Page | 1737 MS-MLPA method for the analysis of the glioma tumor MGMT encoding gene promoter methylation: treatment predictive considerations Anca Amalia Udriste 1,2 , Natalia Cucu 1,3,* , Vlad Constantinescu 4 , Lilia Matei 4 , Octaviana Adriana Dulamea 5 , Ileana Constantinescu 6 , Maria Mirela Iacob 1 , Maria Puiu 7 , Nicoleta Andreescu 7 , Cosmin Arsene 1 , Marius Niculescu 8,9 1 Association for Epigenetics and Metabolomics, Bucharest, Romania 2 Research Center for Studies of Food Quality and Agricultural Products, Bucharest, Romania 3 Genetics Department, University of Bucharest, Bucharest, Romania 4 Taxon Solutions, Bucharest, Romania 5 Neurology Department, Fundeni Clinic Institute, Bucharest, Romania 6 "Carol Davila" University of Medicine and Pharmacy University Bucharest, Immunogenetics Laboratory of Clinical Institute Fundeni, Bucharest, Romania 7 Genetics Department, “Victor Babes” Medical and Pharmaceutical University Timisoara, Romania 8 Colentina Clinical Hospital, Department of Orthopaedics and Traumathology , Bucharest, Romania 9 "Titu Maiorescu" University, Faculty of Medicine, Bucharest, Romania *corresponding author e-mail address:nataliacucu@gmail.com ABSTRACT The epigenotype of a tumor tissue containing methylated promoter of the MGMT encoding gene is considered presently as a good prognostic marker signifying an increase in the sensitivity to its treatment with alkylating agents such as temozolomide (TMZ). The principle of this biomarker application in pharmaco-epigenetics as well as its correct clinical significance is based on the correlation between the silencing methylation process in gene promoters and the DNA damaging activity of the classical alkylating drugs. Two methods that are currently used in genetic laboratories for methylation pattern based biomarkers: Methylation Sensible (MS) – Multiplex ligation-dependent probe amplification (MLPA) and Methylation Specific (MS) -PCR are described comparatively on two tumor types (glioma tumors and Ewing sarcoma-EWS) whose treatment with the alkylating drug TMZ requires an evaluation of its efficiency. The first method was chosen for its semiquantitative capacity as compared with the classical MS-PCR method, whose poor sensibility requires an optimization in order that its only qualitative result to be sensible. The two types of tumors were estimated to behave differently due to their MGMT enzyme activity: for methylated glioma tissues a positive prognostic was established, while for the unmethylated EWS the future evolution of the tumor progression was negative. Key words: epigenetic, MLPA, MGMT, treatment prognostic, glioma. 1. INTRODUCTION Assessment of the methylation status of the O 6 - methylguanine-DNA methyltransferase (MGMT) gene promoter in malignant tumors, such as glioma (G) and Ewing sarcoma (EWS) has been recognized as an important molecular assay in clinical oncology. The landmark study by HEGI [1], followed by numerous clinical trials in glioblastoma of the same research group (HAU [2] and BADY [3]), have confirmed that hypermethylation of the MGMT promoter serves as a strong prognostic factor for progression-free survival and overall survival specifically in glioma tissues. For Ewing's sarcoma this type of methylation pattern in MGMT encoding gene was detected only very rare (12%) (JAHROMI [4]). Also, using this methylation biomarker for the management of EWS patients during their treatment is still a matter of debate since the first studies using the classical toxic drugs, such as temozolomide TMZ.(MISER [5], VIETTI [6], CANGIR [7]). The MGMT gene encodes a native DNA repair enzyme that has a counteracting activity towards the lethal effects of alkylating agents on tumor cells. It acts by removing alkyl adducts from the O 6 -position of guanine (PEGG [8]). The alkylating effect of the classical drugs results in O 6 -alkylated guanine, that causes base mispairing and double-strand breaks, which induce apoptosis and cell death (KARRAN [9]). Based on this DNA repair activity, the MGMT protein is believed to provide resistance against cytotoxic effects of alkylating agents, such as TMZ (KONDO [10]). By contrast, MGMT repair activity should be constantly efficient for the normal cells, however, when its activity is silenced through the epigenetic process of methylation, the neoplasic transformation may be initiated by the increased genomic instability and genetic reprogramming of certain housekeeping or tumor suppressor genes, Therefore, even that MGMT methylation may be dramatic for the primary tumorigenesis through continuously increase in genomic instability it may turn in a benefic state for the late progressed tumor during its treatment with specific, alkylating drugs, which interfere with the repair process catalyzed by the MGMT enzyme. The alkylating drug such az TMZ induces tumor DNA damages which are rapidly repaired by MGMT enzyme. This therapeutically disadvantageous protective effect of the active, unmethylated MGMT gene is thought not to be present in tumors where MGMT is silenced through methylation. Such epigenotype was frequently observed in many human cancers including glioblastoma, head and neck cancers and colon cancers, Volume 6, Issue 6, 2016, 1737 - 1742 ISSN 2069-5837 Open Access Journal Received: 15.02.2016/ Revised: 05.03.2016 / Accepted: 07.03.2016 / Published on-line: 10.11.2016 Original Research Article Biointerface Research in Applied Chemistry www.BiointerfaceResearch.com