Int. J. Cancer: 122, 1391–1399 (2008) ' 2007 Wiley-Liss, Inc.

MGMT in primary and recurrent human glioblastomas after radiation and chemotherapy and comparison with p53 status and clinical outcome
Dorothee Wiewrodt1, Georg Nagel2, Nadine Dreim€ller1, Thomas Hundsberger3, u Axel Perneczky1 and Bernd Kaina2* 1 Neurosurgical Department, University of Mainz, Mainz, Germany 2 Department of Toxicology, University of Mainz, Mainz, Germany 3 Department of Neurology, Kantonsspital St. Gallen, Gallen, Switzerland
The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) plays a pivotal role in alkylating drug resistance. Here, we determined MGMT activity in primary and recurrent glioblastomas (GBM, WHO grade IV) of patients who received radiation therapy (RT) or RT plus chemotherapy with alkylating agents (temozolomide, chloroethylnitrosoureas). The mean MGMT activity of untreated GBM was 37 6 45 (range 0–205) fmol/mg proteins. In the 1st, 2nd and 3rd recurrences, MGMT activity increased from 66 6 50 (13–194) to 68 6 44 (14–143) and 182 6 163 (64–423) fmol/mg protein, respectively. Comparing patients who received RT only with RT plus chemotherapy, a significant increase of MGMT in 1st recurrences was only found after treatment with RT plus chemotherapy, indicating either selection of MGMT expressing cells or induction of the MGMT gene by alkylating agents. The p53 status was not significantly related to the MGMT expression level, although a trend for lower MGMT activity in p53 positively stained tumors was observed. Patients expressing MGMT activity of 30 fmol/mg protein in the pretreatment tumor had a significant better therapeutic response than patients expressing MGMT above this level, which was shown by Kaplan-Meyer curves and the recurrence free interval after primary tumor resection. In patients who received RT only, this correlation was not found. The data revealed a threshold of MGMT expression (30 fmol/mg protein) below which patients respond better to alkylating agents. Therefore, determination of MGMT activity in the primary tumor appears to be useful in predicting the outcome of GBM therapy. ' 2007 Wiley-Liss, Inc. Key words: DNA repair; alkyltransferase; MGMT; glioblastomas; drug resistance; temozolomide

Despite the enormous progress in the treatment of various types of tumors, human gliomas still have a low curative response. In the therapy of gliomas, notably the most severe form glioblastoma multiforme (GBM, WHO grade IV), radiation therapy (RT) or RT concomitant with methylating agents such as temozolomide1 is applied. Methylating and chloroethylating agents such as BCNU (carmustine), CCNU (lomustine) or ACNU (nimustine) are also administered during adjuvant therapy.2 Unfortunately, only a minority of glioblastoma patients respond to temozolomide and chloroethylating nitrosoureas. A likely explanation for this is the development of either primary or acquired glioblastoma cell resistance that leads to protection of the tumor against alkylating drugs. Methylating and chloroethylating agents attack DNA at nucleophilic sites like the O6 position of guanine, forming O6-alkylguanine.3 This lesion is considered to be the major cause of mutations and malignant transformation induced by O6-alkylating agents.4 It also provokes cell death by inducing apoptosis5,6 and, therefore, is considered to be mainly responsible for the antineoplastic effect of O6-alkylating agents. O6-alkylguanine is repaired by the suicide DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) that transfers the alkyl group from the DNA to its own active center.7 Thereby, guanine in DNA is restored and MGMT becomes inactivated. Because of the suicide character of MGMT, the repair capacity of a cell is determined by the amount of preexisting molecules of MGMT and the rate of its resynthesis. MGMT has a profound influence on the cell’s survival and proliferation
Publication of the International Union Against Cancer

