O-methyl-guanine-DNA methyltransferase Methylation in Serum and Tumor DNA Predicts Response to 1,3-Bis(2-Chloroethyl)-1- Nitrosourea but not to Temozolamide Plus Cisplatin in Glioblastoma Multiforme

Purpose: In glioblastoma multiforme (GBM), the cytotoxic effect of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and temozolamide is dependent on O alkylation, which correlates inversely with expression of the DNA repair enzyme O-methyl-guanine-DNA methyltransferase (MGMT). Thus, MGMT assessment can be useful in predicting response in GBM, but the scarcity of neoplastic cells limits the practicality of MGMT assessment in these tumors. Although GBM grows within the skull, we investigated the concordance of methylation in glioma tissue, and paired serum DNA and the potential correlation with response and time to progression. Experimental Design: Using MSP assay, we assessed the methylation of MGMT, p16, DAPK, and RASSF1A in tumor and serum DNA from 28 GBM patients treated with BCNU or with temozolamide plus cisplatin. Results: The concordance between methylation in tumor and serum was highly significant. Overall, response plus stable disease was noted in 10 of 11 (90.9%) patients with MGMT methylation and in 5 of 14 (35.7%) patients without (P 0.01). In the 16 patients treated with temozolamide plus cisplatin, no significant correlation between MGMT methylation status and response was observed, whereas in BCNU-treated patients, a significant difference was observed in favor of those with methylated MGMT. Time to progression was 29.9 weeks in 12 patients with MGMT methylation and 15.7 weeks in 10 patients without (P 0.006). No correlation was observed between response or time to progression and p16, DAPK, or RASSF1A methylation. Conclusions: Methylated MGMT, p16, DAPK, and RASSF1A were found in serum DNA of GBM patients, with a good correlation between serum and primary tumor tissue. Serum MGMT methylation predicted response and time to progression in BCNU-treated GBM patients. The methylation-specific PCR assay in serum DNA could be a good predictive tool for selecting GBM patients to be treated with BCNU or alternatively with the combination of temozolamide plus cisplatin. INTRODUCTION GBM is the highest grade glioma; complete resection is difficult, and even when possible, it is associated with severe neurological damage. Treatment consists of cytoreductive surgery followed by radiotherapy. Median survival time is only 9 months, and 5–10% of patients survive up to 2 years. Metaanalyses have shown that chemotherapy could improve survival with an absolute increase in 1-year survival of 6% (1). Pharmacogenetic research to predict response to chemotherapy has not been fully explored. Until recently, prognostic groups were based on age, Karnofsky performance status, biopsy only, or radiotherapy dose (2). Many genetic abnormalities are involved in gliomagenesis, and relevant mechanisms of resistance to common drugs used in the treatment of GBM have been identified (3). One-step repair (the direct reversal of DNA damage) targets 2-chloroethylnitrosoureas, such as BCNU (carmustine) and temozolamide, both of which involve alkylation of the O site of guanine. It is performed by the repair protein O-alkylguanine-DNA alkyltransferase or MGMT through direct removal of an alkyl group from the O-atom of guanine in the DNA of cells exposed to alkylating agents. With increasing size of the alkyl group ( ethyl), the relative contribution of MGMT to the repair of O-alkylguanines in DNA decreases and excision repair steps in as a backup modality (4). This O-alkylguanine DNA lesion is highly cytotoxic and correlates inversely with the activity of the DNA repair enzyme MGMT, which removes the monofunctional O adduct from the DNA, limiting response to these cytotoxic drugs. MGMT activity in gliomas is Received 8/21/02; revised 11/4/02; accepted 11/6/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by a grant from the Spanish Ministry of Health (FIS 00/0445). 2 To whom requests for reprints should be addressed, at Medical Oncology Service, Hospital Germans Trias i Pujol, Ctra Canyet s/n, 08916 Badalona (Barcelona), Spain. Phone: 34-93-497-8925; Fax: 34-93-4978950; E-mail: [email protected]. 3 The abbreviations used are: GBM, glioblastoma multiforme; MGMT, O-methyl-guanine-DNA methyltransferase; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; MSP, methylation-specific PCR; MRI, magnetic resonance imaging; CI, confidence interval; NSCLC, non-small cell lung cancer; GSTP1, glutathione S-transferase 1. 1461 Vol. 9, 1461–1468, April 2003 Clinical Cancer Research Cancer Research. on October 16, 2017. © 2003 American Association for clincancerres.aacrjournals.org Downloaded from variable; 25% of gliomas have no detectable MGMT activity ( 0.25 fmol/10 cells or 150 molecules/cell), and MGMT activity correlates with aneuploidy (5). Gliomas without MGMT activity responded better to surgery plus alkylating agent-based chemotherapy than to surgery alone (6). MGMT activity is commonly elevated in pediatric brain tumors, raising the hypothesis that elevated MGMT activity could enhance resistance to alkylating agent-based chemotherapy (7). MGMT-predictive chemosensitivity has been reported in a mixed group of BCNUtreated GBM and anaplastic astrocytoma patients by a quantitative indirect immunofluorescence assay. MGMT levels were scored high or low both according to the threshold of 60,000 molecules/nucleus. Time to progression was significantly longer in GBM patients with low MGMT levels (6 months) than in those with high MGMT levels (3 months; P 0.008). Median survival was 12 versus 7 months, respectively (8). The role of MGMT was also examined in temozolamide-treated GBM patients. Responses were observed more frequently in patients with low immunostaining for MGMT ( 20% of cells; Ref. 9). To overcome the one-step repair mechanism of chemoresistance, MGMT inhibitors have been developed, such as O-benzyl-2 deoxyguanosine and O-benzylguanine. Administration of these nonspecific MGMT inhibitors before treatment with either BCNU or temozolamide has shown a benefit in tumors having MGMT activity 45 fmol/mg protein (10). To make matters more complex, it seems that cisplatin can abrogate MGMT activity, which makes novel chemotherapy approaches like temozolamide plus cisplatin particularly attractive in the treatment of GBM (11). With MSP, 40% of brain tumors showed no methylated MGMT, which correlated with better response and survival in BCNU-treated patients. Anaplastic astrocytomas were also included in this study, and striking differences in median time to progression were observed (21 months for methylated versus 8 months for unmethylated gliomas; P 0.001; Ref. 12). However, whereas MGMT transcript is a good predictor of response and survival, GBM is characterized by necrosis, with cells arranged around the edge of the necrotic tissue (pseudopalisading cells), and tumor tissue is often scarce, because in a number of patients only stereotactic tumor biopsy is performed. For this reason, analysis of serum DNA may be a useful method for assessing MGMT methylation status in GBM patients, because the level of free serum DNA was observed to be high in brain tumors. More than 3 decades ago, significant amounts of serum DNA were identified in brain tumors by a radioimmunoassay (13). More recently, several reports indicate that genetic microsatellite alterations in serum DNA mirror those observed in paired tumor samples in head and neck (14), and small-cell lung cancer (15). Similar methylation patterns of p16, DAPK, GSTP1, and MGMT in paired tumor and serum DNA were observed in resected NSCLCs (16). p16, DAPK, and MGMT hypermethylation has also been observed in paired tumor and serum DNA of head and neck cancer patients (17). Interestingly, p16 methylation has been observed in GBM (18). Loss of DAPK expression has been associated significantly with more biologically aggressive pituitary tumors (19). RASSF1A has not yet been explored in adult GBM, although RASSF1A methylation is observed frequently in pediatric tumors, including neuroblastoma (20). RASSF1A contains a region for the DNAdependent ataxia-telangiectasia-mutated and ataxia-telangiectasia-