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The impact of TP53 and RAS mutations on cerebellar glioblastomas
Experimental and Molecular Pathology 97 (2014) 202–207
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The impact of TP53 and RAS mutations on cerebellar glioblastomas Vedrana P. Milinkovic a,⁎, Milica K. Skender Gazibara b, Emilija M. Manojlovic Gacic b, Tatjana M. Gazibara c, Nikola T. Tanic a a b c
University of Belgrade, Institute for Biological Research “Sinisa Stankovic”, Department of Neurobiology, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia University of Belgrade, School of Medicine, Institute of Pathology, Doktora Subotica 1, 11000 Belgrade, Serbia University of Belgrade, School of Medicine, Institute of Epidemiology, Visegradska 26, 11000 Belgrade, Serbia
a r t i c l e
i n f o
Article history: Received 4 July 2014 Available online 16 July 2014 Keywords: Glioblastoma Cerebellar glioblastoma p53 RAS Immunohistochemistry
a b s t r a c t Cerebellar glioblastoma (cGBM) is a rare, inadequately characterized disease, without detailed information on its molecular basis. This is the first report analyzing both TP53 and RAS alterations in cGBM. TP53 mutations were detected in more than half of the samples from our cohort, mainly in hotspot codons. There were no activating mutations in hotspot codons 12/13 and 61 of KRAS and HRAS genes in cGBM samples but we detected alterations in other parts of exons 2 and 3 of these genes, including premature induction of STOP codon. This mutation was present in 3 out of 5 patients. High incidence of RAS mutations, as well as significantly longer survival of cGBM patients compared to those with supratentorial GBM suggest that cGBM may have different mechanisms of occurrence. Our results suggest that inactivation of TP53 and RAS may play an important role in the progression of cerebellar GBM. © 2014 Elsevier Inc. All rights reserved.
Introduction Glioblastoma multiforme (GBM) (World Health Organization, WHO, grade IV) is the most frequent and aggressive primary brain tumor in adults. According to the latest WHO classification (Louis et al., 2007), histologic criteria for the diagnosis of GBM include nuclear atypia, cellular pleomorphism, mitotic activity, microvascular proliferation and necrosis. Majority of glioblastomas develop de novo (primary glioblastomas) without clinical or histological evidence of a less malignant precursor lesion, while others, progressing from low-grade diffuse astrocytoma or anaplastic astrocytoma (AA) represent secondary GBM (Ohgaki and Kleihues, 2007). Alterations of several genes, including TP53, PTEN, CDKN2A, and EGFR, are frequently present in gliomas, usually occurring in a defined order during the progression to a high-grade tumor. A recent genomewide mutational analyses of GBM revealed somatic mutations of the IDH1 and IDH2 (isocitrate dehydrogenase) genes, most frequently in tumors that were known to have evolved from lower-grade gliomas (secondary glioblastomas) (Yan et al., 2009). Glioblastoma occurs most often in the cerebral hemispheres, while cerebellar location is rather rare, reported only in 0.4–3.4% of all GBM (Babu et al., 2013). Almost all previous studies were limited to
⁎ Corresponding author at: Institute for Biological Research, Department of Neurobiology, Laboratory for Molecular Neurobiology, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia. E-mail addresses: [email protected], [email protected] (V.P. Milinkovic).
http://dx.doi.org/10.1016/j.yexmp.2014.07.009 0014-4800/© 2014 Elsevier Inc. All rights reserved.
description of one or a few cases without further analyzes. Although current studies have revealed differences in patient and imaging characteristics between cerebellar and supratentorial GBM (Kuroiwa et al., 1995; Weber et al., 2006), there are also opposed reports (Akimoto et al., 2009; Stark et al., 2010) indicating similarity between tumors within these two locations. Due to its low incidence, little is reported about any genetic abnormalities in cerebellar GBM (cGBM), and there is no consensus on survival compared to supratentorial GBM (Utsuki et al., 2012). Inactivation of p53 tumor suppressor is known to be an early and frequent event in GBM formation, especially in the pathway leading to secondary GBM progression (Furnari et al., 2007; Ohgaki and Kleihues, 2007). A few reports described immunohistochemical studies of p53 in cases of cGBM in adults with exceptionally high immunopositive rates (85.7%) (Kawarabuki et al., 2005; Utsuki et al., 2012) suggesting that cGBMs should be defined as secondary ones. Insight into genetic basis of this extensive p53 immunopositivity is needed for better understanding of its role in cGBM pathogenesis. RAS GTPase family members are crucial players in many signaling networks that regulate cell-cycle progression, growth, migration, cytoskeletal changes, apoptosis, and senescence (Fernandez-Medarde and Santos, 2011). Mutational activation of RAS gene is often present in various tumors in humans, leading to increased invasion and metastasis, and decreased apoptosis due to formation of permanently active GTPbound form of RAS. Specifically, mutations in codon 12, 13, or 61 of one of the three RAS proto-oncogenes (HRAS, KRAS, and NRAS) transform them into oncogenes (Bos, 1989). Incidences of Ras oncogenic
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mutations, however, vary considerably among different types of cancers (Schubbert et al., 2007), and the type of RAS mutation seems to correlate with the tumor type. Besides activating mutations, activation of RAS oncogenes occurs by several other routes, including overexpression, gene amplification, translocations, or insertion of a promoter or enhancer element (Lymbouridou et al., 2009). A few studies, however, have examined the molecular alteration of the RAS genes in gliomas, with general conclusion that somatic mutations in hot-spot codons of RAS genes seem to be very rare in GBMs (Lo, 2010) and slightly more frequent in astrocytomas (Sharma et al., 2005). Anyhow, there are no reports on the presence of alterations of RAS gene family members in cGBM, possibly due to its low incidence in population. The aim of our study was to get better insight into molecular basis of cGBM, a rare but aggressive disease, putting emphasis on specific gene alterations frequently present in other tumor types. Histopatological and immunohistochemical characterization of these tumors was performed and compared with results from previously published studies. Additionally, samples of recurrent cGBM were analyzed in attempt to perceive mechanisms underlying its aggressive nature. Finally, molecular alterations and prognosis of cGBM were compared with supratentorial GBMs, in order to elucidate specific hallmarks of this rare disease. Material and methods Patient characteristics In our database of neurosurgical biopsies from 2002 to 2012 we found seven patients with cGBM. In the present investigation we could analyze five of them, because two were lacking in either clinical or pathological data. Patients were operated at the Neurosurgery Clinic, Clinical Centre of Serbia, Belgrade. All the procedures followed were approved by the Ethical Committee of Clinical Center of Serbia (approval number 3672/1) in accordance with the ethical standards laid down in the 1975 Declaration of Helsinki, laws of Republic of Serbia, as well as GCP guidelines. All available slides of cGBM were reviewed and classified according to the 2007 WHO classification by two pathologists, MSG and EMG. Another set of 30, previously characterized supratentorial high-grade gliomas (Milinkovic et al., 2012), was used for comparison in survival analysis. Mortality data for all patients were obtained from the Statistical Office of