capacity following treatment with O6-alkylating agents like temozolomide, DTIC, BCNU, CCNU, ACNU and fotemustine.8,9 For methylating drugs, MGMT deficiency causes O6-methylguanine lesions to mispair with thymine, which leads to mismatch repair (MMR) mediated apoptosis in gliomas.10 Therefore, for temozolomide and other methylating drugs, MGMT and also MMR and the DNA replication status are critical factors that determine the level of cell resistance. For chloroethylating agents O6-chloroethylguanine is the critical primary lesion that is converted by intra-molecular rearrangement into a DNA interstrand crosslink, which provokes cell death (for review see Ref. 11). Since MGMT repairs the primary DNA lesion induced by both groups of agents before they are processed to trigger apoptosis, MGMT is a very important factor in tumor cell resistance toward methylating and chloroethylating anticancer drugs.12 MGMT is expressed highly variably in tumors, with low expression in brain tumors and malignant melanomas and high expression in ovarian and breast tumors.13–15 For brain tumors, lack of MGMT activity was reported to correlate with the therapeutic response of patients treated with the chloroethylating agent carmustine (BCNU).16,17 Whether a threshold in MGMT activity exists above which alkylating agent therapy is unsuccessful was not yet clear. More recently, MGMT promoter methylation corresponding to silencing of the MGMT gene was found to correlate with increased survival of patients who received radiation plus cisplatin and BCNU18 or radiation and temozolomide therapy.19 To date, MGMT promoter methylation is thought to be a useful predictive marker for glioma therapy. MGMT is an inducible DNA repair gene. Induction of the MGMT gene was shown notably in rat liver cells on glucocorticoid treatment and genotoxic stress, including X-rays and alkylating agents.20,21 It was also demonstrated on the level of the human MGMT promoter and, in the case of genotoxic stress, dependent on functional p53.22 MGMT induction was also reported for gliomas on dexamethasone treatment,23 which others were not able to confirm.24 MGMT gene induction is a transient phenomenon, reaching peak levels 1–2 days after genotoxic treatment.20,25 For human tumors it is unclear whether the MGMT gene becomes up-regulated during therapy. Therefore, studies on MGMT expression in tumor and normal tissue before and after therapeutic treatment are desirable. While previous studies extensively determined the expression of MGMT in pretreatment brain tumor tissue, no information on MGMT on activity level is available in recurrences following chemotherapy. The determination of MGMT expression in pretreatment tumors and their recurrences is important to asses whether and at which level it correlates with the outcome of alkylGrant sponsor: DFG; Grant number: KA 724/13-1, 13/2 and SFB432/ B7. *Correspondence to: Department of Toxicology, University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany. Fax: 10049-6131-393-3421. E-mail: kaina@uni-mainz.de Received 18 April 2007; Accepted after revision 3 September 2007 DOI 10.1002/ijc.23219 Published online 13 November 2007 in Wiley InterScience (www.interscience. wiley.com).

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ating agent therapy, choosing an individual dosing schedule, which might overcome resistance by further chemotherapy. Here, we report for the first time a follow-up study of MGMT activity in primary glioblastomas and its recurrences. We also determined the p53 status and the survival response of the patients and show that, with a few exceptions, MGMT activity increases in recurrences compared to the primary tumor. This increase was most significant in the group of patients who received alkylating agent therapy. We also show that patients with MGMT expression level below 30 fmol/mg protein in the primary tumor responded significantly better than patients with an MGMT expression above this level, demonstrating for the first time the existence of a therapy relevant MGMT threshold level. The data suggest that MGMT activity in primary and recurrent GBM is a useful predictive marker in guiding therapy. Material and methods Patients and tumor samples The study included 40 patients with primary glioblastomas who underwent surgery at the department of neurosurgery of the University of Mainz, Germany, between May 1998 and April 2004. After surgery, patients were treated with the standard protocol, receiving either radiation therapy (RT) (2 Gy, total dose of 60 Gy) or RT plus concomitant chemotherapy. The doses of the agents were as follows: temozolomide at 150–200 mg/m2 for 5 days in 4 week cycles. In the combination therapy with TMZ and CCNU, CCNU was given in a dose of 100 mg/m2 on day 1 and TMZ in a dose of 100–120 mg/m2 from day 2 to 6 in 6 week cycles. ACNU was given in a dose of 90 mg/m2 and VM 26 in a dose of 60 mg/ m2 in 6 weeks cycles. The time between the first operation and the first recurrence was 4–184 weeks, with a median of 28 weeks. Tissue samples were obtained under sterile conditions. Informed consent was obtained from each patient before treatment in accordance with the ethic commission of this university. Samples from tumor tissue (5 3 5 3 5 mm approximately) were shock frozen in liquid nitrogen and stored at 280°C. In parallel, fractions of the samples were formalin fixed and embedded in paraffin. The diagnosis and histological classification of all tumor specimens in this study was confirmed by a neuropathologist. Preparation of cell and tissue extracts For the preparation of tissue extracts, deeply frozen tissue was homogenized by an UltraTurrax homogenizer in buffer containing 20 mM Tris-HCl, pH 8.5, 1 mM EDTA, 1 mM b-mercaptoethanol, 5% glycerol and a cocktail of protease inhibitors (10 lg/ml aprotinin, 10 lM bestatin, 10 lM leupeptin, 1 lM pepstatin and 0.1 mM PMSF). Thereafter, the homogenate was sonified by a Branson sonifyer 250 (2 3 10 pulses, duty cycle 40%, intensity 4.5, on ice). The sonication product was centrifuged to remove debris and the supernatant was snap frozen in liquid nitrogen and stored at 280°C until further use. This procedure did not result in loss of MGMT activity (unpublished data). For the positive control, i.e., cell extracts containing MGMT, HeLa S3 cells were used which express MGMT at high level (750 fmol/mg protein). HeLa MR cells deficient for MGMT served as a negative control and were included in each assay. Extracts were prepared from exponentially growing cells as previously described.26 MGMT activity assay MGMT activity in cell and tissue extracts was determined essentially as previously reported.14 The method is based on the radioactive assay in which transfer of tritium labeled methyl group from the O6-position of guanine to protein in the cell extract is measured.27 Each assay was performed in duplicate or, if sufficient material was available, triplicate. Background experiments showed a very low intraexperimental variation (usually <5%). The detection level was about 2 fmol/mg protein. HeLa MR cell extract served as background control by providing a standard tritium transfer level that was subtracted from the tritium transfer

