 the third operation was performed but the tumour (sample 41) did not carry the deletion in exon 7 ofTP53. As all the resections were subtotal, it might be speculated that during the second surgery the part of the tumour carryingTP53 mutated clone was removed, but the cells withIDH1 mutation were not and they formed the third glioma.Except for the above mutations one synonymous change inIDH1 and 2 aberrations in intron 6 ofTP53 were found. The SNP inIDH1, c. 315G>T, G105G (rs11554137) was located in exon 4 in the same region where the R132 mutation was found. The SNP was detected in 9 patients, one of them harbouring also the mutation R132H. The aberrations found in intron 6 ofTP53 were rs17884607 (g.7578129T>C) and rs34949160 (g.7578146C>T).


## 3.2. Association of Mutation Status with Clinical Features

Table2 shows the distribution of mutations inIDH1/IDH2 andTP53 among patients according to their clinical characteristics. Mutations were predominantly found in young patients (age ≤ 56 years) with higher KPS (>70). This trend was more pronounced in the group withIDH1/IDH2 mutations than in the one withTP53 mutations. In confirmation of the previous publicationsIDH1/IDH2 mutations were more frequent in secondary than in primary GBM. Also, median OS was longer in patients harbouring aberrations, 30.9 months forIDH1/IDH2 mutated and 9.1 months forTP53 mutated, compared to 6.2 months for the group without mutations.Table 2
Clinical features of the patients with mutations inIDH1/IDH2 and TP53 genes.
Clinical characteristics
N (n
=
106)
IDH1/IDH2 mutated (n
=
14)
TP53 mutated (n
=
37)
Gender
Male
53
8 (15.1%)
20 (37.7%)
Female
53
6 (11.3%)
17 (32.1%)
Age
<56 years old
51
12 (23.5%)
22 (43.1%)
≥56 years old
55
2 (0.36%)
15 (27.3%)
Karnofsky Scalea
KPS ≤70
56
3 (0.5%)
15 (26.8%)
KPS >70
45
10 (22.2%)
21 (46.6%)
Resectiona
Subtotal or partial
54
10 (18.5%)
21 (38.8%)
Total
50
4 (0.8%)
16 (32%)
Glioblastoma type
Primary
93
10 (10.7%)
29 (31.2%)
Secondary
13
4 (30.7%)
8 (61.5%)
Overall survivala (months)
Median
7.7
30.9
9.1
aMissing data for several patients.
N: number of patients.Clinical data of the patients with homozygousIDH1/IDH2 mutations were analyzed. The patient with homozygousIDH1 mutation was 53 years old and had OS of 34.6 months; the tumour was a primary GBM and surgery was followed by both chemo- and radiotherapy. The clinical features seemed similar to the others carrying heterozygousIDH1 mutations. The patient with the novel mutation inIDH2 gene had long OS (30.3 months), as expected for a secondary GBM, although he was 74 years old. Because of the long OS this case was added to those with mutations inIDH1 for further analysis.Univariate analysis using log-rank test was used to evaluate the prognostic value ofIDH1/IDH2 mutations; the patients carrying mutations had better OS (P
=
0.001) (Figure 1(a)). Although genetic aberrations inIDH1/IDH2 have been correlated with those inTP53 [14], no significant difference in survival was found between mutated and nonmutatedTP53 groups (median survival of 9.1 versus 7.6 months) (Figure 1(b)). Other factors with prognostic value were also examined. The younger patients showed longer OS (P
