1 International Agency for Research on Cancer, F-69372 Lyon, France and
2 Department of Neurosurgery, University Hospital Zürich, CH-8091 Zürich, Switzerland
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Abstract |
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Abbreviations: BCNU, 1,3-bis-(2-chloroethyl)-1-nitrosourea; CCNU, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea; MGMT, O6-Methylguanine-DNA methyltransferase.
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Introduction |
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O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that directly and specifically removes pro-mutagenic DNA lesions at the O6 position of guanine by transferring the alkyl group to one of its own cysteine residues, without base excision (12). Intracellular levels of MGMT vary among tissues and appear to be inversely correlated with tissue-specific tumorigenesis induced by alkylating agents in rats (12,14).
MGMT activity varies significantly between tumors, over an ~300-fold range (15). Approximately 2025% of human tumours lack detectable MGMT activity and carry the methyl repair-deficient (Mer) phenotype (15,16). Neoplastic cell lines with the Mer phenotype have been found to show methylation of CpG sequences in the promoter of the MGMT gene; methylation was not detected in MGMT-expressing cells (12,1719). MGMT methylation has been found in ~40% of gliomas and colorectal carcinomas, less frequently (~25%) in non-small cell lung carcinomas, lymphomas and head and neck carcinomas, and rarely in non-glial brain tumours, breast cancer, endometrial cancer and leukemias (20).
The objective of the present study was to compare the frequency of MGMT methylation in primary glioblastomas with that in low-grade astrocytomas and secondary glioblastomas derived from them. We further correlated MGMT methylation with the frequency and pattern of TP53 mutations.
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Materials and methods |
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Thirty-six patients with primary glioblastoma (20 males, 16 females, mean age 56.8 ± 11.2 years, range 3473 years) had a preoperative clinical history of <3 months (mean, 1.5 months) and a histological diagnosis of glioblastoma at the first biopsy, without any evidence of a less malignant precursor lesion. Sixteen patients with secondary glioblastoma (eight males, eight females, mean age 39.7 ± 8.8 years, range 2653 years) had at least two biopsies, with clinical and histological evidence of progression from low-grade diffuse astrocytoma (WHO grade II). Low-grade diffuse astrocytomas (15 cases) from the same patients with secondary glioblastomas and 39 additional low-grade diffuse astrocytomas (31 males, 23 females, mean age 36.3 ± 10.5 years, range 2262 years) were also examined. DNA was extracted from typical tumor areas that were manually microdissected from paraffin sections as described previously (22).
Peritumoral normal brain tissue was available in sections of 11 low-grade astrocytomas and was also carefully microdissected manually and analysed.
Methylation-specific PCR for MGMT methylation
DNA methylation in the CpG islands of the MGMT gene was determined by methylation-specific PCR (23). Sodium bisulfite modification was performed using the CpGenomeTM DNA Modification Kit (Intergen, Oxford, UK) as described previously (24). Control methylated DNA (Intergen) and unmethylated DNA (normal blood) were treated with bisulfite by the same method. Primer sequences for the methylated and unmethylated reactions have been reported by Esteller et al. (20,25). Sense primers were located at the 3' end of exon 1 and antisense primers were at the 5' end of intron 1, the region where the presence of an enhancer element has been reported (17). PCR was carried out in a 10 µl volume containing PCR buffer (20 mM Tris pH 8.4, 50 mM KCl), 2 mM MgCl2, dNTPs (250 mM each), primers (0.5 mM each), 0.5 U PLATINUM® Taq DNA polymerase (Gibco-BRL, Cergy Pontoise, France) and ~40 ng bisulfite-modified DNA. Amplification was carried out in a Robocycler (Stratagene) with initial denaturing at 95°C for 5 min followed by 35 cycles of denaturing at 95°C for 50 s, annealing for 50 s at 59°C and extension for 50 s at 72°C, and then a final extension for 2 min at 72°C. Amplified products were electrophoresed on a 3% agarose gel, and were visualized by ethidium bromide. For each PCR reaction, we included methylated and unmethylated DNA, and normal blood DNA without bisulfite modification, as positive and negative controls.