level of the tumor sample. With the conditions used, the assay was not substrate saturated and the tritium transfer was linear depending on the amount of the cell extract protein. The protein concentration was determined by the method of Bradford.28 For the assays at least 100 lg of cell extract protein were used. In each assay, a negative and positive sample was included of HeLa MR (not detectable MGMT) and HeLa S3 (750 fmol/mg protein) cell extract respectively, which was stored frozen at 280°C in batches. For the preparation of 3H-MNU (specific activity 18.0 Ci/mmol, Amersham, NL) labeled substrate containing O6-methylguanine, calf thymus DNA (Sigma) was used. The incubation of cell extract together with 3H-MNU labeled DNA containing O6-methylguanine (total 120.000 cpm/sample) occurred in buffer containing 700 mM Hepes-KOH (pH 7.8), 10 mM dithiothreitol, 50 mM EDTA for 90 min, which was the optimal time span for the reaction to be completed (as demonstrated in background experiments; unpublished data). Data are expressed as fmol of radioactivity transferred from 3H-labelled DNA to protein per milligram of protein within the sample. Immunohistochemistry The expression of p53 in tumor samples was determined by immunohistochemistry using the p53 monoclonal mouse antihuman antibody DO-7 (DakoCytomation, Swiss). Anti-mouse Ig biotinylated protein from sheep was used as secondary antibody. Slides were stained with diaminobenzidine tetrahydrochloride. The number of positive-stained tumor cells was counted under the microscope. In accordance with previous work with other tumors,29,30 sections containing <10% of positively stained cells were considered to be p53 wild-type whereas >10% staining were considered to be mutant for p53. Statistics Tumors were classified according to MGMT activity and p53 staining intensity, which was assayed on shock frozen samples and parafinized slides obtained from the same tumor, respectively. Progression-free survival (PFS) was defined as the time from resection of the primary tumor to the detection of tumor regrowth by MRI, and overall survival (OS) is defined as the time from diagnosis to the death of the patient. For a few patients who were still alive at the time of study, OS was defined as the time from diagnosis to the current stage. Mean MGMT expression levels were compared by Student’s t test. Kaplan-Meyer curves were drawn using a computer program (SPSS), comparing patients with different MGMT cut-off levels (10 fmol/mg intervals). Statistical comparison of outcome (PFS and OS) was based on the exact log-rank test. Time to the first progress (Table I) was compared with Student’s t test (single sided unequal variance test). Results MGMT activity in primary and recurrent glioblastomas In this study, all tumors investigated were from patients with glioblastomas (GBM, WHO grade IV). The MGMT activity in pretreatment tumors (N 5 40) was neither correlated with age of the patient (Fig. 1a) nor with gender (Fig. 1b). The MGMT level in paired samples (obtained from the same patient on tumor resection) of primary tumors and the first recurrences is shown in Figure 2. There is a considerable variability in MGMT activity ranging between 0 and 81 fmol/mg protein in primary tumors, and 14– 194 fmol/mg in the 1st recurrences. The comparison of paired samples revealed that the MGMT activity in recurrences is usually higher than in the primary tumor. In only 3 out of 16 cases MGMT activity was lower in the recurrence than in the primary tumor (Fig. 2). In Figure 3, we compared the MGMT expression level in primary GBMs to 1st, 2nd and 3rd recurrences (this collection includes the paired samples shown in Fig. 2). The data demonstrate that MGMT activity, on average, increases during the clinical course. The difference between the average MGMT value of untreated tumors (N 5 40) and 1st recurrence (N 5 18) was statis-