the Republic of Serbia (Statistical Office of the Republic of Serbia http://webrzs.stat.gov.rs/WebSite/). Immunohistochemistry For each tumor, tissue samples were fixed in 10% buffered formalin, embedded in paraffin blocks and cut into 5 μm thick sections used for routine histology (hematoxylin–eosin stain) and immunohistochemistry. Immunohistochemical stains were performed manually using the following antibodies according to the manufacturer instructions: Glial Fibrillary Acidic Protein (GFAP) (1:100, polyclonal, DAKO, Denmark), S-100 protein (1:200, polyclonal, DAKO, Denmark), Vimentin (1:300, clone V9, DAKO, Denmark), Synaptophysin (1:20, polyclonal, DAKO, Denmark), Cytokeratin (CK) (1:100, clone AE1/AE3, DAKO, Denmark), p53 (1:450, clone DO7, Thermo Scientific, Fremont, CA, USA) and Ki67 (1:50, clone MIB-1, DAKO, Denmark). The antibodies were detected by Ultra Vision Detection System, Large Volume Anti-Polyvalent, HRP (Thermo Scientific, Fremont, CA, USA). The sections were visualized with 3,3′-diaminobenzidine. Nuclear counterstaining was performed with Mayer's hematoxylin. Analysis of Ki-67 labeling index and p53 expression Areas with the highest number of positive nuclei were captured using Olympus camera DP70 with ×400 magnification. Counting of nuclei was performed manually, using ImageJ program. We counted a
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minimum of 1000 cells per case (ranging from 1000 to 1417), except for Case 1B, where counting was done on 500 cells because of paucity of tissue available for the analysis. Only intensely stained nuclei were taken into account. Results were expressed as percentage of tumor cells with positive nuclei (labeling index). PCR amplification and sequencing DNA from paraffin-embedded tumor material was extracted using Kapa Express Extract DNA extraction kit (Kapa Biosystems, United States). The quality of the isolated DNA was verified by electrophoresis on 0.8% agarose gel. The DNA concentration was assessed spectrophotometrically. Frequently mutated exons of TP53 gene (5–9) were amplified and screened for mutations by PCR-SSCP (Single Strand Conformation Polymorphism) analysis according to Orita et al. (1989). Codons 12, 13 and 61 of RAS gene were also amplified and subjected to sequencing. Primers and PCR conditions for RAS gene (H-RAS and K-RAS) are shown in Table 1. Detected mutations were confirmed by sequencing with Applied Biosystems Incorporated dye terminator sequencing kit according to the manufacturer's specifications on an ABI Prism 3130 automated sequencer (Applied Biosystems, Foster City, USA). Sequencing was carried out in both directions. The obtained sequences were analyzed and compared with wild-type TP53 and RAS sequences using BLAST software in the NCBI GenBank database. Statistical analysis The survival time of 5 cerebellar and 30 supratentorial GBM patients analyzed in our previous study (Milinkovic et al., 2012) was compared. Survival analyses were performed using Kaplan and Meier productlimit method. The log rank test was used to assess the significance of the difference between pairs of survival probabilities. Overall survival was calculated from the day after surgery to the last follow-up examination or death of the patient. Statistical differences were considered significant when p was b 0.05 (*). Results Clinical data of each patient with cGBM are summarized in Table 2. All patients were males. The mean age at diagnose was 46.4 years (ranging from 36 to 64 years). Duration of symptoms (nausea—4 cases, headaches—3 cases, unstable gait—3 cases, weakness of one side of body and speech disturbances—1 case), from onset to diagnoses ranged from one week to one month. Two patients had cGBM recurrences (patient 1 and patient 5). Both of them had two recurrences (labeled by 1B, 1C, 5B and 5C, respectively). However, the tissue from the second recurrence of patient 1 (1C) was not available for immunohistochemical and molecular analysis. In patient 2 cGBM appeared 1 year after surgery of supratentorial diffuse low grade astrocytoma (Case 2S) without any evidence of supratentorial recurrence. All patients died within 5 years upon diagnosis, except for patient 4 who is still alive. Median survival was 33.2 months. Histopathological and immunohistochemical features Typical histopathological features of GBM were observed in all cases (Fig. 1A). On immunohistochemistry, all tumors showed moderate to intensive GFAP (Fig. 1B), Vimentin and S-100 immunopositivity, while they were negative on CK and Synaptophysin. Ki-67 labeling index showed great variability, ranging from 0.3% to 40.65%, with a tendency to increase along recurrences, in spite of adjuvant therapy. Percentage of p53 positive nuclei was also heterogeneous, ranging from 1.71% to 40.62%, with variable trends in recurrences (Table 3, Fig. 1C and D).
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Table 1 Ras gene oligonucleotide primers and PCR conditions. Ras gene
Exon/codon
Primer sequence
PCR conditions
K Ras
exon-2 12/13 exon-3 61 exon-2 12/13 exon-3 61
(f) 5′-ATGACTGAATATAAACTTGT-3′ (r) 5′-CTCTATTGTTGGATCATATT-3′ (f) 5′-AAGTAGTAATTGATGGAGAA-3′ (r) 5′-AGAAAGCCCTCCCCAGTCCT-3′ (f) 5′-ATGACGGAACATAAGCTGGT-3′ (r) 5′-CGCCAGGCTCACCTCTATA-3′ (f) 5′-AGGTGGTCATTGATGGGGAG-3′ (r) 5′-AGGAAGCCCTCCCCGGTGCG-3′
95 35 95 56 65
K Ras H Ras H Ras
PCR reaction mixture (25 μl)
°C (5 min) cycles: °C (1 min) °C (1 min) °C (1 min)
1.5 mM MgCl2; 2 mM dNTP; 0.6 μM each (f) and (r) primer; 1 U/reaction Taq polymerase
65 °C (7 min)
Analysis of genetic alterations
Survival analyses
All primary and recurrent tumor samples from 5 patients were further analyzed for the presence of DNA alterations in TP53 and RAS genes. We analyzed 9 samples in total, one from Cases 3 and 4 each, two from Case 1 (1A and 1B), two from Case 2 (2 and 2S) and three samples from Case 5 (5A, 5B and 5C). A PCR-SSCP analysis revealed TP53 aberrations in 5 out of 9 samples, which were further sequenced to confirm and identify mutations. DNA sequencing confirmed all 5 mutations in 3 patients (Fig. 2A). The exon locations, codon positions, nucleotide changes, and predicted effects on protein primer structure of all 5 mutations are summarized in Table 4. In Case 1, TP53 was altered only in recurrent glioblastoma sample (1B), bearing mutation in known hotspot mutation site in codon 273. We detected splice site deletion in supratentorial diffuse low grade astrocytoma (Case 2S) which was not present in cerebellar glioblastoma of the same patient. Samples 5 and 5B shared the same mutation in codon 176 exon 5 of TP53 gene, while there were no mutations in this gene in the second recurrent sample from the same patient (5C). Detected mutations in TP53 gene were confirmed in all samples with higher percentage of p53 positive nuclei. We further performed a systematic mutation analysis of the KRAS and HRAS genes with the special emphasis on known mutation hotspot sites in these genes. The exon locations, codon positions, nucleotide changes, and predicted effects are summarized in Table 5. There were no alterations in hot spot codons 12, 13 and 61 in any of the analyzed samples. All patients had alterations in KRAS or HRAS gene, but identified mutations found in these genes were not previously reported in cancer databases. The most frequent alteration was missense mutation in codon 4 of HRAS gene, present in five samples from four patients. We also detected single nucleotide insertions in codon 3 of KRAS gene in 2 samples, and in codon 4 of HRAS gene in all 3 samples from patient 5. Both insertions lead to premature induction of STOP codon, preventing the synthesis of a full-length RAS protein. Finally, we identified frameshift mutation due to single nucleotide insertion in codon 60 of KRAS gene, present in only one sample.
