Low-grade gliomas cause considerable morbidity and most will recur after initial therapy. At recurrence, low-grade gliomas can undergo transformation to high-grade gliomas (grade III or grade IV), which are associated with worse prognosis. Temozolomide (TMZ) provides survival benefit in patients with glioblastomas, but its value in patients with low-grade gliomas is less clear. A subset of TMZ-treated, isocitrate dehydrogenase‒mutant, low-grade astrocytomas recur as more malignant tumors with thousands of de novo, coding mutations bearing a signature of TMZ-induced hypermutation. Preliminary studies raise the hypothesis that TMZ-induced hypermutation may contribute to malignant transformation, although with highly variable latency. On the other hand, hypermutated gliomas have radically altered genomes that present new opportunities for therapeutic intervention. In light of these findings and the immunotherapy clinical trials they inspired, how do patients and providers approach the risks and benefits of TMZ therapy? This review discusses what is known about the mechanisms and consequences of TMZ-induced hypermutation and outstanding questions regarding its clinical significance.

Abstract

INTRODUCTION

Diffuse low-grade gliomas (DLGG) treated with temozolomide (TMZ) can develop somatic hypermutation. We present data on the incidence and prognostic importance of somatic hypermutation in IDH-mutant DLGGs.

METHODS

We analyzed 120 patients treated on a phase II clinical trial of TMZ for sub-totally resected DLGGs to estimate the risk of recurrence and transformation after TMZ. To understand the prognostic significance of somatic hypermutation, we determined hypermutation status by exome or targeted sequencing on tumors from 81 patients with recurrent IDH-mutant DLGGs. 63/81 patients received TMZ before recurrence, including 28 patients treated on-trial.

RESULTS

With median follow-up of 8.7 years, 89 patients from the phase II trial progressed, 60 underwent 1 re-operation, and 36 had histologically confirmed transformation. The 8-year freedom from transformation was 48.2% and 59.9% for IDH-mutant astrocytomas and oligodendrogliomas, respectively; risk of transformation increased with pre-TMZ tumor volume (HR 2.5 per 100cc, p<0.001). In the recurrent glioma cohort, 65/81 patients transformed to grade III or IV; hypermutation was identified at transformation in 30/53 (57%) treated with TMZ. Hypermutation occurred in 31 patients all had received TMZ and 30/31 had developed transformed tumors. Analyzing by specimen, hypermutation was associated with transformation (bootstrapped logistic regression p < 0.001). After transformation to grade III disease (n=47), hypermutation was associated with diminished survival (HR 5.6, p=0.007), controlling for molecular subtype and age at diagnosis. Patients with transformation to glioblastoma (n=18) had poor prognosis regardless of hypermutation (p=0.53). Four cases of spinal dissemination were identified, all of whom had hypermutated gliomas. CONCLUSIONS: Somatic hypermutation is common in transformed, initially low grade IDH-mutant diffuse gliomas treated with TMZ. After anaplastic transformation, somatic hypermutation is associated with reduced survival, independent of molecular subtype. These data have implications for the management of newly diagnosed and recurrent DLGG, and indicate a potential role for immunotherapy.

Reactivation of telomerase reverse transcriptase (TERT) expression enables cells to overcome replicative senescence and escape apoptosis, which are fundamental steps in the initiation of human cancer. Multiple cancer types, including up to 83% of glioblastomas (GBMs), harbor highly recurrent TERT promoter mutations of unknown function but specific to two nucleotide positions. We identified the functional consequence of these mutations in GBMs to be recruitment of the multimeric GA-binding protein (GABP) transcription factor specifically to the mutant promoter. Allelic recruitment of GABP is consistently observed across four cancer types, highlighting a shared mechanism underlying TERT reactivation. Tandem flanking native E26 transformation-specific motifs critically cooperate with these mutations to activate TERT, probably by facilitating GABP heterotetramer binding. GABP thus directly links TERT promoter mutations to aberrant expression in multiple cancers.

Background

Chemotherapy improves overall survival after surgery and radiotherapy for newly diagnosed high-risk IDH-mutant low-grade gliomas (LGGs), but a proportion of patients treated with temozolomide (TMZ) will develop recurrent tumors with TMZ-induced hypermutation. We aimed to determine the prevalence of TMZ-induced hypermutation at recurrence and prognostic implications.