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FIGURE 1 – MGMT activity in primary GBM as a function of age of the patient (a) and gender (b). In Figure 1a, there is no significant correlation between MGMT activity and patient’s age (R2 5 0.2426); in Figure 1b, difference between male and female MGMT is not significant (p 5 0.1279). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

FIGURE 2 – MGMT activity in primary GBM and 1st recurrences of the same patient. Paired samples are connected by line.

tically significant (mean values of 37.1 and 65.8 fmol/mg protein, respectively; p < 0.05). For the 2nd (N 5 7) and 3rd recurrences (N 5 4), the available data indicate a trend for further increase of MGMT activity (Fig. 3). Because of the low number of samples a statistic evaluation was not performed. It is interesting that 17.5% (7/40) of untreated (primary) tumors completely lacked detectable MGMT activity (0 fmol/mg protein) and 30% (12/40) expressed very low levels (<10 fmol/mg protein). In contrast, in the 1st, 2nd and 3rd recurrences all tumors displayed MGMT activity (>10 fmol/mg), supporting the conclusion that MGMT becomes upregulated during GBM progression and/or because of the therapy. To elucidate whether MGMT expression levels are randomly distributed within the collection of GBMs, we compared primary tumors and 1st recurrences as to the percentages of tumors expressing MGMT at increasing levels. As shown in Figure 4, about 33% of primary tumors and 17% of 1st recurrences expressed MGMT at levels ranging between 0–20 and 21– 40 fmol/mg protein. Six percent of primary tumors expressed 41– 60 fmol/mg compared to 23% of 1st recurrences. Again, the figure shows a general increase in the MGMT level in 1st recurrences. MGMT activity did not show a Poisson distribution. In both groups, there was a subgroup of tumors exhibiting quite high MGMT activity. This subgroup ameliorates in the 1st recurrences. Overall, the data show that MGMT activity increases in GBM recurrences, with a decrease in the proportion of low expressing and an increase in the proportion of high expressing tumors. MGMT in tumors during therapy Increase of MGMT activity in GBM recurrences could be a general phenomenon related to tumor progression. Alternatively,

FIGURE 3 – MGMT activity in primary GBM and in the 1st, 2nd, and 3rd recurrences. Each tumor is symbolized by circle. Bar represents mean value in the given tumor group. Mean values are for primary GBMs 37.1; 1st recurrences 65.8; 2nd recurrences 68.4; 3rd recurrences 181.8 fmol/mg protein. The difference between primary tumors and 1st recurrences is significant (p 5 0.0225). Because of low sample size, statistical comparison of primary tumors and 2nd and 3rd recurrences was not performed.

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FIGURE 4 – Distribution profile of MGMT activity in primary GBM and 1st recurrences. Data are based on a collection of 60 tumors.

