Evolution of DNA repair defects during malignant progression of low-grade gliomas after temozolomide treatment

Abstract

Temozolomide (TMZ) increases the overall survival of patients with glioblastoma (GBM), but its role in the clinical management of diffuse low-grade gliomas (LGG) is still being defined. DNA hypermethylation of the O (6) -methylguanine-DNA methyltransferase (MGMT) promoter is associated with an improved response to TMZ treatment, while inactivation of the DNA mismatch repair (MMR) pathway is associated with therapeutic resistance and TMZ-induced mutagenesis. We previously demonstrated that TMZ treatment of LGG induces driver mutations in the RB and AKT-mTOR pathways, which may drive malignant progression to secondary GBM. To better understand the mechanisms underlying TMZ-induced mutagenesis and malignant progression, we explored the evolution of MGMT methylation and genetic alterations affecting MMR genes in a cohort of 34 treatment-naïve LGGs and their recurrences. Recurrences with TMZ-associated hypermutation had increased MGMT methylation compared to their untreated initial tumors and higher overall MGMT methylation compared to TMZ-treated non-hypermutated recurrences. A TMZ-associated mutation in one or more MMR genes was observed in five out of six TMZ-treated hypermutated recurrences. In two cases, pre-existing heterozygous deletions encompassing MGMT, or an MMR gene, were followed by TMZ-associated mutations in one of the genes of interest. These results suggest that tumor cells with methylated MGMT may undergo positive selection during TMZ treatment in the context of MMR deficiency.

Fig. 1

Comparison of MGMT methylation in different assays and across tumor regions. a Position of the MGMT bisulfite amplicon (light blue) encompassing the 23 CpGs assessed in this study, the position of the MGMT CpG island (green), and the enhancer region encompassing CpGs 10–16 (dark blue). TSS= transcription start site. b Distribution of histological subtypes and grades of the recurrent tumors in the three groups. c Comparison of the binary outcome of MSP (x-axis) to MGMT methylation level of CpGs 10 to 16 determined by bisulfite, PCR and sequencing of 10 or more independent clones (y-axis). d The degree of variation in methylation levels was determined in replicate experiments from independent aliquots of the same genomic DNA isolation (y-axis) with bisulfite and sequencing of the MGMT promoter in individual samples (x-axis). e MGMT methylation levels (y-axis) in spatially distinct regions of individual tumors (x-axis). Sample designations are the patient (p) number followed by .1 for initial tumor and .2 for the recurrent tumor.

Fig. 2

Significantly elevated MGMT methylation in TMZ-associated hypermutated recurrent tumors that arise from LGG. MGMT methylation levels in initial (green) and recurrent (yellow) tumors of three patient subgroups; non-TMZ= patients not treated with TMZ, TMZ-non-HM= patients treated with TMZ without a hypermutated recurrent tumor, TMZ-HM= patients treated with TMZ with a hypermutated recurrent tumor.

Fig. 3

Sequential acquisition of DNA repair deficiency exclusively in LGG patients that were treated with TMZ and had a hypermutated recurrence. a Exome derived copy number status and germ-line variant allele frequency across chromosome 2 encompassing MSH2 and MSH6 in the initial and recurrent tumor of patient 18. b Methylation level of MGMT, mutations and copy number status of MGMT and MMR-related genes in initial LGG and paired recurrent tumors. The panel includes patients for whom exome sequencing data was available, * indicates samples with a proportion of tumor cells lower than 50%.

Fig. 4

A working model of the effect of an impaired MMR system on clonal outgrowth of MGMT methylated cells during acquisition of the TMZ-associated hypermutator phenotype and TMZ-associated malignant progression. Left: When MMR is intact, TMZ treatment induces cell death in MGMT methylated cells. The histology of the recurrent tumor is variable. Right: When MMR is deficient, TMZ treatment fails to induce cell death and MGMT methylated tumor cells may expand, become hypermutated, and undergo malignant progression to GBM.