Median OS of patients with cGBM was 33.2 months (range 1– 78 months). Four out of five patients died of this disease. Median time to recurrence was 10 months, and at the time of the analysis two patients had experienced recurrence. The median survival of the 30 patients with supratentorial high-grade glioma was 11 months, significantly shorter compared to cGBM patients (log rank test, p = 0.04, Fig. 3). Discussion Cerebellar GBMs in adults are rare tumors that are insufficiently characterized in terms of incidence, natural history, prognostic factors and the presence of genetic alterations. Majority of previously published reports were case reports or retrospective studies focused on better characterization of outcome and identification of different factors which affect survival, being insufficient in explaining molecular mechanisms underlying this disease. We wanted to further characterize genetic alterations in cGBM with the aim to compare detected changes with alterations in supratentorial GBM. Besides, we were able to analyze recurrent tumors from the same patient in order to compare their histological and genetic profile with matching primary tumor. First of all, we examined mutational status of TP53 tumor suppressor gene, most frequently altered in various tumor types, in order to evaluate its impact on development of cGBM. Clinical studies identifying TP53 mutations in low-grade glioma, along with the well-established role of p53 in cancer, have suggested that TP53 is a potent driver mutation in GBM, predominantly in the pathway leading to secondary glioblastoma progression (England et al., 2013). The presence of mutations in samples of three out of five analyzed patients suggests that p53 has an important role in cGBM progression as well. Besides, almost all detected mutations in our samples were either previously reported in glioma (Meyer-Puttlitz et al., 1997) or positioned in hotspot codons (England et al., 2013), indicating that these mutations seriously affect p53
Table 2 Clinical data of patients with cGBM. Patient number
Age (years)
Sex
Cerebellar location
Surgical treatment
RT (Gy)
CT
Recurrent (months)
Survival (months)
1
42
M
Right hemisphere
Partial resection
60
–
Died (63)
2
41
M
Vermis
Partial resection
60
–
1B local (12) 1C local (48) None
3
64
M
Right hemisphere
Total resection
–
–
None
4
36
M
Vermis
Total resection
60
BCNU
None
5
49
M
Right hemisphere
Partial resection
60
BCNU Temo-zolomide
5B local (8) 5C local (16)
Died (1) Died (6) Alive (78) Died (18)
Legend: M — male, B — sample from the second surgery/first recurrence, C — sample from the third surgery/second recurrence, RT — radiation therapy, CT — chemotherapy.
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Fig. 1. Histopathological and immunohistochemical features of cGBM sample (Case 5). (A) Highly cellular tumor tissue with prominent microvascular proliferation (arrows) and multiple small pseudopalisading necroses. Zone of partially preserved cerebellar folia is present at the right corner (H–E ×400); (B) Diffuse GFAP immunoreactivity in tumor cells. Vascular “glomeruloid tufts” are negative. (×400); (C) High Ki-67 labeling index (×100); (D) Nuclear accumulation of p53 protein in tumor cells but not in endothelial cell (×200).
function in cerebellar glioblastoma. In contrast to primary GBM, where TP53 mutations were almost equally distributed, the majority of TP53 mutations in secondary glioblastomas were located in codons 248 and 273 (Ohgaki et al., 2004), as it was the case in one of our samples, supporting the assumptions that cGBMs are more similar to secondary glioblastomas. We found it interesting that mutational patterns of TP53 tumor suppressor gene were not repeated in recurrent samples of the same patient. Tumor heterogeneity and infiltrative growth pattern seem to be the most plausible explanation for this finding (Marusyk and Polyak, 2010). Specifically, one subclone in the tumor carried a TP53 mutation in contrast to another parent clone, which persisted through the clinical course of the patient and was finally sampled in the third tumor removed from this patient. In case of patient 2, both supratentorial and cerebellar tumor samples carried mutations in TP53. Differences in identified mutations confirm our assumption that these two tumors represent two distinct tumor entities. Table 3 Ki-67 and p53 labeling indices in cGBM cases. Case no.
Ki-67 (%)
P53 (%)
1A 1B 1C 2 3 4 5A 5B 5C
3.46 4.44 NA 0.3 5.94 5.48 31.94 41.61 40.65
4.72 14.02 NA 1.71 4.41 0.62 30 59.1 5.02
Legend: NA — not available, A — sample from the first surgery, B — sample from the second surgery/first recurrence, C — sample from the third surgery/second recurrence.