FIGURE 6 – MGMT activity in relation to the percentage of p53 positively stained cells in primary GBM (N 5 40). Bars represent mean MGMT values in the particular subgroup, which are 58.8, 47.0, 29.4 and 14.8 fmol/mg protein for the groups of 0, 10, 20 and 30% cells positively stained with p53, respectively.

and first recurrences of patients that received RT plus alkylating agent therapy (p < 0.05). The data revealed that chemotherapy with O6-alkylating agents provokes a significant up-regulation of MGMT expression in the tumor tissue. This could be because of gene induction and/or selection of high MGMT expressing cells during TMZ, CCNU or ACNU therapy. p53 in GBM Previously it was reported that p53 is involved in the regulation of MGMT.22,31 It was also reported that p53 has an impact on the sensitivity of glioma cells to TMZ, ACNU and BCNU in vitro.10,24,52 Therefore, we determined the p53 status of tumors and compared it to the MGMT expression level. The p53 status was determined by immunohistochemistry as described.15 Specimens in which >10% of tumor cells were positively stained were defined as p53 expressing and thus mutated (p53mt). According to this definition, 49% of primary GBMs (18/37) and 67% of 1st recurrences (9/13) were p53 negative (p53wt). In 2nd and 3rd recurrences 66% (4/6) were p53 negative. Comparing MGMT to the p53 status, there was a tendency for lower MGMT activity in tumor sections where the amount of p53 stained cells was high (Fig. 6). With a cut-off level of >20% p53 stained cells in the tumor, the proportion of p53 negative tumors was similar in primary GBM and 1st recurrences (76 and 77%, respectively), indicating that the p53 status in primary GBM appears not to change during tumor growth. MGMT and therapeutic outcome To elucidate the response of GBM patients expressing different levels of MGMT, we compared tumor MGMT to the progression free survival (PFS) and overall survival (OS) of the patients after primary tumor resection. Kaplan-Meier curves are shown in Figure 7 (left panel, PFS; right panel, OS). We found a statistical significant difference between patients exhibiting 30 fmol/mg MGMT activity and patients exhibiting MGMT activity above this level in their tumors for both PFS (Fig. 7a) and OS (Fig. 7b). Interestingly, in the group of patients treated only with radiation following surgery, there was no significant difference in the PFS (Fig. 7c) and OS (Fig. 7d), showing MGMT has no impact on RT. In contrast, the subgroup of patients who received radiation with concomitant alkylating agent chemotherapy, the difference between

FIGURE 5 – MGMT expression in primary GBM (N 5 40) and in 1st recurrences after RT only (N 5 16) or RT combined with chemotherapy with temozolomide and/or CCNU or ACNU (N 5 24). Difference between mean values of primary tumors and 1st recurrences treated with RT only: p 5 0.1324; between primary tumors and 1st recurrences treated with RT1TMZ/CCNU/ACNU: p 5 0.0269.

the data could be explained on the basis of the assumption that during tumor therapy selection occurred for tumor cells expressing high MGMT levels. Although evidence for human tumors is still lacking, it should also be taken into account that the MGMT gene can be up-regulated by ionizing radiation, alkylating agents and glucocorticoids.21 Therefore, the increase in MGMT activity in recurrences could be a result of MGMT gene activation caused by ionizing radiation or/and chemotherapy. In Figure 5, we compared the MGMT activity in primary GBMs versus 1st recurrences of patients who received radiation therapy only (RT) and RT with concomitant chemotherapy (TMZ alone or TMZ plus CCNU or ACNU). Interestingly, in both groups, the MGMT activity was, on average, higher than in primary tumors (median values: 28 fmol/ mg in primary tumors versus 35 and 69 fmol/mg protein in 1st recurrent GBMs following RT and RT 1 chemotherapy, respectively). Most interestingly, the subpopulation of MGMT negative tumors was completely lacking in both groups of recurrences, irrespective of whether patients were treated with RT alone or RT plus alkylating agents. The difference between primary tumors and recurrences of patients that received RT only was not statistically significant. It was significant, however, for primary tumors

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FIGURE 7 – Correlation between MGMT enzyme expression and therapeutic response of patients. Kaplan-Meier curves are shown for patients expressing MGMT at low level (30 fmol/mg protein) and high level (>30 fmol/mg protein) in the primary tumor. Progression free survival (a,c,e) and overall survival (b,d, f ) for all patients who received RT or RT in combination with alkylating agents (a,b) and for those who received RT only (c,d) or RT 1 alkylating agents (e, f ). Blue line: MGMT activity in the primary tumor was <30 fmol/mg; green line: MGMT activity in the primary tumor was >30 fmol/mg protein. In case of significant difference, the p value was given. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

low and high MGMT expression tumors (cut-off level 30 fmol/mg protein) was again significant with the end point PFS (Fig. 7e), but not OS (Fig. 7f). Overall, the data demonstrate that patients expressing less than 30 fmol/mg protein MGMT in the primary tumor

show a significantly better therapeutic response than patients exhibiting more than 30 fmol/mg protein. This benefit to therapy for patients expressing low MGMT activity was also observed if the patient’s response was compared on