TP53 mutations
TP53 mutations in all glioblastomas and most low-grade diffuse astrocytomas except for nine cases were reported previously (5,8,9). For these nine cases, pre-screening for mutations by PCRSSCP analysis was carried out in exons 58 of the TP53 gene. Sequencing primers used were as follows: 5'-TCT GTC TCC TTC CTC TTC CTA C-3' (sense) and 5'-AAC CAG CCC TGT CGT CTC TCC A-3' (antisense) for exon 5; 5'-CTG GGG CTG GAG AGA CGA CA-3' (sense) and 5'-GCC ACT GAC AAC CAC CCT TA-3' (antisense) for exon 6; 5'-TGC CAC AGG TCT CCC CAA GG-3' (sense) and 5'-GGG TCA GAG GCA AGC AGA GG-3' (antisense) for exon 7; 5'-TCC TTA CTG CCT CTT GCT TC-3' (sense) and 5'-TCT CCT CCA CCG CTT CTT GT-3' (antisense) for exon 8. Samples which showed mobility shifts in SSCP analysis were further analyzed by DNA sequencing. After PCR amplification with the same set of primers, PCR products were sequenced on a Genetic Analyzer (ABI PRISMTM 310, Perkin-Elmer Biosystems) using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kits (Perkin-Elmer Applied Biosystems).
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Results |
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Correlation between MGMT methylation and TP53 mutations
The majority of low-grade astrocytomas with MGMT methylation (24/26, 92%) contained a TP53 mutation, whereas only 11 out of 28 (39%) low-grade astrocytomas without MGMT methylation contained a TP53 mutation (Table I P, < 0.0001). G:C
A:T transition mutations at CpG sites were significantly more frequent in low-grade astrocytomas with MGMT methylation (15/26, 58%) than in those without MGMT methylation (3/28, 11%: P < 0.0004, Table I
). In secondary glioblastomas, TP53 mutations were significantly more frequent in cases with MGMT methylation (92%) than in those without (25%; P = 0.027). In primary glioblastomas, the difference was not significant, probably because this glioblastoma subtype rarely contains TP53 mutations.
Correlation between MGMT methylation and age of patients
There was no significant difference in age of patients with low-grade astrocytomas between those with and without MGMT methylation. The patients with glioblastomas with MGMT methylation were significantly younger (47 ± 13 years) than those without MGMT methylation (55 ± 13 years, P = 0.0267).
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Discussion |
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We have also shown that MGMT methylation is often associated with a TP53 mutation in secondary glioblastomas and low-grade astrocytomas. Except for two cases, all low-grade astrocytomas with MGMT methylation contained a TP53 mutation, the majority of which were G:C A:T transition mutations (18/26, 69%) and of these, most were at CpG sites (15/18, 83%). In contrast, only 11 out of 28 (39%) low-grade astrocytomas lacking MGMT methylation contained a TP53 mutation; in these cases, G:C
A:T mutations were also less frequent (14%). For comparison, the frequency of G:C
A:T TP53 mutations in 749 human brain tumours listed in the IARC TP53 database is 52%, of which 66% are located at CpG sites (www.iarc.fr/p53/index.html); this frequency is lower than that of our cases with MGMT methylation.
A similar correlation has been reported for colon carcinomas (25), for which a strong association was found between MGMT methylation and the presence of G A mutations in the K-ras gene: 71% (36/51) of the tumours with a G
A mutation had MGMT methylation, whereas only 32% (12/37) of those with other K-ras mutations (not involving G
A transitions) and 35% (55/156) of the tumours without K-ras mutations had MGMT methylation (25). These and our results suggest that epigenetic silencing of MGMT by promoter methylation may lead to preferential occurrence of G:C
A:T transitions in transformation-associated genes in human neoplasms. It is of interest that spontaneous G:C
A:T transitions are detected more frequently in the adenine phosphoribosyl transferase gene of Chinese hamster ovary cells lacking MGMT activity (28%) than in those expressing MGMT (5%) (26).
The aetiology of human brain tumours is still largely unknown. With the exception of the very rare causation by therapeutic irradiation (22), epidemiological studies have failed to unequivocally identify environmental carcinogens that operate in the evolution of gliomas. Several endogenous pathways may lead to G:C A:T transitions at CpG sites, which are the most frequently observed TP53 mutations in human brain tumours (27). The best-characterized underlying mechanism is the deamination to thymine of 5-methylcytosine that is clustered at CpG sites. This is considered to occur spontaneously or to be factor-mediated, e.g. through the action of oxygen radicals or by nitric oxide produced by nitric oxide synthase in conditions of chronic inflammation (28). The possibility exists that the loss of MGMT-mediated repair due to methylation causes accumulation of G:C
A:T mutations resulting from O6-alkylG
T mispairing. However, analyses of tumour samples do not allow unequivocal distinction between the two major underlying mechanisms, i.e. C
T transitions in the transcribed strand of the gene resulting from either cytosine mutation (following deamination) or from mutation of the paired guanine on the complementary strand due to O6-alkylguanine mispairing with T.