Methods

We sequenced recurrent tumors from 82 patients with initially low-grade IDH-mutant gliomas who underwent reoperation and correlated hypermutation status with grade at recurrence and subsequent clinical outcomes.

Results

Hypermutation was associated with high-grade disease at the time of reoperation (OR 12.0 95% CI 2.5-115.5, P = .002) and was identified at transformation in 57% of recurrent LGGs previously exposed to TMZ. After anaplastic (grade III) transformation, hypermutation was associated with shorter survival on univariate and multivariate analysis (HR 3.4, 95% CI 1.2-9.9, P = .024), controlling for tumor grade, subtype, age, and prior radiotherapy. The effect of hypermutation on survival after transformation was validated in an independent, published dataset. Hypermutated (HM) tumors were more likely to develop discontiguous foci of disease in the brain and spine (P = .003). To estimate the overall incidence of high-grade transformation among low-grade IDH-mutant tumors, data from a phase II trial of TMZ for LGG were analyzed. Eight-year transformation-free survival was 53.8% (95% CI 42.8-69.2), and 61% of analyzed transformed cases were HM.

Conclusions

TMZ-induced hypermutation is a common event in transformed LGG previously treated with TMZ and is associated with worse prognosis and development of discontiguous disease after recurrence. These findings impact tumor classification at recurrence, prognostication, and clinical trial design.

Cancer-associated fibroblasts (CAFs) were presumed absent in glioblastoma given the lack of brain fibroblasts. Serial trypsinization of glioblastoma specimens yielded cells with CAF morphology and single-cell transcriptomic profiles based on their lack of copy number variations (CNVs) and elevated individual cell CAF probability scores derived from the expression of 9 CAF markers and absence of 5 markers from non-CAF stromal cells sharing features with CAFs. Cells without CNVs and with high CAF probability scores were identified in single-cell RNA-Seq of 12 patient glioblastomas. Pseudotime reconstruction revealed that immature CAFs evolved into subtypes, with mature CAFs expressing actin alpha 2, smooth muscle (ACTA2). Spatial transcriptomics from 16 patient glioblastomas confirmed CAF proximity to mesenchymal glioblastoma stem cells (GSCs), endothelial cells, and M2 macrophages. CAFs were chemotactically attracted to GSCs, and CAFs enriched GSCs. We created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF and TGF-β as mediators of GSC effects on CAFs and osteopontin and HGF as mediators of CAF-induced GSC enrichment. CAFs induced M2 macrophage polarization by producing the extra domain A (EDA) fibronectin variant that binds macrophage TLR4. Supplementing GSC-derived xenografts with CAFs enhanced in vivo tumor growth. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target.

Abstract Temozolomide, a commonly used alkylating agent, can induce somatic hypermutation in gliomas. The prevalence and implications of this phenomenon are not well characterized. Using targeted and whole exome sequencing from a cohort of 82 patients with recurrent IDH-mut LGG, we evaluated the clinical implications of hypermutation. Hypermutation was identified at transformation in 57% of recurrent gliomas exposed to TMZ and occurred for both IDH-mutant astrocytomas (52%) and oligodendrogliomas (64%). Among astrocytomas, receipt of radiotherapy prior to transformation was associated with decreased risk of hypermutation (11% vs 70%, p=0.0052), but this trend was not observed for oligodendrogliomas (78% vs 54%, p=NS). Among hypermutated tumors, 94% were transformed to higher WHO grades. Hypermutation was associated with transformation to higher WHO grade (OR 12.0 95% CI 2.5-115.5, p=0.002) and shorter survival after transformation (HR 2.1, 95% CI 1.1-4.0, p=0.018) compared with non-hypermutated transformed tumors. It remained prognostic (controlling for grade, molecular subtype, age, and prior radiotherapy. Patients with transformation to glioblastoma had poor survival regardless of hypermutation (p=0.78). Multivariate models were validated using an external, independent dataset (Harrel’s C=0.72). Strikingly, hypermutated tumors were also associated with development of discontiguous disease after transformation (3-year CI 41% vs 8% p=0.005), including ependymal and leptomeningeal distributions and four cases of spinal dissemination that were not observed in non-hypermutated tumors. These data have significant implications for management of IDH-mut LGG at recurrence.