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the basis of the time of appearance of the first recurrence after resection of the primary tumor. As shown in Table I, the progression free interval is clearly higher if RT plus alkylating agents were applied, compared to RT only. Even more interesting, the time to the first progress was significantly higher in the group of patients whose primary tumor expressed MGMT at a low level of 30 fmol/mg protein. The difference was nearly 2-fold between low and high MGMT expressers (Table I). p53 status and therapeutic response With all patients included in the study who received RT or RT in combination with chemotherapy, we neither found a correlation between p53 status and PFS (Fig. 8a) nor with p53 status and OS (Fig. 8b). If we consider the low MGMT expressing (30 fmol/ mg protein) subgroup of patients who received chemotherapy with O6-alkylating agents, a difference between p53 negative and p53
TABLE I – TIME TO THE FIRST PROGRESS AFTER RESECTION OF THE PRETREATMENT TUMOR (WEEKS WITH RANGE) IN PATIENTS WHOSE TUMOR EXPRESSED 30 FMOL/MG AND >30 FMOL/MG PROTEIN MGMT (fmol/mg protein) Radiation only Radiation plus chemotherapy

positive tumors becomes apparent, with p53 negative tumors (p53wt) responding slightly better (Figs. 8c and 8d). The difference was, however, not statistically significant. 

30 >30

22.1 6 15.5 21.0 6 10.9

59.7 6 47.0 30.6 6 12.4*

*The difference between low and high MGMT expressions in the group who received radiation plus chemotherapy with alkylating agents was statistically significant (p 5 0.0142).

Discussion Expression and regulation of MGMT This study was aimed at determining the MGMT repair activity in newly diagnosed primary GBM and in their recurrences after therapy. Furthermore, the tumor MGMT activity was set in relation to the p53 status as well as PFS and OS of the patients. We showed that 17.5% of primary untreated GBMs were completely lacking MGMT activity, 30% expressed a very low level within the range of 0–10 fmol/mg protein, and 62.5% expressed a level 30 fmol/mg protein. This is in accordance with previous work of this and other groups reporting up to 24% of gliomas lacking detectable MGMT (previously defined as Mer2).26,32–34 Interestingly, the frequency of MGMT lacking GBMs does not correspond with the frequency of MGMT promoter methylation, for which 45% of untreated GBM harbor epigenetic silencing of the MGMT gene.19 Nevertheless, the frequency of 17.5% is clearly higher than in other groups of solid tumors for which maximally 5% were reported to be MGMT deficient.12 Surprisingly, no data are available on MGMT expression (as determined on the level of activity of the repair protein) in primary compared to 1st–3rd recurrent GBM after therapy. Information about MGMT in recurrent GBM would be even more important