It has been shown that O6-methylation of the guanine moiety at CpG islands is not efficiently repaired by MGMT if normal 5-methylcytosine is present in the TP53 sequence (27). This raises the possibility that TP53 mutations at CpG sites are not a result of deamination of 5-methylcytosine alone. They may, in addition, result from endogenous or exogenous factors that produce DNA adducts at the O6 position of guanine. A great variety of adducts at this position have been shown to be substrates for repair by MGMT (12). Such adducts typically result from exposure to N-nitrosamides and related alkylating agents that cause brain tumours in rats (29) but there is no evidence that these carcinogens are involved in the aetiology of human brain tumours.
If MGMT methylation is directly associated with preferential occurrence of TP53 mutations, one would expect that normal tissue surrounding tumours might also show MGMT methylation. Lack of MGMT activity (Mer status) has been found in ~60% of histologically normal brain samples adjacent to a primary brain tumour (15,16,30). The incidence of Mer status in non-neoplastic cerebral tissue from brain tumour patients was age-dependent, increasing from 21% in children (<19 years) to 75% in adults over 50 (30). In contrast, the Mer status was found in only 12% of normal brain specimens from patients operated for conditions other than primary brain tumours, and was not age-dependent (30). In the present study, we assessed 11 normal brain tissue samples (five of which were from an area adjacent to a low-grade astrocytoma with MGMT methylation), but no single case showed MGMT methylation.
One possibility would be that MGMT methylation occurs after the development of low-grade astrocytomas but before acquisition of a TP53 mutation. TP53 mutations constitute an early event in the pathway leading to secondary glioblastomas; >65% of low-grade astrocytomas contain TP53 mutations (5,9,31) and this fraction does not increase during malignant progression. In a clonal assay, it was shown that in low-grade astrocytomas, the fraction of mutated cells is low (821%) and increases during progression, reaching values of >95% in glioblastomas (32), suggesting that TP53 mutation is first present in a small fraction of tumour cells which, because of their growth advantage, then gradually expand during progression (32,33). However, it cannot be excluded that the lower fraction of mutated cells in low-grade astrocytomas is a result of contamination with non-neoplastic reactive astrocytes.
As the present results are based on DNA from biopsies rather than cultured cells, identification of MGMT methylation and TP53 mutation does not necessarily imply that both events occurred in the same tumour cell. In fact, methylation was more frequent in secondary glioblastomas than in low-grade astrocytomas from which they were derived.
The activity of MGMT can be modified in several ways. O6-benzylguanine efficiently inactivates the MGMT protein, thereby increasing the chemotherapeutic effectiveness of methylating and chloroethylating agents in vitro and in human tumour xenograft models (34). Other factors that may affect levels and activity of MGMT include polymorphisms in the MGMT gene, exposure to formaldehyde, presence of metal ions and the extent of DNA depurination (12,35).
In an analysis of 152 adult gliomas, MGMT activity was inversely correlated with the age of patients (16). In the present study, the mean age of patients with low-grade astrocytoma with MGMT methylation was similar to that of patients with tumours lacking methylation. The glioblastoma patients with MGMT methylation were younger than those without MGMT methylation, but this is likely to reflect the younger age of patients with secondary glioblastoma compared to primary glioblastoma (5).
O6-Alkylguanine adducts are produced by cytostatic monomethylating agents (procarbazine, temozolomide, dacarbazine), bifunctional chloroethylnitrosoureas that are used for the treatment of gliomas including 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl- 1-nitrosourea (CCNU), and related cancer therapeutic drugs (36). Brain tumours expressing low levels of MGMT have been found to be more sensitive to chemotherapy, and the therapeutic efficacy was enhanced by depletion of MGMT by O6-benzylguanine in vitro and in patients (12,34,37). Patients with malignant gliomas expressing high levels of MGMT had a shorter time to treatment failure and death following radiation plus adjuvant chemotherapy with BCNU (38). Furthermore, the frequency of the Mer phenotype among tumours recurring after surgery, radiation and alkylating agent-based chemotherapy was 7-fold lower than in tumours treated with surgery alone and 6-fold lower than in tumours recurring after surgery and radiation (15). Recently, Esteller et al. (39) assessed MGMT promoter methylation in anaplastic astrocytomas and glioblastomas that were subsequently treated with BCNU, and showed that MGMT promoter methylation was significantly associated with longer overall survival and time till progression.
The present study shows that MGMT methylation is significantly more frequent in secondary glioblastomas that progressed from low-grade astrocytomas than in primary glioblastomas (75 versus 36%). It is still a matter of controversy whether the prognosis of patients with secondary glioblastoma is better than (40) or similar to (41) that of patients with primary (de novo) glioblastomas. It thus remains to be shown whether susceptibility to chemotherapy differs between primary and secondary glioblastomas and whether this correlates with MGMT methylation.
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Notes |
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Acknowledgments |
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References |
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