Ras genes (HRAS, KRAS and NRAS) are frequently mutated in human cancers. Their protein products are GTPases that function as molecular switches in regulating pathways responsible for proliferation and cell survival (Prior et al., 2012). Since there is no evidence of activating mutations in hotspot codons 12/13 and 61 of Ras genes in gliomas (Ghazali et al., 2005; Gomori et al., 1999), RAS activation must be due to other alterations, such as amplification and overexpression of growth factor receptor genes or aberrations in yet other RAS pathway genes (Knobbe et al., 2004). When analyzing alterations in Ras genes, we discovered what may be fairly novel mutations that are not typical of cancers. There were no activating mutations in hot spot codons of KRAS and HRAS genes in our set of cGBM samples, but we detected alterations in other parts of exons 2 and 3 of these genes. The most peculiar finding was premature induction of STOP codon in both RAS genes present in three samples from five patients. Single nucleotide insertions in the codon 3 of KRAS and in the codon 4 of HRAS were found, leading to formation of truncated forms of these proteins. This represents the first evidence of this type of genetic alteration in HRAS gene, while there were a few reports demonstrating the presence of nonsense mutations in KRAS gene. Palmirotta et al. (2009) detected novel nonsense mutation in the codon 22 of KRAS gene in colorectal cancer, while a single nucleotide deletion of one G in codon 12 in squamous cell carcinoma resulted in a stop codon formation several bases downstream (Vachtenheim et al., 1995), both determining the synthesis of a completely inactive truncated KRAS protein. These novel types of RAS mutations might be more frequent than we presently know, probably because current conventional analytical techniques have focused exclusively on the identification of conventional mutations (van Krieken et al., 2008). Although RAS is a positive regulator of cell growth, differentiation and survival, inactivation of this gene was present in primary tumors which later gave rise to recurrences. In support of our finding, Lymbouridou et al. (2009) reported significant decrease in KRAS and HRAS mRNA levels in glioblastoma samples,
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Fig. 2. DNA sequencing of TP53 and RAS genes. (A) Part of exon 5 DNA sequence of TP53 gene bearing a point mutation at codon 176 with missense effect (p.C176R). The arrow indicates the mutation site in Case 5A sample. (B) Fragment of exon 2 DNA sequence of HRAS gene showing a point mutation at codon 4 with missense effect (p.Y4H). The arrow indicates the mutation site (shown is the sequence of the antisense strand of Case 1B).
Table 4 Identified mutations in TP53 tumor suppressor gene. Patient number
Case number
Exon/intron
Codon number
Nucleotide change
Predicted effect
1 2 2 5 5
1B 2S 2 5A 5B
Exon 8 Exon 9 Exon 8 Exon 5 Exon 5
273
CGT → CCT (CpG) del 13987–13992 GAG → GAC TGC → CGC TGC → CGC
Arg → Pro Splice site deletion Glu → Asp Cys → Arg Cys → Arg
298 176 176
Legend: S — supratentorial diffuse low grade astrocytoma, A — sample from the first surgery, B — sample from the second surgery/first recurrence.
confirmed by western blot analysis showing no detectable levels of RAS protein in analyzed samples. Same authors also reported mutations in codons 16 and 26 of KRAS gene in analyzed samples, but without further explanation of their effect. Further, we detected missense mutations in the codon 4 region of HRAS gene (TAT N CAT; Tyr4His), present in all samples without recurrence, one recurrent sample and supratentorial GBM. To our knowledge, this is the first report describing alterations in the region of the 4th codon of HRAS gene, and further evaluations are needed to confirm their effect. We also detected single nucleotide insertion in codon 60 of KRAS gene in one of analyzed samples from a patient with single cerebellar tumor, which is otherwise commonly present in patients with Noonan syndrome (NS) (Fernandez-Medarde and Santos, 2011).
Survival of cGBM patients was significantly longer in comparison with the group of 30 patients with supratentorial GBM analyzed in our previous study (Milinkovic et al., 2012). Proliferation indices (Ki-67) were remarkably low in majority of cGBM samples, with values exceeding 18% in samples from only one patient (Case 5). That specific patient had three recurrent tumors, suggesting that higher values of Ki-67 labeling index could be used as a potential marker of cGBM recurrence. Other studies have attempted to correlate survival with Ki-67 proliferation index and reported contradictory findings (Bredel et al., 2002; Kuriyama et al., 2002; Reavey-Cantwell et al., 2001), leading to a general conclusion that association between Ki-67 and clinical outcome has not been demonstrated (Moskowitz et al., 2006). Although limited by the number of analyzed samples, we also concluded that Ki-67 labeling index could not represent a reliable marker of cGBM
Table 5 Identified mutations in RAS gene. Patient number
Case number
Exon
Codon number
Nucleotide change
Predicted effect
KRAS 1 2 4
1A 2 4
2 2 3
3 3 60
GAA TAT AAA → GTaA ATA TAA GAA TAT AAA → GTA ATA TAA GGT CAA → AbGG TCA
Glu Tyr Lys → Val Ile STOP Glu Tyr Lys → Val Ile STOP Frameshift
HRAS 1 2 2 3 4 5 5 5
1B 2S 2 3 4 5A 5B 5C
2 2 2 2 2 2 2 2
4 4 4 4 4 4 4 4
TAT → CAT TAT → CAT TAT → CAT TAT → CAT TAT → CAT TAT AAG → CcTA TAA TAT AAG → CTA TAA TAT AAG → CTA TAA
Tyr → His Tyr → His Tyr → His Tyr → His Tyr → His Tyr Lys → Leu STOP Tyr Lys → Leu STOP Tyr Lys → Leu STOP
Legend: S — see legend of Table 4, A — sample from the first surgery, B — sample from the second surgery/first recurrence, C — sample from the third surgery/second recurrence, S — supratentorial diffuse low grade astrocytoma. a Insertion of T in the 3rd codon. b Insertion of A in the 60th codon. c Insertion of C in the 4th codon.
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References
Fig. 3. Kaplan–Meier survival curves. Survival of patients with cerebellar glioblastoma was significantly longer compared to survival of supratentorial GBM patients (log rank test, p = 0.04).
outcome. Similar to our findings, Saito et al. (2006) didn't find any significant correlation between survival and Ki-67 LI in cerebellar GBM. At present, there is no consensus on impact of p53 alterations on patients' survival. Although limited by the small number of cGBM samples analyzed in this study, our results could suggest that the presence of TP53 alterations might represent a negative predictor of this disease outcome, especially having in mind that the only alive patient had no alterations in this gene and had the lowest number of p53 immunopositive cells. Conclusion This is the first report analyzing specific genetic alterations in cGBM, as well as morphological features of this rare disease. Although a small number of samples were analyzed, we postulate that TP53 mutations might be responsible for the progression of this tumor type. Besides, younger age at onset, longer follow-up, as well as high immunopositive rate and specific p53 mutational pattern suggest that cerebellar GBMs resemble secondary GBMs. Premature induction of STOP codon in both KRAS and HRAS gene, as well as other novel alterations in these genes, suggest that RAS signaling is impaired in cGBM. Mutations of RAS family genes might be a specific feature of this tumor type, especially when we know that these alterations have not been detected in supratentorial GBM. Further investigation should follow to clarify the impact of detected gene alterations on RAS function. High incidence of RAS mutations, as well as significantly longer survival, suggest that cerebellar GBM may have different mechanisms of occurrence and progression in comparison with supratentorial GBM. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This study was supported by Grant III41031 from the Ministry of Education, Science, and Technical Development, Republic of Serbia.