FIGURE 8 – p53 status and therapeutic response of patients. Kaplan-Meier curves are shown for patients with p53 positive (>10% stained cells in the tumor section) and p53 negative tumor. Progression free survival (a,c) and overall survival (b,d) for all patients (a,b) and for those who expressed low MGMT in the primary tumor (30 fmol/mg protein) and received RT 1 alkylating agents (c,d). Blue line: p53 negative tumor (<10% positively stained cells); green line, p53 positive tumor (>10% positively stained cells). p values for the curves shown in Figures 8a–8d are 0.660, 0.396, 0.266 and 0.240, respectively. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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than in the primary tumor because the recurrences are subject of therapy with O6-alkylating agents. In a recent study it was shown, comparing 37 pretreatment glioblastomas and 11 recurrent tumors by immunohistochemistry, that the number of MGMT positively stained tumors did not clearly increase following treatment with radiation and temozolomide, whereas an amelioration was found for the number of MSH6 deficient tumors.35 Although the overall MMR capacity was not determined, the data indicate that MMR was down-regulated during therapy. The authors could not detect a correlation between MGMT status of the pretreatment tumor and tumor growth following temozolomide treatment.35 We should note, however, that in this study MGMT was determined by immunohistochemistry, which is a semiquantitative method. The variation in the MGMT activity level is quite small in GBM, compared to other tumor groups. Therefore, determination of MGMT activity is obviously a reliable method to discriminate between responders and nonresponders. Comparing paired samples (original tumor versus recurrences from the same patient) we found, with only a few exceptions, augmentation of MGMT activity in the recurrences. We also show that the average level of MGMT activity was higher in the 1st recurrence compared to the primary tumor. There was a tendency of further increase of MGMT in the 2nd and 3rd recurrences (mean values for untreated GBM 37 fmol/mg, 1st recurrences 66 fmol/ mg, 2nd recurrences 68 fmol/mg, 3rd recurrences 182 fmol/mg). Interestingly, the subgroup of tumors devoid of detectable MGMT activity (17%) was completely lacking in the first and subsequent recurrences. Thus, the data show a trend for increase of MGMT expression in GBM during tumor progression and therapy. We should note that a significant increase in MGMT activity was also reported during tumor progression in ovarian carcinomas, although these tumors were not treated with O6-alkylating agents.15 Increase in the MGMT expression level could be due to a change in gene expression pattern that occurs during tumor growth and progression or it might be therapy-related. The MGMT gene has been shown to be subject of regulation on transcription factor level. Thus, RT and alkylating agent chemotherapy are able to stimulate the rodent MGMT gene and the human MGMT promoter that leads to increased MGMT transcript and protein levels.13,20,21,36,37 Whether or not the MGMT gene can be induced in human normal and tumor tissue (in vivo) is still unknown. This, however, is a highly important question since RT could counteract concomitant chemotherapy with O6-alkylating agents if RT would activate the MGMT gene. Comparing 1st recurrences of patients who received RT only or RT concomitant with O6-alkylating agents (TMZ, CCNU, ACNU), we found a significant increase in the MGMT level not in the group of patients who received only RT, but in the group of patients who received RT plus O6-alkylating agents. Therefore, the data suggests that during alkylating agent therapy selection occurred for preexisting tumor cells expressing high MGMT. We can, however, not entirely exclude the possibility that alkylating agents actively stimulate the MGMT gene, leading to increased MGMT levels. This, however, is less likely than selection for high MGMT expressing cells because RT did not significantly increase the MGMT level in recurrences, even though it was shown that RT can activate the human MGMT promoter (which was shown to occur already with a radiation dose of 2 Gy).21 We should note that MGMT gene induction provoked by RT or alkylating agents is a transient phenomenon, lasting up to several days, which is short compared to the time interval between RT and 2nd resection (more than 1 month) during which transient gene induction would be turned off. Nevertheless, the unresolved question of MGMT gene induction by RT in GBM is still of utmost importance since induction of the MGMT gene would clearly counteract the therapeutic effect of O6-alkylating agents administered concomitantly with RT. The regulation of MGMT is complex. It also involves p53, which has been shown to be required for induction and also for down-regulation of the basal level of MGMT.22,31,38 Furthermore,

