Introduction. O6-methylguanine-DNA methyltransferase (MGMT) gene encodes a ubiquitously expressed DNA repair enzyme that counteracts the normally lethal effects of alkylating agents by removing alkyl adducts from the O6-position of guanine. Due to this DNA repair activity, the MGMT protein is believed to provide resistance against cytotoxic effects of alkylating agents. Therefore assessment of the methylation status of the O6-methylguanine-DNA is routinely assessed in malignant gliomas as a predictive factor for response to chemotherapy with alkylating drugs as well as a prognostic parameter. The aim of the present study was to analyze MGMT promoter methylation status in different portions of surgically resected glioblastomas (GBM) in order to verify its homogeneity across the surgical specimen. Materials and methods Thirteen patients with GBM submitted to surgical resection between 2012 and 2015 were selected from the files of the Department of Human Pathology of the University of Messina, Italy, and included in the study. Only those patients with at least two paraffin blocks corresponding to different topographical portions of the tumors were considered. One of the GBM had arisen as the progression of a grade II diffuse astrocytoma (DA), while two of the cases developed a recurrent GBM. Formalin fixed and paraffin embedded tumor tissue was available for DA as well as for the recurrent GBM. Finally a total of sixteen tumors (1 DA, 13 GBM and 2 recurrent GBM) were analyzed for MGMT promoter methylation status. All samples were microdissected to select exclusively tumor cells and for each sample DNA was extracted by FFPE extraction kit. Methylation status was investigated by Real Time PCR utilizing AlphaReal MGMT kit (Alphagenics Biotechnologies). Immunohistochemical data on isocitrate dehydrogenase 1 (IDH1) mutated (R132H) protein, p53 and Ki-67 expression were available in all of the cases. Results MGMT promoter methylation status was unmethylated in all of the analyzed tissue blocks in 6 GBM. On the other hand, 7 GBM were MGMT promoter methylated. Of these, two cases showed different and not-uniform pattern of methylation. In detail, one GBM displayed MGMT promoter methylation in the central, but not in the superficial portion of the tumor and one GBM showed MGMT promoter methylation in the central and intermediate portions but not in the superficial part of the tumor. Of these cases, one had R132H mutant IDH1, and one IDH1 wild type. One of the GBM with heterogeneous MGMT promoter methylation status represented the progression of DA, which was MGMT unmethylated and had IDH1 R132H expression. MGMT methylation status was unchanged in the recurrences of the GBM. In detail, one of the recurrent GBM was methyalated and one unmethylated as were the primitive tumors. Conclusions Our data suggest that MGMT promoter methylation may be heterogeneous through GBM and may depend on the site of surgical sample collection. We may speculate that methylated phenotype might be acquired during tumor progression from low grade astrocytomas.

MGMT promoter methylation status in different portions of surgically resected glioblastomas

GIUFFRE', Giuseppe;SIMONE, Angela;SCARFI', Rosa;IENI, ANTONIO;CAFFO, Maria;BARRESI, Valeria
2015

Abstract

Introduction. O6-methylguanine-DNA methyltransferase (MGMT) gene encodes a ubiquitously expressed DNA repair enzyme that counteracts the normally lethal effects of alkylating agents by removing alkyl adducts from the O6-position of guanine. Due to this DNA repair activity, the MGMT protein is believed to provide resistance against cytotoxic effects of alkylating agents. Therefore assessment of the methylation status of the O6-methylguanine-DNA is routinely assessed in malignant gliomas as a predictive factor for response to chemotherapy with alkylating drugs as well as a prognostic parameter. The aim of the present study was to analyze MGMT promoter methylation status in different portions of surgically resected glioblastomas (GBM) in order to verify its homogeneity across the surgical specimen. Materials and methods Thirteen patients with GBM submitted to surgical resection between 2012 and 2015 were selected from the files of the Department of Human Pathology of the University of Messina, Italy, and included in the study. Only those patients with at least two paraffin blocks corresponding to different topographical portions of the tumors were considered. One of the GBM had arisen as the progression of a grade II diffuse astrocytoma (DA), while two of the cases developed a recurrent GBM. Formalin fixed and paraffin embedded tumor tissue was available for DA as well as for the recurrent GBM. Finally a total of sixteen tumors (1 DA, 13 GBM and 2 recurrent GBM) were analyzed for MGMT promoter methylation status. All samples were microdissected to select exclusively tumor cells and for each sample DNA was extracted by FFPE extraction kit. Methylation status was investigated by Real Time PCR utilizing AlphaReal MGMT kit (Alphagenics Biotechnologies). Immunohistochemical data on isocitrate dehydrogenase 1 (IDH1) mutated (R132H) protein, p53 and Ki-67 expression were available in all of the cases. Results MGMT promoter methylation status was unmethylated in all of the analyzed tissue blocks in 6 GBM. On the other hand, 7 GBM were MGMT promoter methylated. Of these, two cases showed different and not-uniform pattern of methylation. In detail, one GBM displayed MGMT promoter methylation in the central, but not in the superficial portion of the tumor and one GBM showed MGMT promoter methylation in the central and intermediate portions but not in the superficial part of the tumor. Of these cases, one had R132H mutant IDH1, and one IDH1 wild type. One of the GBM with heterogeneous MGMT promoter methylation status represented the progression of DA, which was MGMT unmethylated and had IDH1 R132H expression. MGMT methylation status was unchanged in the recurrences of the GBM. In detail, one of the recurrent GBM was methyalated and one unmethylated as were the primitive tumors. Conclusions Our data suggest that MGMT promoter methylation may be heterogeneous through GBM and may depend on the site of surgical sample collection. We may speculate that methylated phenotype might be acquired during tumor progression from low grade astrocytomas.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11570/3068571
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