O-Methylguanine-DNA Methyltransferase in Pediatric Primary Brain Tumors: Relation to Patient and Tumor Characteristics

The DNA repair protein O-methylguanine-DNA methyltransferase (MGMT) confers resistance to methylating and chloroethylating agents in pediatric medulloblastomaand glioma-derived cell lines and xenografts. Here, we assayed MGMT activity in 110 pediatric brain tumors to establish correlates with patient and tumor characteristics. We also assayed MGMT in histologically normal brain adjacent to 22 tumors to characterize changes in activity accompanying neurocarcinogenesis. MGMT activity was detected in 94% of tumors, ranging ca. 1,500-fold from 0.34 to 498 fmol/10 cells (;205–300,000 molecules/cell). Mean activity was 25 6 66 fmol/10 cells, including six specimens with undetectable activity (Mer phenotype; <0.25 fmol/10 cells or 151 molecules/cell). MGMT content varied 10-fold among diagnostic groups and was associated with degree of malignancy, as evidenced by a 4-fold difference in activity between highand low-grade tumors (P 5 0.03). Tumor MGMT content was age dependent, being 5-fold higher in children 3–12 years old than in infants (P 5 0.015) and adolescents (P 5 0.015). Mean activity in tumors was 9-fold higher than in adjacent histologically normal brain (21 6 44 versus 2.4 6 4.0 fmol/10 cells; P 5 0.05). By comparing tumor and adjacent normal tissue from the same patient, we found that 68% of cases exhibited an elevation of tumor activity that ranged from 2to >590-fold. Moreover, 67% of Mer normal tissue was accompanied by Mer tumor. These observations indicate that MGMT activity is frequently elevated during pediatric neurocarcinogenesis. Significantly, enhanced MGMT activity may heighten resistance to alkylating agents, suggesting a potential role for MGMT inhibitors in therapy. INTRODUCTION Primary CNS tumors are the most common solid malignancy of childhood, occurring in ;2200 children annually (1). Whereas the majority (60–70%) are gliomas (astrocytomas, oligodendrogliomas, and ependymomas) histologically similar to those found in adults (2, 3), a sizable minority consists of diagnostic types uncommon in adults, including PNETs and mixed neuronal-glial tumors (2, 3). Moreover, unlike adult gliomas, which occur predominantly in the cerebral hemispheres, ;50% occur in the cerebellum and brain stem (3), suggesting that a subset of childhood tumors may have a unique pathogenesis. Although survival rates for some tumor types have improved in the last three decades, the prognosis for malignant pediatric CNS tumors remains grim, with a 5-year survival rate of 50%. As a consequence, brain tumors account for 26% of all pediatric cancer deaths (4). In light of evidence that the incidence of childhood brain tumors has been increasing at an annual rate of 2% (5, 6), development of more effective therapies remains an urgent priority. Chloroethylating and methylating agents, when used in single agent or combination chemotherapy, are among the most effective antitumor drugs for treatment of malignant pediatric and adult brain tumors (7–9). However, intrinsic and acquired resistance to alkylating agents limits their usefulness. A large body of work with pediatric brain tumor-derived cell lines and xenografts has demonstrated that the DNA repair protein MGMT can contribute to alkylating agent resistance (10–14). MGMT exerts its protective effect by removing cytotoxic chloroethyl and methyl adducts from the O position of guanine to an internal cysteine, yielding guanine and S-alkylcysteine (15). Because the alkyl receptor site is not regenerated, the number of O-alkylguanine adducts that can be removed from DNA in vivo is limited by the number of MGMT molecules and the rate of synthesis of the protein. Recent evidence that MGMT limits the cytotoxicity of cyclophosphamide (16), an agent frequently used to treat newly diagnosed and progressive childhood gliomas and medulloblastomas (17, 18), suggests an even wider role for MGMT in pediatric CNS tumor resistance. Most neoplastic human tissue specimens, including those Received 8/16/00; revised 12/14/00; accepted 12/15/00. 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 by grants from the American Cancer Society (RPG-97-019 to J. R. S.) and the NIH (CA 719397 to M. S. Bo., CA 10382 and CA 81454 to J. R. G., and CA 707090 to J. R. S.). Additional support was from the Neurooncology Gift Fund and Jessie’s Perfect Peach Fund of Children’s Hospital and Regional Medical Center. 2 To whom requests for reprints should be addressed, at Division of Neurosurgery, Department of Surgery, Children’s Hospital and Regional Medical Center, Seattle, Washington 98105. Phone: (206) 5262046; Fax: (206) 527-3925; E-mail: [email protected]. 3 The abbreviations used are: CNS, central nervous system; DNT, dysembryoplastic neuroepithelial tumor; Mer, methyl repair; MGMT, Omethylguanine-DNA methyltransferase; PNET, primitive neuroectodermal tumor. 613 Vol. 7, 613–619, March 2001 Clinical Cancer Research Research. on May 4, 2017. © 2001 American Association for Cancer clincancerres.aacrjournals.org Downloaded from from brain, express MGMT activity (19–22), raising the possibility that MGMT contributes to the drug resistance of tumors in vivo. In a survey of 152 adult gliomas, we found a 300-fold range of detectable MGMT activity (21). Importantly, 24% of specimens had no detectable activity [i.e., were Mer (methyl repair deficient)], suggesting that this appreciable fraction of brain tumors may be more sensitive to alkylating agents. In accord, data from our laboratory suggest that Mer adult glioma cells are preferentially killed by alkylators in vivo (22). To expand our analysis to childhood tumors, we have now assayed MGMT activity in 110 pediatric primary brain tumors and in histologically normal brain adjacent to 22 tumors. Our analysis of the largest collection of such tissue samples examined to date revealed that pediatric brain tumors have a wide range of MGMT activity and a low frequency of Mer phenotype. We also demonstrate that tumorigenesis in pediatric brain is most often accompanied by an increase in MGMT activity. Our findings may be significant for understanding clinical response to alkylating agent therapy, and they suggest a therapeutic role for MGMT inhibitors. MATERIALS AND METHODS Tissue. Tumors were resected at Children’s Hospital and Medical Research Center and the University of Washington Medical Center from 1991 to 1999. Ninety-five tumors were newly diagnosed, and 15 were recurrent after prior therapy (Table 1). None of the recurrent tumors was from patients from whom a newly diagnosed specimen was obtained. Care was taken to ensure that tumor was distant from the interface with normal tissue. All tissues were reviewed by a panel of neuropathologists affiliated with the University of Washington. Diagnosis, along with an S-phase fraction determined by flow cytometry of some unselected tumors, was obtained from the final neuropathology report. The S-phase cell fraction of some, unselected tumors was determined by flow cytometry. Normal tissue was microscopically free of hypercellularity, infiltrating tumor, endothelial proliferation, edema, and gliosis. Demographic information together with course and response to therapy was obtained from medical records. The precautions taken to preserve tissue viability and enzymatic activities during transport of specimens, and the procedure for determining cell number are described elsewhere (21, 22). The MGMT activity of 27 tumors and 13 normal brain samples was reported previously