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Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
The impact of TP53 and RAS mutations on cerebellar glioblastomas Vedrana P. Milinkovic a,⁎, Milica K. Skender Gazibara b, Emilija M. Manojlovic Gacic b, Tatjana M. Gazibara c, Nikola T. Tanic a a b c
University of Belgrade, Institute for Biological Research “Sinisa Stankovic”, Department of Neurobiology, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia University of Belgrade, School of Medicine, Institute of Pathology, Doktora Subotica 1, 11000 Belgrade, Serbia University of Belgrade, School of Medicine, Institute of Epidemiology, Visegradska 26, 11000 Belgrade, Serbia
a r t i c l e
i n f o
Article history: Received 4 July 2014 Available online 16 July 2014 Keywords: Glioblastoma Cerebellar glioblastoma p53 RAS Immunohistochemistry
a b s t r a c t Cerebellar glioblastoma (cGBM) is a rare, inadequately characterized disease, without detailed information on its molecular basis. This is the first report analyzing both TP53 and RAS alterations in cGBM. TP53 mutations were detected in more than half of the samples from our cohort, mainly in hotspot codons. There were no activating mutations in hotspot codons 12/13 and 61 of KRAS and HRAS genes in cGBM samples but we detected alterations in other parts of exons 2 and 3 of these genes, including premature induction of STOP codon. This mutation was present in 3 out of 5 patients. High incidence of RAS mutations, as well as significantly longer survival of cGBM patients compared to those with supratentorial GBM suggest that cGBM may have different mechanisms of occurrence. Our results suggest that inactivation of TP53 and RAS may play an important role in the progression of cerebellar GBM. © 2014 Elsevier Inc. All rights reserved.
Introduction Glioblastoma multiforme (GBM) (World Health Organization, WHO, grade IV) is the most frequent and aggressive primary brain tumor in adults. According to the latest WHO classification (Louis et al., 2007), histologic criteria for the diagnosis of GBM include nuclear atypia, cellular pleomorphism, mitotic activity, microvascular proliferation and necrosis. Majority of glioblastomas develop de novo (primary glioblastomas) without clinical or histological evidence of a less malignant precursor lesion, while others, progressing from low-grade diffuse astrocytoma or anaplastic astrocytoma (AA) represent secondary GBM (Ohgaki and Kleihues, 2007). Alterations of several genes, including TP53, PTEN, CDKN2A, and EGFR, are frequently present in gliomas, usually occurring in a defined order during the progression to a high-grade tumor. A recent genomewide mutational analyses of GBM revealed somatic mutations of the IDH1 and IDH2 (isocitrate dehydrogenase) genes, most frequently in tumors that were known to have evolved from lower-grade gliomas (secondary glioblastomas) (Yan et al., 2009). Glioblastoma occurs most often in the cerebral hemispheres, while cerebellar location is rather rare, reported only in 0.4–3.4% of all GBM (Babu et al., 2013). Almost all previous studies were limited to
⁎ Corresponding author at: Institute for Biological Research, Department of Neurobiology, Laboratory for Molecular Neurobiology, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia. E-mail addresses: [email protected], [email protected] (V.P. Milinkovic).
http://dx.doi.org/10.1016/j.yexmp.2014.07.009 0014-4800/© 2014 Elsevier Inc. All rights reserved.
description of one or a few cases without further analyzes. Although current studies have revealed differences in patient and imaging characteristics between cerebellar and supratentorial GBM (Kuroiwa et al., 1995; Weber et al., 2006), there are also opposed reports (Akimoto et al., 2009; Stark et al., 2010) indicating similarity between tumors within these two locations. Due to its low incidence, little is reported about any genetic abnormalities in cerebellar GBM (cGBM), and there is no consensus on survival compared to supratentorial GBM (Utsuki et al., 2012). Inactivation of p53 tumor suppressor is known to be an early and frequent event in GBM formation, especially in the pathway leading to secondary GBM progression (Furnari et al., 2007; Ohgaki and Kleihues, 2007). A few reports described immunohistochemical studies of p53 in cases of cGBM in adults with exceptionally high immunopositive rates (85.7%) (Kawarabuki et al., 2005; Utsuki et al., 2012) suggesting that cGBMs should be defined as secondary ones. Insight into genetic basis of this extensive p53 immunopositivity is needed for better understanding of its role in cGBM pathogenesis. RAS GTPase family members are crucial players in many signaling networks that regulate cell-cycle progression, growth, migration, cytoskeletal changes, apoptosis, and senescence (Fernandez-Medarde and Santos, 2011). Mutational activation of RAS gene is often present in various tumors in humans, leading to increased invasion and metastasis, and decreased apoptosis due to formation of permanently active GTPbound form of RAS. Specifically, mutations in codon 12, 13, or 61 of one of the three RAS proto-oncogenes (HRAS, KRAS, and NRAS) transform them into oncogenes (Bos, 1989). Incidences of Ras oncogenic
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mutations, however, vary considerably among different types of cancers (Schubbert et al., 2007), and the type of RAS mutation seems to correlate with the tumor type. Besides activating mutations, activation of RAS oncogenes occurs by several other routes, including overexpression, gene amplification, translocations, or insertion of a promoter or enhancer element (Lymbouridou et al., 2009). A few studies, however, have examined the molecular alteration of the RAS genes in gliomas, with general conclusion that somatic mutations in hot-spot codons of RAS genes seem to be very rare in GBMs (Lo, 2010) and slightly more frequent in astrocytomas (Sharma et al., 2005). Anyhow, there are no reports on the presence of alterations of RAS gene family members in cGBM, possibly due to its low incidence in population. The aim of our study was to get better insight into molecular basis of cGBM, a rare but aggressive disease, putting emphasis on specific gene alterations frequently present in other tumor types. Histopatological and immunohistochemical characterization of these tumors was performed and compared with results from previously published studies. Additionally, samples of recurrent cGBM were analyzed in attempt to perceive mechanisms underlying its aggressive nature. Finally, molecular alterations and prognosis of cGBM were compared with supratentorial GBMs, in order to elucidate specific hallmarks of this rare disease. Material and methods Patient characteristics In our database of neurosurgical biopsies from 2002 to 2012 we found seven patients with cGBM. In the present investigation we could analyze five of them, because two were lacking in either clinical or pathological data. Patients were operated at the Neurosurgery Clinic, Clinical Centre of Serbia, Belgrade. All the procedures followed were approved by the Ethical Committee of Clinical Center of Serbia (approval number 3672/1) in accordance with the ethical standards laid down in the 1975 Declaration of Helsinki, laws of Republic of Serbia, as well as GCP guidelines. All available slides of cGBM were reviewed and classified according to the 2007 WHO classification by two pathologists, MSG and EMG. Another set of 30, previously characterized supratentorial high-grade gliomas (Milinkovic et al., 2012), was used for comparison in survival analysis. Mortality data for all patients were obtained from the Statistical Office of the Republic of Serbia (Statistical