<
0.001) (Figure 1(c)). Further, the secondary GBM was the favourable type (P
=
0.001) (Figure 1(d)). KPS over 70 predicted longer OS as well (Figure 1(e)). The impact of theIDH1 polymorphism was also analysed, but no association with OS was found (P
=
0.454) (Figure 1(f)).Kaplan-Meier plots of glioma patients showing the association of the following factors with overall survival: (a)IDH1/IDH2 mutations, (b)TP53 mutations, (c) age, (d) primary versus secondary GBM, (e) KPS, and (f)IDH1 rs11554137.
(a)
(b)
(c)
(d)
(e)
(f)Cox proportional hazards model was used to calculate the hazard ratios for each of the examined factors (Table3). Univariate Cox regression demonstrated significant association of OS with the age (per year), type of GBM (primary versus secondary), andIDH1/IDH2 mutation status. In the multivariate analysis only age andIDH1/IDH2 mutation status showed statistical significance as independent prognostic factors. While age was a predictor of poor survival,IDH1/IDH2 mutations were predicting longer OS (Table 3).Table 3
Univariate and multivariate Cox analyses of the association between the features of the patients and overall survival.
Univariate Cox regression
Multivariate Cox regression
HR
95% CI
P
HR
95% CI
P
Age (per year)
1.048
1.029–1.067
<0.001
1.039
1.018–1.061
<0.001
Gender (female versus male)
0.884
0.575–1.361
0.576
*
*
*
Secondary versus primary GBM
0.271
0.123–0.597
0.001
0.487
0.215–1.106
0.086
Resection (total versus others)
0.854
0.559–1.303
0.463
*
*
*
KPS (per 10 points)
0.990
0.979–1.001
0.083
*
*
*
IDH1/IDH2 (mutated versus nonmutated)
0.274
0.124–0.604
0.001
0.237
0.157–0.791
0.011
IDH1 SNP (SNP versus no SNP)
0.745
0.343–1.617
0.456
*
*
*
TP53 (mutated versus nonmutated)
0.764
0.489–1.196
0.240
*
*
*
*Not included in multivariate analysis.
HR: hazard ratio; CI: confidence interval;P: P value; IDH1 SNP refers to rs11554137.We investigated further the impact ofIDH1/IDH2 mutations on survival in groups stratified by the type of GBM (Figure 2). When the patients were divided into primary and secondary GBM the most favourable group included patients with secondary GBM andIDH1/IDH2 mutations (median OS: 68.6 months) while the worst was primary GBM with noIDH1/IDH2 aberrations (median OS: 5.5 months). It was also observed that the group with secondary GBMs without IDH1/IDH2 mutations had a worse overall survival than the group with primary GBM but with mutated IDH1/IDH2 (Figure 2(a)).Kaplan-Meier plots of overall survival in groups divided by two factors:IDH1/IDH2 mutation status and (a) primary versus secondary GBM and (b) age.
(a)
(b)Coevaluation of age andIDH1/IDH2 mutation status showed that both factors contribute to prognosis (Figure 2(b)). Younger patients with mutatedIDH1/IDH2 had the most favourable clinical course while those over 56 and carrying wild-typeIDHgenes were with the worst prognosis (P
<
0.001).


