What single-cell RNA sequencing taught us about MGMT expression in glioblastoma.

Background: The promoter methylation status of O-6-methylguanine-DNA methyltransferase (MGMTp) is an important prognostic marker in GBM. Previous studies showed that the expression of MGMT based on immunohistochemistry did not correlate with survival. This is partly because nontumor cells express MGMT. Single-cell sequencing assesses gene expression in tumor cells specifically.

Methods: We used publicly available data from 3 recent single-cell/nucleus sequencing GBM studies that included MGMTp methylation status data for patients to evaluate MGMT expression at the single-cell level.

Results: In the CPTAC study, a median of 0.82% and 5.7% of tumor cells expressed MGMT in the MGMTp methylated group and MGMTp unmethylated group, respectively (P-value .001). In the Neftel study, a median of 0.59% and 14.01% of tumor cells expressed MGMT in the MGMTp methylated group and MGMTp unmethylated group, respectively (P-value .01). Three unmethylated samples (out of 16) had MGMT expression <2%. It is unclear if this is due to technical inaccuracies as the Neftel paper did not specify the detection method for MGMTp methylation. Alternatively, the percentage of GBM cells expressing MGMT as a continuous variable may be more relevant than the dichotomous MGMTp status. The Wang study confirmed that MGMT expression can increase or decrease between paired primary and recurrent samples. The gene set enrichment analysis shows that MGMT expressing and negative cells are enriched with mesenchymal and proneural genes, respectively.

Conclusion: Single-cell data suggest that MGMT expression falls on a continuous spectrum. A smaller percentage of tumor cells express MGMT when MGMTp is methylated.