MGMT Gene Promoter Methylation Status – Assessment of Two Pyrosequencing Kits and Three Methylation-specific PCR Methods for their Predictive Capacity in Glioblastomas

Abstract

Background: Although methylation of the O 6-methylguanine-DNA methyltransferase (MGMT) gene promoter predicts response to temozolomide in patients with glioblastoma, no consensus exists as to which assay is best for its detection. Materials and Methods: Methylation of MGMT promoter was examined by methylation-specific polymerase chain reaction (MSP), quantitative real-time MSP, methylation-sensitive high-resolution melting analysis, and two commercial pyrosequencing (PSQ) kits. Survival was compared among 48 patients with glioblastoma according to assay results. Results: Only PSQ and MSP significantly separated patients who benefited from temozolomide, with PSQ being the superior method. For PSQ analysis, the cutoff value that best correlated with prognostic outcome was 7% methylation of MGMT. Median survival in patients with MGMT promoter methylation above this cutoff value was 7.8 months longer compared to those with less than 7% methylation. Two-year overall survival for the two groups was 42% and 7.4%, respectively. Conclusion: PSQ is the method of choice for MGMT promoter methylation analysis in routine clinical practice.

Current status of development of methylation biomarkers for in vitro diagnostic IVD applications

“…Clinical performance of the "targeted methylation assay" was evaluated in another CCGA nested study that included 2301 participants (1422 cancer [> 20 tumor types, all stages], 879 noncancer). The sensitivity of the assay across all cancer types ranged from 59 to 86% (at 99% specificity), with 34% (95% CI 27-43) sensitivity in stage I (n = 151), 77% (95% CI 70-83) in stage II (n = 171), 84% (95% CI [79][80][81][82][83][84][85][86][87][88][89] in stage III (n = 204), and 92% (95% CI 88-95) in stage IV (n = 281). Moreover, the assay assigned tissue of origin to 94% of cancers in the study and for 90% of these cases, the assignment of TOO was correct [70].…”

“…The performance of that test was evaluated in a study that included 48 primary GBM samples, using four meningioma samples as negative controls. The study showed that the therascreen® MGMT Pyro® Kit, accurately detects MGMT promoter methylation and can be used for patients stratification according to OS (hazard ratio (HR) of 2.3348 (95% CI 1.1918-4.5724; p = 0.013) [82].…”

A novel Cas9-targeted long-read assay for simultaneous detection of IDH1/2 mutations and clinically relevant MGMT methylation in fresh biopsies of diffuse glioma

“…2c and 4b). However, some patients who are below this arbitrary cutoff value (e.g., 10%) do respond to TMZ therapy [31][32][33], placing them in a "gray zone" and producing a clinical quandary. With this in mind, Chai et al developed a novel CpG averaging model for pyrosequencing data that defines the MGMT promoter as being methylated when at least 3 CpGs exceed their respective cutoff values; this allows clinicians to better stratify patients with very low levels of methylation (e.g., < 10%) [34].…”

Assessment of MGMT methylation status using high-performance liquid chromatography in newly diagnosed glioblastoma

“…Previous studies have identified two regions (the promoter and gene body) that are significantly correlated with MGMT expression [ 11 ]. However, due to the difficulty of designing a primer for the GC-rich promoter area and the lack of a method that enables analysis of long PCR products at low cost, the region of the gene body is generally selected as the target for MGMT methylation analysis [ 12 ]. To demonstrate the effectiveness of methylation analysis in the promoter region, a new method for analyzing longer PCR products is required.…”