## 4. Discussion

In the present study we focused on the investigation of prognostic markers for patients with glioblastomas, especially genetic aberrations inIDH1, IDH2, andTP53 genes. Mutations in R132 of IDH1 were detected in only 12.3% of patients, probably due to the high number of primary GBM (93 cases) in the study (Table 2). The mutations were more frequent in secondary glioblastomas (30.7%). This frequency is still much lower than reported [12–14, 16, 17, 20, 29] and this could be due to the small number of secondary GBMs in our study group. Also, this was the first investigation in Bulgaria evaluating the frequency of the IDH1/IDH2 mutations in GBM patients and there was no data, which could be used as a reference for Bulgarian population frequencies. Furthermore, we classified tumours as primary GBM only on the basis of lack of previously detected glial tumour, and thus a few secondary tumours might have been missed.In accordance with previous publications, 64% ofIDH1/IDH2 mutated tumours harboured also a mutation inTP53 [7, 14, 23].Normally both cytosolic IDH1 and mitochondrial IDH2 exist as homodimers [30]. Analysis of the effect of heterozygousIDH1 mutations on glioma cells has shown that mutant IDH1 affects the ability of the enzyme to reduce α-ketoglutarate to 2-hydroxyglutarate (2HG) [31]. The 2HG-producing IDH mutants can prevent the histone demethylation required for the differentiation of lineage-specific progenitor cells. In glioma patientsIDH mutations have been associated with a gene expression profile enriched for genes expressed in neural progenitor cells, which were correlated with increased histone methylation [32]. It has recently been shown that glioblastomas with the CpG island methylator phenotype (CIMP) are associated with the proneural subgroup of tumours and are driven byIDH1 mutation [33, 34].In the present study we found a homozygous mutation in codon 132 (R132H) in a GBM patient. Deletion of the normal allele was suspected, as both the mutation and the polymorphism inIDH1 gene were in a homozygous state. Recently homozygousIDH1 aberrations in gliomas were reported for the first time in 2 patients with secondary GBM [35] and in a patient with astrocytoma [36]. Previously, homozygousIDH1 mutations have been detected in leukemia [37] and thyroid cancer patients [38]. Jin et al. have reported lower 2HG levels in the cells lacking wild-typeIDH1 allele compared to the ones with heterozygous mutations which might hamper application of 2HG-level modulators as a potential therapeutic strategy for IDH1-mutated tumours [35]. In line with the current finding that 2-HG production, but not dominant negative function, is conferred by glioma-derived NADP+-dependentIDH mutations [39], coupled with their role to block the histone demethylation and associate with CIMP [32], we could speculate that in the homozygous R132H carrier the extent of methylation will be greatly increased compared to the heterozygote.The role ofIDH2 mutations alone is still unclear because of the rare occurrence in gliomas: 1-2% [28]. It is more common in acute myeloid leukemia (AML), about 15%, where hot spots for mutations are codons 140 and 172 [40]. Although the examined cohort included 106 patients we did not find any mutations in the reported codons but found a novel aberration in codon 129, c.386A>G, K129R. Both lysine and arginine are basic amino acids, but the change affected a part of a conservative domain in IDH2. Also, the second allele was lost. However, analyses of the variant with SIFT, PolyPhen, and SNPs&GO showed that most likely the change is not pathogenic.Several previous studies have demonstrated the important role ofIDH1/IDH2 mutations for determining the prognosis of glioblastoma patients [6, 17, 20]. Our group of patients was with short median overall survival of 7.7 months, which was lower than the most published data [41, 42]. One of the reasons for this could be the predominant number of primary GBM in our cohort. Ohgaki and coauthors reported lower median survival in primary GBM (4.7 months) than in secondary GBM (7.8 months) [26], and even though only half of their patients underwent surgical treatment, while all of our patients had surgery, their data were close to ours; median OS was 6.2 and 25.8 months for primary and secondary GBMs, respectively [26]. Another reason could be the changes in radio- and/or chemotherapy strategy through the years. Bleeker et al. published data of tumours, which have been under treatment before 2006 and were with a similar median OS to ours, 8.7 months [43]. However, we confirmed that patients with IDH1/2 mutations had much longer OS than those with no mutation (Figure 1(a)).In addition,IDH1/IDH2 mutations were found predominantly in younger patients. Although 64% ofIDH1/IDH2 mutated tumours carried alsoTP53 mutations, an investigation over OS of groups with and withoutTP53 mutations did not show any significant difference. Moreover, no favourable prognosis was found in the group of patients with affected codon 248 or 273 of theTP53 in accordance with previous reports [25].The impact of the polymorphism inIDH1 (rs11554137) on survival has been investigated in AML and thyroid carcinoma. Although the SNP has not been significant for thyroid carcinogenesis [44], in AML patients it has been associated with poor prognosis and higher expression ofIDH1 mRNA [45]. This aberration has been suspected to alter IDH1 activity by changing RNA stability, folding, and splicing [45]. However, in our study rs11554137 did not show association with OS of glioma patients (Table 3).Multivariate regression analysis determined age andIDH1/IDH2 mutation status as independent prognostic factors (Table 3). According to Hartmann et al.,IDH1 mutations are better predictors of survival than histopathology [46]. Although we studied only GBM cases, our results showed that IDH1/IDH2 mutations were more powerful prognostic factors than primary/secondary GBM classification; we observed better OS in primary GBM with mutatedIDH1/IDH2 compared to the secondary GBM with no mutation (Figure 2(a)). It is likely thatIDH1 mutations are favourable for prognosis independently of the GBM type. On the other hand, coevaluation of age andIDH1/IDH2 mutation status revealed predictive value ofIDH1/IDH2 only in the group of patients below 56 years of age(Figure 2(b)). This might be a result of the small number of mutations in older patients (Table 2).


## 5. Conclusions

Even though in gliomas mutations inIDH1 andIDH2genes are usually heterozygous, homozygous ones also exist.IDH1/IDH2 aberrations, but not the ones inTP53, together with age might be applied in clinical practice for prediction of survival in patients with glioblastomas.



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*Source: 654727-2014-04-24.xml*