BACKGROUND: Glioblastoma is the commonest malignant brain tumor and has a very poor prognosis. Reduced expression of the MGMT gene (10q26.3), influenced primarily by the methylation of two differentially methylated regions (DMR1 and DMR2), is associated with a good response to temozolomide treatment. However, suitable methods for detecting the methylation of the MGMT gene promoter and setting appropriate cutoff values are debated. RESULTS: A cohort of 108 patients with histologically and genetically defined glioblastoma was retrospectively examined with methylation-specific Sanger sequencing (sSeq) and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) methods. The DMR2 region was methylated in 29% of samples, whereas DMR1 was methylated in 12% of samples. Methylation detected with the MS-MLPA method using probes MGMT_215, MGMT_190, and MGMT_124 from the ME012-A1 kit (located in DMR1 and DMR2) correlated with the methylation of the corresponding CpG dinucleotides detected with sSeq (p = 0.005 for probe MGMT_215; p < 0.001 for probe MGMT_190; p = 0.016 for probe MGMT_124). The threshold for methylation detection with the MS-MLPA method was calculated with a ROC curve analysis and principal components analysis of the data obtained with the MS-MLPA and sSeq methods, yielding a weighted value of 0.362. Thus, methylation of the MGMT gene promoter was confirmed in 36% of samples. These patients had statistically significantly better overall survival (p = 0.003). CONCLUSIONS: Our results show that the threshold for methylation detection with the MS-MLPA method determined here is useful from a diagnostic perspective because it allows the stratification of patients who will benefit from specific treatment protocols, including temozolomide. Detailed analysis of the MGMT gene promoter enables the more-precise and personalized treatment of patients with glioblastoma.

• Klíčová slova
• MGMT, Glioblastoma, MS-MLPA, Methylation, Sanger sequencing, Stupp protocol,
• MeSH
• DNA modifikační methylasy genetika   MeSH
• dospělí MeSH
• enzymy opravy DNA * genetika   MeSH
• glioblastom * genetika   farmakoterapie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• metylace DNA * MeSH
• multiplexová polymerázová řetězová reakce * metody   MeSH
• nádorové supresorové proteiny genetika   MeSH
• nádory mozku * genetika   farmakoterapie   MeSH
• promotorové oblasti (genetika) MeSH
• retrospektivní studie MeSH
• sekvenční analýza DNA * metody   MeSH
• senioři MeSH
• temozolomid MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• validační studie MeSH
• Názvy látek
• DNA modifikační methylasy MeSH
• enzymy opravy DNA * MeSH
• MGMT protein, human MeSH Prohlížeč
• nádorové supresorové proteiny MeSH
• temozolomid MeSH

BACKGROUND: Diffuse astrocytomas are characterized by their highly variable biological behavior. The possibility that tumors develop novel aberrations, with relevant biological properties, is often neglected. In this study, we present two cases of diffuse astrocytoma in which additional cytogenetic and epigenetic markers with potential influence on cell proliferation or differentiation were detected at relapse. FINDINGS: The biopsies taken from the primary and recurrent tumors of two patients were analyzed with molecular methods to detect copy number variations (CNVs), gene mutations and epigenetic changes. Both cases were characterized by the R132H mutation in the isocitrate dehydrogenase 1 (IDH1) gene. Features typical of astrocytomas, such as copy-neutral loss of heterozygosity at 17p and the deletion of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, were also detected in both cases. These markers were present in the primary and recurrent lesions. Other aberrations, predominantly deletions or amplifications of chromosomal segments and the hypermethylation of gene promoters, were detected in the recurrent lesions. CONCLUSIONS: The IDH1 mutation was the primary event, as previously reported. According to our observations, the methylation of promoters constituted later events, which may have further disrupted cell proliferation and/or differentiation, together with additional CNVs.

• Klíčová slova
• Clonality, Diffuse astrocytoma, Isocitrate dehydrogenase 1, MutL homolog 3,
• Publikační typ
• časopisecké články MeSH