Assessment of Quantitative and Allelic MGMT Methylation Patterns as a Prognostic Marker in Glioblastoma

Abstract

Methylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene is a predictive and prognostic marker in newly diagnosed glioblastoma patients treated with temozolomide but how MGMT methylation should be assessed to ensure optimal detection accuracy is debated. We developed a novel quantitative methylation-specific PCR (qMSP) MGMT assay capable of providing allelic methylation data and analyzed 151 glioblastomas from patients receiving standard of care treatment (Stupp protocol). The samples were also analyzed by immunohistochemistry (IHC), standard bisulfite pyrosequencing, and genotyped for the rs1690252 MGMT promoter single nucleotide polymorphism. Monoallelic methylation was observed more frequently than biallelic methylation, and some cases with monoallelic methylation expressed the MGMT protein whereas others did not. The presence of MGMT methylation was associated with better overall survival (p = 0.006; qMSP and p = 0.002; standard pyrosequencing), and the presence of the protein was associated with worse overall survival (p = 0.009). Combined analyses of qMSP and standard pyrosequencing or IHC identified additional patients who benefited from temozolomide treatment. Finally, low methylation levels were also associated with better overall survival (p = 0.061; qMSP and p = 0.02; standard pyrosequencing). These data support the use of both MGMT methylation and MGMT IHC but not allelic methylation data as prognostic markers in patients with temozolomide-treated glioblastoma.

Keywords: Alkylating agents; Allele-specific; Glioblastoma; Immunohistochemistry; MGMT; Methylation; Temozolomide..

FIGURE 1.

Overview of the quantitative methylation-specific PCR (qMSP)-pyrosequencing workflow. First, genomic DNA is used for genotyping the single nucleotide polymorphism (SNP) in the region between the MSP primers. Then, for the methylation analysis, genomic DNA is bisulfite converted. Next, 2 separate real-time PCRs are performed; one with MSP primers (one of which is biotinylated) targeting the region of interest, and one with primers capable of amplifying both methylated and unmethylated templates targeting a control region (Alu sequences depleted of CpG sites). The data obtained are used to calculate the level of methylation in the samples relative to in vitro methylated DNA. Melting analysis is performed as an integrated part of the real-time PCR runs to confirm that the observed amplification is specific. Subsequently, amplification products are pyrosequenced to allow allelic methylation analysis for the patients being heterozygous for the SNP found between the MSP primers.

FIGURE 2.

The quantitative methylation results using quantitative methylation-specific PCR (qMSP) and standard pyrosequencing. (A) Methylation levels obtained by qMSP plotted for each of the samples. (B) Methylation levels obtained by standard pyrosequencing plotted for each of the samples. (C) Examples of individual results from the MGMT qMSP assay and the Alu assay. The red curves represent the in vitro methylated DNA; the other curves represent individual samples that were analyzed in duplicate. (D) Examples of individual results from the standard pyrosequencing assay that interrogates 4 individual CpG sites.

FIGURE 3.

Representative examples of MGMT immunohistochemistry (IHC) on glioblastomas (World Health Organization [WHO], grade IV). (A, C, E) There are characteristic pleomorphic glial tumor cells, necrosis with pseudopalisading and endothelial cell proliferation. Hematoxylin and eosin stain. (B, C, F) Variable amounts of staining by IHC for MGMT of endothelial cells, lymphocytes, microglia, and tumor cells. (B) Only a minority of tumor cells is positive (0%–10%). (D) There is clear staining of endothelial cells in tumor vessels and diffusely in tumor cells (11%–25%). (F) Many endothelial cells and tumor cells are positive (26%–50%). The staining of tumor cells is heterogeneous, varying from area to area in the tissue. Magnification: x200 for all panels.

FIGURE 4.

Overall survival analyses according to MGMT methylation status, immunohistochemistry (IHC) data, and rs16906252 genotypes. (A) Overall survival according to methylation status assessed by quantitative methylation-specific PCR. (B) Overall survival according to methylation status assessed by standard pyrosequencing. (C) Overall survival according to MGMT protein expression assessed by IHC. (D) Overall survival according to rs16906252 genotypes.

FIGURE 5.

Overall survival analyses according to MGMT methylation levels and combined methylation and immunohistochemistry (IHC) analyses. (A) Overall survival according to methylation levels determined by quantitative methylation-specific PCR (qMSP). (B) Overall survival according to methylation status determined by qMSP and standard pyrosequencing. (C) Overall survival according to methylation status determined by qMSP and IHC data. (D) Overall survival according to methylation status determined by standard pyrosequencing and IHC data.