Office of the Republic of Serbia http://webrzs.stat.gov.rs/WebSite/). Immunohistochemistry For each tumor, tissue samples were fixed in 10% buffered formalin, embedded in paraffin blocks and cut into 5 μm thick sections used for routine histology (hematoxylin–eosin stain) and immunohistochemistry. Immunohistochemical stains were performed manually using the following antibodies according to the manufacturer instructions: Glial Fibrillary Acidic Protein (GFAP) (1:100, polyclonal, DAKO, Denmark), S-100 protein (1:200, polyclonal, DAKO, Denmark), Vimentin (1:300, clone V9, DAKO, Denmark), Synaptophysin (1:20, polyclonal, DAKO, Denmark), Cytokeratin (CK) (1:100, clone AE1/AE3, DAKO, Denmark), p53 (1:450, clone DO7, Thermo Scientific, Fremont, CA, USA) and Ki67 (1:50, clone MIB-1, DAKO, Denmark). The antibodies were detected by Ultra Vision Detection System, Large Volume Anti-Polyvalent, HRP (Thermo Scientific, Fremont, CA, USA). The sections were visualized with 3,3′-diaminobenzidine. Nuclear counterstaining was performed with Mayer's hematoxylin. Analysis of Ki-67 labeling index and p53 expression Areas with the highest number of positive nuclei were captured using Olympus camera DP70 with ×400 magnification. Counting of nuclei was performed manually, using ImageJ program. We counted a
203
minimum of 1000 cells per case (ranging from 1000 to 1417), except for Case 1B, where counting was done on 500 cells because of paucity of tissue available for the analysis. Only intensely stained nuclei were taken into account. Results were expressed as percentage of tumor cells with positive nuclei (labeling index). PCR amplification and sequencing DNA from paraffin-embedded tumor material was extracted using Kapa Express Extract DNA extraction kit (Kapa Biosystems, United States). The quality of the isolated DNA was verified by electrophoresis on 0.8% agarose gel. The DNA concentration was assessed spectrophotometrically. Frequently mutated exons of TP53 gene (5–9) were amplified and screened for mutations by PCR-SSCP (Single Strand Conformation Polymorphism) analysis according to Orita et al. (1989). Codons 12, 13 and 61 of RAS gene were also amplified and subjected to sequencing. Primers and PCR conditions for RAS gene (H-RAS and K-RAS) are shown in Table 1. Detected mutations were confirmed by sequencing with Applied Biosystems Incorporated dye terminator sequencing kit according to the manufacturer's specifications on an ABI Prism 3130 automated sequencer (Applied Biosystems, Foster City, USA). Sequencing was carried out in both directions. The obtained sequences were analyzed and compared with wild-type TP53 and RAS sequences using BLAST software in the NCBI GenBank database. Statistical analysis The survival time of 5 cerebellar and 30 supratentorial GBM patients analyzed in our previous study (Milinkovic et al., 2012) was compared. Survival analyses were performed using Kaplan and Meier productlimit method. The log rank test was used to assess the significance of the difference between pairs of survival probabilities. Overall survival was calculated from the day after surgery to the last follow-up examination or death of the patient. Statistical differences were considered significant when p was b 0.05 (*). Results Clinical data of each patient with cGBM are summarized in Table 2. All patients were males. The mean age at diagnose was 46.4 years (ranging from 36 to 64 years). Duration of symptoms (nausea—4 cases, headaches—3 cases, unstable gait—3 cases, weakness of one side of body and speech disturbances—1 case), from onset to diagnoses ranged from one week to one month. Two patients had cGBM recurrences (patient 1 and patient 5). Both of them had two recurrences (labeled by 1B, 1C, 5B and 5C, respectively). However, the tissue from the second recurrence of patient 1 (1C) was not available for immunohistochemical and molecular analysis. In patient 2 cGBM appeared 1 year after surgery of supratentorial diffuse low grade astrocytoma (Case 2S) without any evidence of supratentorial recurrence. All patients died within 5 years upon diagnosis, except for patient 4 who is still alive. Median survival was 33.2 months. Histopathological and immunohistochemical features Typical histopathological features of GBM were observed in all cases (Fig. 1A). On immunohistochemistry, all tumors showed moderate to intensive GFAP (Fig. 1B), Vimentin and S-100 immunopositivity, while they were negative on CK and Synaptophysin. Ki-67 labeling index showed great variability, ranging from 0.3% to 40.65%, with a tendency to increase along recurrences, in spite of adjuvant therapy. Percentage of p53 positive nuclei was also heterogeneous, ranging from 1.71% to 40.62%, with variable trends in recurrences (Table 3, Fig. 1C and D).
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Table 1 Ras gene oligonucleotide primers and PCR conditions. Ras gene
Exon/codon
Primer sequence
PCR conditions
K Ras
exon-2 12/13 exon-3 61 exon-2 12/13 exon-3 61
(f) 5′-ATGACTGAATATAAACTTGT-3′ (r) 5′-CTCTATTGTTGGATCATATT-3′ (f) 5′-AAGTAGTAATTGATGGAGAA-3′ (r) 5′-AGAAAGCCCTCCCCAGTCCT-3′ (f) 5′-ATGACGGAACATAAGCTGGT-3′ (r) 5′-CGCCAGGCTCACCTCTATA-3′ (f) 5′-AGGTGGTCATTGATGGGGAG-3′ (r) 5′-AGGAAGCCCTCCCCGGTGCG-3′
95 35 95 56 65
K Ras H Ras H Ras
PCR reaction mixture (25 μl)
°C (5 min) cycles: °C (1 min) °C (1 min) °C (1 min)
1.5 mM MgCl2; 2 mM dNTP; 0.6 μM each (f) and (r) primer; 1 U/reaction Taq polymerase
65 °C (7 min)
Analysis of genetic alterations
Survival analyses
All primary and recurrent tumor samples from 5 patients were further analyzed for the presence of DNA alterations in TP53 and RAS genes. We analyzed 9 samples in total, one from Cases 3 and 4 each, two from Case 1 (1A and 1B), two from Case 2 (2 and 2S) and three samples from Case 5 (5A, 5B and 5C). A PCR-SSCP analysis revealed TP53 aberrations in 5 out of 9 samples, which were further sequenced to confirm and identify mutations. DNA sequencing confirmed all 5 mutations in 3 patients (Fig. 2A). The exon locations, codon positions, nucleotide changes, and predicted effects on protein primer structure of all 5 mutations are summarized in Table 4. In Case 1, TP53 was altered only in recurrent glioblastoma sample (1B), bearing mutation in known hotspot mutation site in codon 273. We detected splice site deletion in supratentorial diffuse low grade astrocytoma (Case 2S) which was not present in cerebellar glioblastoma of the same patient. Samples 5 and 5B shared the same mutation in codon 176 exon 5 of TP53 gene, while there were no mutations in this gene in the second recurrent sample from the same patient (5C). Detected mutations in TP53 gene were confirmed in all samples with higher percentage of p53 positive nuclei. We further performed a systematic mutation analysis of the KRAS and HRAS genes with the special emphasis on known mutation hotspot sites in these genes. The exon locations, codon positions, nucleotide changes, and predicted effects are summarized in Table 5. There were no alterations in hot spot codons 12, 13 and 61 in any of the analyzed samples. All patients had alterations in KRAS or HRAS gene, but identified mutations found in these genes were not previously reported in cancer databases. The most frequent alteration was missense mutation in codon 4 of HRAS gene, present in five samples from four patients. We also detected single nucleotide insertions in codon 3 of KRAS gene in 2 samples, and in codon 4 of HRAS gene in all 3 samples from patient 5. Both insertions lead to premature induction of STOP codon, preventing the synthesis of a full-length RAS protein. Finally, we identified frameshift mutation due to single nucleotide insertion in codon 60 of KRAS gene, present in only one sample.
