MGMT in primary and recurrent human glioblastomas after radiation and chemotherapy and comparison with p53 status and clinical outcome Dorothee Wiewrodt 1 , Georg Nagel 2 , Nadine Dreim€ uller 1 , Thomas Hundsberger 3 , Axel Perneczky 1 and Bernd Kaina 2 * 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 O 6 -methylguanine-DNA methyltransfer- ase (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 ac- tivity 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 sig- nificant increase of MGMT in 1st recurrences was only found af- ter treatment with RT plus chemotherapy, indicating either selec- tion 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 pre- treatment 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 pre- dicting 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 temozolomide 1 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 mi- nority of glioblastoma patients respond to temozolomide and chloroethylating nitrosoureas. A likely explanation for this is the development of either primary or acquired glioblastoma cell re- sistance that leads to protection of the tumor against alkylating drugs. Methylating and chloroethylating agents attack DNA at nucleo- philic sites like the O 6 position of guanine, forming O 6 -alkylgua- nine. 3 This lesion is considered to be the major cause of mutations and malignant transformation induced by O 6 -alkylating agents. 4 It also provokes cell death by inducing apoptosis 5,6 and, therefore, is considered to be mainly responsible for the antineoplastic effect of O 6 -alkylating agents. O 6 -alkylguanine is repaired by the suicide DNA repair protein O 6 -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 preex- isting molecules of MGMT and the rate of its resynthesis. MGMT has a profound influence on the cell’s survival and proliferation capacity following treatment with O 6 -alkylating agents like temo- zolomide, DTIC, BCNU, CCNU, ACNU and fotemustine. 8,9 For methylating drugs, MGMT deficiency causes O 6 -methylguanine lesions to mispair with thymine, which leads to mismatch repair (MMR) mediated apoptosis in gliomas. 10 Therefore, for temozolo- mide 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 O 6 -chloroethylgua- nine is the critical primary lesion that is converted by intra-molec- ular rearrangement into a DNA interstrand crosslink, which pro- vokes 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 chloroethy- lating anticancer drugs. 12 MGMT is expressed highly variably in tumors, with low expres- sion in brain tumors and malignant melanomas and high expres- sion 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 car- mustine (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 corre- sponding to silencing of the MGMT gene was found to correlate with increased survival of patients who received radiation plus cis- platin and BCNU 18 or radiation and temozolomide therapy. 19 To date, MGMT promoter methylation is thought to be a useful pre- dictive marker for glioma therapy. MGMT is an inducible DNA repair gene. Induction of the MGMT gene was shown notably in rat liver cells on glucocorti- coid treatment and genotoxic stress, including X-rays and alkyl- ating agents. 20,21 It was also demonstrated on the level of the human MGMT promoter and, in the case of genotoxic stress, de- pendent 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 phenom- enon, 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 expres- sion in tumor and normal tissue before and after therapeutic treat- ment 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 pre- treatment tumors and their recurrences is important to asses whether and at which level it correlates with the outcome of alkyl- Grant 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). Int. J. Cancer: 122, 1391–1399 (2008) ' 2007 Wiley-Liss, Inc. Publication of the International Union Against Cancer