O⁶‐methylguanine‐DNA methyltranspherase gene expression in gliomas by means of real‐time quantitative RT‐PCR and clinical response to nitrosoureas

Abstract

Key words: MGMT; real-time RT-PCR; ACNU; gliomaNitrosoureas are alkylating agents that cause cell death by binding to DNA. Among nitrosoureas, 1-(4-amino-2-methyl-5-pyrimidynyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) is widely used in Japan to treat gliomas because of its ability to permeate the blood-brain-barrier and excellent clinical effects in combination with radiation and interferon (IFN)-␤. [1][2][3] Drug-resistance genes are some of the most important elements of tumors themselves in determining drug-resistance, 4 and O 6 -methylguanine-DNA methyltransferase (MGMT) is a drug-resistance gene for nitrosoureas. 5,6 The cross-linking of doublestranded DNA by alkylating agents is inhibited by the cellular DNA-repair protein MGMT. MGMT rapidly reverses alkylation at the O 6 position of guanine, thereby averting lethal cross-linking. 7 This is the mechanism by which MGMT induces resistance to alkylating agents such as ACNU.Protocol studies for malignant gliomas have not provided encouraging therapeutic results because of the heterogeneity and various drug-sensitivities of the tumors. 8,9 Individualization of glioma therapy is recommended. We carried out a new adjuvant therapy that was individualized based on the results of the reverse transcription-polymerase chain reaction (RT-PCR) for MGMT to treat malignant gliomas. 2 Our preliminary individual adjuvant therapies (IAT) based on RT-PCR results regarding MGMT expression seemed to be more effective than conventional therapies for malignant gliomas.The level of MGMT varies widely according to the type of tumor, and even varies among tumors of the same histological classification. 5,10 Approximately 30 -50% of gliomas lack MGMT. 11 The fact that more than 50% of gliomas express MGMT limits the indications for ACNU. In our preliminary IAT, platinum (Pt)-compounds were used instead of ACNU even though Ptcompounds had not been accepted as being effective in gliomas. 12,13 To ensure IAT for gliomas, quantitative evaluation is needed for a higher initial response and prolongation of survival.We carried out the present study to determine more appropriate indications for the use of ACNU in gliomas. Real-time quantitative RT-PCR was used to re-evaluate the treated gliomas. MATERIAL AND METHODS Real-time quantitative RT-PCRIn 100 frozen sample of neuroepithelial tumors (19 low-grade neuroepithelial tumors, 28 Grade III gliomas, 41 glioblastomas, and 12 medulloblastomas), MGMT was quantitated by real-time quantitative RT-PCR using SYBR Green dye. RT-PCR was basically carried out as described previously. 14 -16 Briefly, total RNA was extracted from frozen tissue specimens weighing about 1 g, which had been homogenized using a glass Teflon homogenizer, via the guanidinium thiocyanate-phenol-chloroform single-step extraction method with Isogen (Nippon Gene, Toyama, Japan). 17 After ethanol precipitation, about 100 g of total RNA was extracted. Forty microliters of complementary deoxyribonucleic acid (cDNA) solution was synthesized from 2 g of total RNA wi...

Promoter methylation and expression analysis of MGMT in advanced pediatric brain tumors

“…Tumors with a relative quantitation value, expressed as 2 -ddC , of MGMT ≥1 should not be treated by ACNU. These results showed a close relationship between MGMT expression and the prognosis of HGG patients treated with ACNU (23). The role of methylation status of this gene has been also studied in medulloblastoma/PNET.…”

Biopsy of Brain Stem Glioma Using Motor-Evoked Potential Mapping by Direct Peduncular Stimulation and Individual Adjuvant Therapy-Case Report-

“…19) Recently, real-time RT-PCR has been used to quantify MGMTmRNA. 20) MGMT expression is closely related to resistance to ACNU. 14) In our retrospective study, no patients with MGMT RQV of more than 1 responded to adjuvant therapy using ACNU.…”

“…8,9,18) We describe a case of diffuse brain stem glioma which developed into intrinsic brain stem glioma which was treated by surgery using motor-evoked potential (MEP) mapping by direct peduncular stimulation, and then by individual adjuvant therapy (IAT) based on the results of real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis of the surgical specimen. 19,20) Case Report A 23-year-old man was admitted to The Kitasato Institute Medical Center Hospital on May 23, 2000, with a 6-month history of progressive right hemiparesis and diplopia. He noticed that he could not play his guitar as well as before in December 1999.…”

“…7). 20) Postoperative IAT based on the results of the real-time RT-PCR of MGMTmRNA was begun on July 26, 2002. 19) He received two courses of 40 mg of cis-platinum and 120 mg of etoposide for 5 consecutive days followed by 6 × 10 6 IU of IFN-b three times a week for 5 weeks given over 3 months because the RQV of MGMTmRNA was more than 1.…”

“…19) He received two courses of 40 mg of cis-platinum and 120 mg of etoposide for 5 consecutive days followed by 6 × 10 6 IU of IFN-b three times a week for 5 weeks given over 3 months because the RQV of MGMTmRNA was more than 1. 20) He was discharged on October 1, 2002, and continues to receive maintenance adjuvant therapy of 6 × 10 6 IU of IFN-b once a week. Subsequently, 600 mg of carboplatin was added beginning on October 17, 2002.…”