Median OS of patients with cGBM was 33.2 months (range 1– 78 months). Four out of five patients died of this disease. Median time to recurrence was 10 months, and at the time of the analysis two patients had experienced recurrence. The median survival of the 30 patients with supratentorial high-grade glioma was 11 months, significantly shorter compared to cGBM patients (log rank test, p = 0.04, Fig. 3). Discussion Cerebellar GBMs in adults are rare tumors that are insufficiently characterized in terms of incidence, natural history, prognostic factors and the presence of genetic alterations. Majority of previously published reports were case reports or retrospective studies focused on better characterization of outcome and identification of different factors which affect survival, being insufficient in explaining molecular mechanisms underlying this disease. We wanted to further characterize genetic alterations in cGBM with the aim to compare detected changes with alterations in supratentorial GBM. Besides, we were able to analyze recurrent tumors from the same patient in order to compare their histological and genetic profile with matching primary tumor. First of all, we examined mutational status of TP53 tumor suppressor gene, most frequently altered in various tumor types, in order to evaluate its impact on development of cGBM. Clinical studies identifying TP53 mutations in low-grade glioma, along with the well-established role of p53 in cancer, have suggested that TP53 is a potent driver mutation in GBM, predominantly in the pathway leading to secondary glioblastoma progression (England et al., 2013). The presence of mutations in samples of three out of five analyzed patients suggests that p53 has an important role in cGBM progression as well. Besides, almost all detected mutations in our samples were either previously reported in glioma (Meyer-Puttlitz et al., 1997) or positioned in hotspot codons (England et al., 2013), indicating that these mutations seriously affect p53
Table 2 Clinical data of patients with cGBM. Patient number
Age (years)
Sex
Cerebellar location
Surgical treatment
RT (Gy)
CT
Recurrent (months)
Survival (months)
1
42
M
Right hemisphere
Partial resection
60
–
Died (63)
2
41
M
Vermis
Partial resection
60
–
1B local (12) 1C local (48) None
3
64
M
Right hemisphere
Total resection
–
–
None
4
36
M
Vermis
Total resection
60
BCNU
None
5
49
M
Right hemisphere
Partial resection
60
BCNU Temo-zolomide
5B local (8) 5C local (16)
Died (1) Died (6) Alive (78) Died (18)
Legend: M — male, B — sample from the second surgery/first recurrence, C — sample from the third surgery/second recurrence, RT — radiation therapy, CT — chemotherapy.
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Fig. 1. Histopathological and immunohistochemical features of cGBM sample (Case 5). (A) Highly cellular tumor tissue with prominent microvascular proliferation (arrows) and multiple small pseudopalisading necroses. Zone of partially preserved cerebellar folia is present at the right corner (H–E ×400); (B) Diffuse GFAP immunoreactivity in tumor cells. Vascular “glomeruloid tufts” are negative. (×400); (C) High Ki-67 labeling index (×100); (D) Nuclear accumulation of p53 protein in tumor cells but not in endothelial cell (×200).
function in cerebellar glioblastoma. In contrast to primary GBM, where TP53 mutations were almost equally distributed, the majority of TP53 mutations in secondary glioblastomas were located in codons 248 and 273 (Ohgaki et al., 2004), as it was the case in one of our samples, supporting the assumptions that cGBMs are more similar to secondary glioblastomas. We found it interesting that mutational patterns of TP53 tumor suppressor gene were not repeated in recurrent samples of the same patient. Tumor heterogeneity and infiltrative growth pattern seem to be the most plausible explanation for this finding (Marusyk and Polyak, 2010). Specifically, one subclone in the tumor carried a TP53 mutation in contrast to another parent clone, which persisted through the clinical course of the patient and was finally sampled in the third tumor removed from this patient. In case of patient 2, both supratentorial and cerebellar tumor samples carried mutations in TP53. Differences in identified mutations confirm our assumption that these two tumors represent two distinct tumor entities. Table 3 Ki-67 and p53 labeling indices in cGBM cases. Case no.
Ki-67 (%)
P53 (%)
1A 1B 1C 2 3 4 5A 5B 5C
3.46 4.44 NA 0.3 5.94 5.48 31.94 41.61 40.65
4.72 14.02 NA 1.71 4.41 0.62 30 59.1 5.02
Legend: NA — not available, A — sample from the first surgery, B — sample from the second surgery/first recurrence, C — sample from the third surgery/second recurrence.
