Absence of MGMT promoter methylation in diffuse midline glioma, H3 K27M-mutant
© The Author(s). 2017
Received: 24 November 2017
Accepted: 24 November 2017
Published: 15 December 2017
To clarify the frequency of MGMT promoter methylation in DMG we analyzed a retrospective series of 143 astrocytic midline tumors for H3F3A and HIST1H3B codon 27 mutations by pyrosequencing (MHH ethic board vote #1707–2013 & #6960). We identified H3F3A K27M mutations in 46/143 tumors including 25 males (54%) and 21 females (46%) with a median age of 23 at diagnosis and a range of 1–68 years. No HIST1H3B mutation was found. Next, we tested these 46 DMG for MGMT promoter methylation. For this purpose, DNA underwent bisulfite treatment and 14 CpG sites in the distal promoter region were analyzed by pyrosequencing (Fig. 1). A mean methylation level of 10% was defined as threshold for hypermethylation. Not a single DMG showed MGMT promoter hypermethylation. To compare this result with the methylation rate in none-DMG GBM we evaluated MGMT-promoter methylation in 40 cases of midline GBM without H3 K27M-alterations showing a hypermethylation status in 14 cases (35%) and absence of methylation in 26 tumors (65%). We, moreover, performed the same analysis in another control group of 247 patients with supratentorial GBM, IDH-wildtype that revealed hypermethylation in 94 tumors (38%) vs. 153 cases (62%) lacking MGMT hypermethylation.
Only few reports are available analyzing MGMT status in DIPG or DMG. First, Babu et al. published a series of five adult patients with DIPG. Using immunohistochemistry, they found MGMT expression in all tumors . Later, the same group reported an MGMT expression frequency of 64.7% in a cohort of 34 patients with DIPG , while the H3 mutation status remained unknown. These findings might imply that in most adult DIPG patients the tumor carries no MGMT methylation. In another study, Reyes-Botero et al. found no MGMT promoter methylation in three adult patients with infratentorial DMG . Using the Illumina 450 K methylation platform, 3/69 pediatric patients with DMG (4%) exhibited hypermethylation of the CpG site in the MGMT promoter . However, only two of the 98 CpG sites of the MGMT CpG island are subject of analysis by the commonly applied algorithm  to filter 450 K data for MGMT promoter methylation (Fig. 1). A direct comparison between such 450 K based MGMT data and results of MS-PCR has demonstrated a good correlation . Nevertheless, these two CpG sites are not located within the DHMR which is commonly analyzed by most neuropathology departments by MS-PCR or pyrosequencing  and has been shown to be strongly associated with the predictive role of MGMT promoter methylation according to the responsible CpG sites in this area (Fig. 1) . To overcome this technical limitation of 450 K MGMT analysis we designed a pyrosequencing assay focusing on the DHMR  encompassing 4/6 CpG sites found to highly correlate with MGMT mRNA expression . Therefore, we assume that the results of our study are more comparable with MGMT promoter methylation results of those neurooncology laboratories where 450 K technology is not available yet.
We had a median age of 23 years at diagnosis in DMP patients of our study with 9 patients aged 40 years or older including one 54- and one 68-year-old patient. These older ages of tumor occurrence in H3 K27M-mutant tumors are unusual, as these tumors have been reported to occur mostly at younger ages [10, 14].
In summary, our results and the published data clearly indicate that MGMT promoter methylation is a rare event in DMG patients supporting the idea that analyzing the MGMT promoter status would only be recommended in H3 K27M-wildtype GBM. Based on the concept that MGMT promoter hypermethylation is associated with a better response to TMZ through reduced expression of MGMT protein, our observation might also explain the failure of clinical trials administrating TMZ to patients with DIPG .
We wish to thank Wiebke Schulze for skillful technical assistance.
RB conceptual design, database retrieval, molecular analysis, analyzing the data, writing the manunscript. AC molecular analysis, writing the manunscript. SB molecular analysis, writing the manunscript. UL molecular analysis, writing the manunscript. CH conceptual design, database retrieval, analyzing the data, writing the manunscript.
The authors declare that they have no competing interests.
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