RESUMO
BACKGROUND: The utility of O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status as a prognostic marker in patients with glioblastoma (GBM) has been established. However, the number of CpG sites that must be methylated to cause transcriptional silencing remains unclear, and no significant consensus exists on the optimal method of assessing MGMT methylation. We developed a new high-performance liquid chromatography (HPLC) method that enables accurate analysis of DNA methylation levels using long PCR products. In the present study, we analyzed the MGMT methylation status of 28 isocitrate dehydrogenase-wild-type GBMs treated with temozolomide using ion-exchange HPLC and set the optimal cutoff values. RESULTS: We designed three primers for separate regions (regions 1-3) that had 21 to 38 CpGs for PCR and validated the MGMT promoter methylation status using frozen samples. There was a strong correlation between HPLC and bisulfite sequencing results (R = 0.794). The optimal cutoff values for MGMT methylation in HPLC were determined to allow differentiation of patient prognosis by receiver operating characteristic curve analysis. The cutoff values were 34.15% for region 1, 8.84% for region 2, and 36.72% for region 3. Kaplan-Meyer curve analysis estimated that the most differentiated prognosis was enabled in the setting of 8.84% methylation of MGMT in region 2. Progression-free survival and overall survival were significantly longer for patients in this setting of region 2 methylation (p = 0.00365 and p = 0.00258, respectively). CONCLUSIONS: The combination of our HPLC method and the original primer setting provides a new standard method for determination of MGMT methylation status in patients with GBM and is useful for refining MGMT-based drug selection.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Glioblastoma/genética , Regiões Promotoras Genéticas/genética , Proteínas Supressoras de Tumor/genética , Idoso , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/patologia , Ilhas de CpG , Metilação de DNA , Epigenômica , Feminino , Glioblastoma/diagnóstico , Glioblastoma/tratamento farmacológico , Humanos , Masculino , Reação em Cadeia da Polimerase/métodos , Prognóstico , Intervalo Livre de Progressão , Proteínas Repressoras/genética , Temozolomida/uso terapêuticoRESUMO
Evaluating peritoneal elastic laminal invasion (ELI) has been proposed as an additional assessment for pT3 colorectal cancers (CRC). Its clinical significance has not yet been established. We performed a meta-analysis to investigate the prognostic impact of ELI assessment for subcategorisation of pT3 CRC. We performed a search in three electronic databases. HR and its 95% CI for overall survival (OS) and disease-free survival (DFS) were calculated using the random effects model weighted by the inverse variance method. We identified six studies that met inclusion criteria out of an original 703 studies found with our database search terms. Our meta-analysis included 1925 patients with pT3 and pT4a CRCs. The presence of ELI in pT3 CRC was associated with shortened OS compared with ELI negative pT3 CRC (HR=1.76; 95% CI 1.21 to 2.55); whereas the DFS was not statistically significant (HR=1.79; 95% CI 0.91 to 3.52). Furthermore, pT4a patients' OS (HR=1.84; 95% CI 1.41 to 2.40) and DFS (HR=1.88; 95% CI 1.17 to 3.04) were even worse than the OS and DFS of pT3 ELI (+) patients. ELI is a useful marker for stratifying patients with pT3 or pT4a CRCs into three prognostically distinct groups. We recommend the subcategorisation of pT3 CRC by ELI for better prognostic assessment and treatment strategy of patients with CRC.