Ras genes (HRAS, KRAS and NRAS) are frequently mutated in human cancers. Their protein products are GTPases that function as molecular switches in regulating pathways responsible for proliferation and cell survival (Prior et al., 2012). Since there is no evidence of activating mutations in hotspot codons 12/13 and 61 of Ras genes in gliomas (Ghazali et al., 2005; Gomori et al., 1999), RAS activation must be due to other alterations, such as amplification and overexpression of growth factor receptor genes or aberrations in yet other RAS pathway genes (Knobbe et al., 2004). When analyzing alterations in Ras genes, we discovered what may be fairly novel mutations that are not typical of cancers. There were no activating mutations in hot spot codons of KRAS and HRAS genes in our set of cGBM samples, but we detected alterations in other parts of exons 2 and 3 of these genes. The most peculiar finding was premature induction of STOP codon in both RAS genes present in three samples from five patients. Single nucleotide insertions in the codon 3 of KRAS and in the codon 4 of HRAS were found, leading to formation of truncated forms of these proteins. This represents the first evidence of this type of genetic alteration in HRAS gene, while there were a few reports demonstrating the presence of nonsense mutations in KRAS gene. Palmirotta et al. (2009) detected novel nonsense mutation in the codon 22 of KRAS gene in colorectal cancer, while a single nucleotide deletion of one G in codon 12 in squamous cell carcinoma resulted in a stop codon formation several bases downstream (Vachtenheim et al., 1995), both determining the synthesis of a completely inactive truncated KRAS protein. These novel types of RAS mutations might be more frequent than we presently know, probably because current conventional analytical techniques have focused exclusively on the identification of conventional mutations (van Krieken et al., 2008). Although RAS is a positive regulator of cell growth, differentiation and survival, inactivation of this gene was present in primary tumors which later gave rise to recurrences. In support of our finding, Lymbouridou et al. (2009) reported significant decrease in KRAS and HRAS mRNA levels in glioblastoma samples,
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Fig. 2. DNA sequencing of TP53 and RAS genes. (A) Part of exon 5 DNA sequence of TP53 gene bearing a point mutation at codon 176 with missense effect (p.C176R). The arrow indicates the mutation site in Case 5A sample. (B) Fragment of exon 2 DNA sequence of HRAS gene showing a point mutation at codon 4 with missense effect (p.Y4H). The arrow indicates the mutation site (shown is the sequence of the antisense strand of Case 1B).
Table 4 Identified mutations in TP53 tumor suppressor gene. Patient number
Case number
Exon/intron
Codon number
Nucleotide change
Predicted effect
1 2 2 5 5
1B 2S 2 5A 5B
Exon 8 Exon 9 Exon 8 Exon 5 Exon 5
273
CGT → CCT (CpG) del 13987–13992 GAG → GAC TGC → CGC TGC → CGC
Arg → Pro Splice site deletion Glu → Asp Cys → Arg Cys → Arg
298 176 176
Legend: S — supratentorial diffuse low grade astrocytoma, A — sample from the first surgery, B — sample from the second surgery/first recurrence.
confirmed by western blot analysis showing no detectable levels of RAS protein in analyzed samples. Same authors also reported mutations in codons 16 and 26 of KRAS gene in analyzed samples, but without further explanation of their effect. Further, we detected missense mutations in the codon 4 region of HRAS gene (TAT N CAT; Tyr4His), present in all samples without recurrence, one recurrent sample and supratentorial GBM. To our knowledge, this is the first report describing alterations in the region of the 4th codon of HRAS gene, and further evaluations are needed to confirm their effect. We also detected single nucleotide insertion in codon 60 of KRAS gene in one of analyzed samples from a patient with single cerebellar tumor, which is otherwise commonly present in patients with Noonan syndrome (NS) (Fernandez-Medarde and Santos, 2011).
Survival of cGBM patients was significantly longer in comparison with the group of 30 patients with supratentorial GBM analyzed in our previous study (Milinkovic et al., 2012). Proliferation indices (Ki-67) were remarkably low in majority of cGBM samples, with values exceeding 18% in samples from only one patient (Case 5). That specific patient had three recurrent tumors, suggesting that higher values of Ki-67 labeling index could be used as a potential marker of cGBM recurrence. Other studies have attempted to correlate survival with Ki-67 proliferation index and reported contradictory findings (Bredel et al., 2002; Kuriyama et al., 2002; Reavey-Cantwell et al., 2001), leading to a general conclusion that association between Ki-67 and clinical outcome has not been demonstrated (Moskowitz et al., 2006). Although limited by the number of analyzed samples, we also concluded that Ki-67 labeling index could not represent a reliable marker of cGBM
Table 5 Identified mutations in RAS gene. Patient number
Case number
Exon
Codon number
Nucleotide change
Predicted effect
KRAS 1 2 4
1A 2 4
2 2 3
3 3 60
GAA TAT AAA → GTaA ATA TAA GAA TAT AAA → GTA ATA TAA GGT CAA → AbGG TCA
Glu Tyr Lys → Val Ile STOP Glu Tyr Lys → Val Ile STOP Frameshift
HRAS 1 2 2 3 4 5 5 5
1B 2S 2 3 4 5A 5B 5C
2 2 2 2 2 2 2 2
4 4 4 4 4 4 4 4
TAT → CAT TAT → CAT TAT → CAT TAT → CAT TAT → CAT TAT AAG → CcTA TAA TAT AAG → CTA TAA TAT AAG → CTA TAA
Tyr → His Tyr → His Tyr → His Tyr → His Tyr → His Tyr Lys → Leu STOP Tyr Lys → Leu STOP Tyr Lys → Leu STOP
Legend: S — see legend of Table 4, A — sample from the first surgery, B — sample from the second surgery/first recurrence, C — sample from the third surgery/second recurrence, S — supratentorial diffuse low grade astrocytoma. a Insertion of T in the 3rd codon. b Insertion of A in the 60th codon. c Insertion of C in the 4th codon.
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References
Fig. 3. Kaplan–Meier survival curves. Survival of patients with cerebellar glioblastoma was significantly longer compared to survival of supratentorial GBM patients (log rank test, p = 0.04).
outcome. Similar to our findings, Saito et al. (2006) didn't find any significant correlation between survival and Ki-67 LI in cerebellar GBM. At present, there is no consensus on impact of p53 alterations on patients' survival. Although limited by the small number of cGBM samples analyzed in this study, our results could suggest that the presence of TP53 alterations might represent a negative predictor of this disease outcome, especially having in mind that the only alive patient had no alterations in this gene and had the lowest number of p53 immunopositive cells. Conclusion This is the first report analyzing specific genetic alterations in cGBM, as well as morphological features of this rare disease. Although a small number of samples were analyzed, we postulate that TP53 mutations might be responsible for the progression of this tumor type. Besides, younger age at onset, longer follow-up, as well as high immunopositive rate and specific p53 mutational pattern suggest that cerebellar GBMs resemble secondary GBMs. Premature induction of STOP codon in both KRAS and HRAS gene, as well as other novel alterations in these genes, suggest that RAS signaling is impaired in cGBM. Mutations of RAS family genes might be a specific feature of this tumor type, especially when we know that these alterations have not been detected in supratentorial GBM. Further investigation should follow to clarify the impact of detected gene alterations on RAS function. High incidence of RAS mutations, as well as significantly longer survival, suggest that cerebellar GBM may have different mechanisms of occurrence and progression in comparison with supratentorial GBM. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This study was supported by Grant III41031 from the Ministry of Education, Science, and Technical Development, Republic of Serbia.
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