p53 determines the efficiency of execution of apoptosis in glioma cells upon TMZ or CNU treatment.10,52 Therefore, we determined the p53 status of the tumors, which was performed by means of immunohistochemistry,15 and set it in relation to MGMT and therapeutic response. We found a tendency of decline of MGMT activity with increasing fraction of p53 positively stained cells in the tumor (Fig. 6), which could be taken to indicate that accumulation of (mutated) p53 in GBM may have an impact on MGMT expression. It is also conceivable that tumor precursor cells expressing low MGMT activity are more vulnerable for accumulating mutations in the p53 gene, which might explain the tendency for an inverse relationship between MGMT activity and p53 mutant status of the tumor. Therapeutic response of patients We compared patients expressing different levels of MGMT in the primary tumors and found, with a cut-off level of 30 fmol/mg protein, a significant difference both in PFS and OS: patients who expressed 30 fmol/mg displayed a longer median PFS and OS time than patients expressing >30 fmol/mg in the primary tumor. Comparing patients who received RT only, an influence of MGMT (irrespective of cut-off levels) on survival was not observed. In contrast, comparing the subgroup of patients who received RT in combination with chemotherapy, we found a significant difference concerning PFS; for OS significance was not detected. This could be because of the fact that MGMT activity increases in the 2nd and 3rd recurrences, which therefore became more resistant to alkylating agent reducing the overall survival. Therefore, the impact of MGMT is more significant if the comparison is based on PFS instead of OS (Fig. 7). The time to the first progress was also MGMT dependent (Table I). It was generally longer in the group of patients who received RT plus alkylating agents compared to RT only. An effect of MGMT was not seen in the RT group, but in the RT plus chemotherapy group in which the recurrence free interval was 2-fold enhanced if MGMT was expressed below the threshold of 30 fmol/mg protein (59.7 versus 30.6 weeks). Overall, we observed a significant correlation between low MGMT activity and increased therapeutic response. We conclude that MGMT activity in the primary tumor is a reliable predictive marker in GBM therapy with O6-alkylating agents, with a threshold level of 30 fmol/mg protein MGMT activity. Several studies have been conducted to correlate MGMT with the therapeutic response of patients. The majority of trials showed a relationship between low MGMT and better therapeutic response in patients with malignant gliomas on treatment with BCNU16,17,39,40 or temozolomide.19,41,42 Some studies, however, failed to show a correlation.33,43 It should be noted that MGMT was determined in previous studies by different methods such as mRNA or protein quantification, MGMT activity, or MGMT promoter methylation by MS-PCR. Most of these methods are semiquantitative, providing a ‘‘yes or no’’ answer. Since the expression of MGMT in gliomas is, on average, quite low compared to most of the normal tissue and other tumor groups, a precise quantitative determination is required. The MGMT activity assay is highly sensitive and quantitative. Unfortunately, the activity assay does not take into account the intratumor heterogeneity. Nevertheless, it is able to identify tumors completely lacking MGMT or expressing it at very low level. In a recent study, it has been shown that the outcome of different MGMT assays (activity, immunohistochemistry, promoter methylation) is not necessarily interchangeable.44 Therefore, it is clear that further studies are warranted to establish a reliable method of MGMT quantification that predicts the outcome of therapy with methylating and chloroethylating agents more precisely. Here, we define for the first time a threshold level of MGMT activity in the pretreatment GBM of 30 fmol/mg protein, below which tumors respond better to a temozolomide/CNU based therapy. For p53, we did not find a significant correlation between p53 status and therapeutic response if all patients were included in the

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study. If the comparison is based on the subgroup of patients who was p53wt (here defined as 10% of p53 stained tumor cells) and treated with RT 1 TMZ/CCNU, ACNU a better but still insignificant PFS and OS were observed for patients who are p53 negative. It should be noted that p53wt sensitizes glioma cells by stimulating apoptosis via the Fas/CD95/Apo-1 system on treatment with TMZ.10,24 Further, there is a report on children with malignant gliomas who had a better outcome if p53 was not overexpressed in the tumor.45 Thus, is appears that the p53 status might also be predictive in alkylating agent based glioma therapy. Clearly, further studies are required with a larger group of patients with a well-defined MGMT and p53 status receiving TMZ or chloroethylnitrosourea therapy. The standard therapy of GBM consists of tumor resection followed by radiotherapy (total dose of 60 Gy) and treatment with alkylating agents (methylating agents such as TMZ and/or chlorethylnitrosoureas) at recurrence. Concomitant and sequential treat-

ment with RT plus TMZ showed significantly longer 2-year-survial compared to RT alone.1 Therefore, postoperative RT and concomitant and sequential administration of TMZ up to 6 cycles is now recommended as first-line therapy of GBM. MGMT promoter methylation has been suggested to be a predictive factor in gliomblastoma therapy.18,19,46 Whether MGMT promoter hypermethylation correlates with lack of MGMT activity in the tumor is, however, still an intriguing question. Recently, it was reported that this is not the case.44 Therefore, determining MGMT activity in GBM may provide an additional source of information supporting individualized cancer therapy. To overcome MGMT-related chemotherapy resistance in gliomas, a wide range of therapeutic strategies are currently under trial such as intensified and continuous TMZ regiments47–49 and treatment with MGMT inhibitors.8,50,51 It is conceivable that knowledge on MGMT level of a given tumor will be guiding therapy as to dose and time course of administration of O6-alkylating drugs as well as MGMT inhibitors.

References
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