Improved prognostic stratification of patients with isocitrate dehydrogenase-mutant astrocytoma.

Prognostic factors and standards of care for astrocytoma, isocitrate dehydrogenase (IDH)-mutant, CNS WHO grade 4, remain poorly defined. Here we sought to explore disease characteristics, prognostic markers, and outcome in patients with this newly defined tumor type. We determined molecular biomarkers and assembled clinical and outcome data in patients with IDH-mutant astrocytomas confirmed by central pathology review. Patients were identified in the German Glioma Network cohort study; additional cohorts of patients with CNS WHO grade 4 tumors were identified retrospectively at two sites. In total, 258 patients with IDH-mutant astrocytomas (114 CNS WHO grade 2, 73 CNS WHO grade 3, 71 CNS WHO grade 4) were studied. The median age at diagnosis was similar for all grades. Karnofsky performance status at diagnosis inversely correlated with CNS WHO grade (p < 0.001). Despite more intensive treatment upfront with higher grade, CNS WHO grade was strongly prognostic: median overall survival was not reached for grade 2 (median follow-up 10.4 years), 8.1 years (95% CI 5.4-10.8) for grade 3, and 4.7 years (95% CI 3.4-6.0) for grade 4. Among patients with CNS WHO grade 4 astrocytoma, median overall survival was 5.5 years (95% CI 4.3-6.7) without (n = 58) versus 1.8 years (95% CI 0-4.1) with (n = 12) homozygous CDKN2A deletion. Lower levels of global DNA methylation as detected by LINE-1 methylation analysis were strongly associated with CNS WHO grade 4 (p < 0.001) and poor outcome. MGMT promoter methylation status was not prognostic for overall survival. Histomolecular stratification based on CNS WHO grade, LINE-1 methylation level, and CDKN2A status revealed four subgroups of patients with significantly different outcomes. In conclusion, CNS WHO grade, global DNA methylation status, and CDKN2A homozygous deletion are prognostic in patients with IDH-mutant astrocytoma. Combination of these parameters allows for improved prediction of outcome. These data aid in designing upcoming trials using IDH inhibitors.

A clinically compatible in vitro drug-screening platform identifies therapeutic vulnerabilities in primary cultures of brain metastases.

PURPOSE: Brain metastases represent the most common intracranial tumors in adults and are associated with a poor prognosis. We used a personalized in vitro drug screening approach to characterize individual therapeutic vulnerabilities in brain metastases. METHODS: Short-term cultures of cancer cells isolated from brain metastasis patients were molecularly characterized using next-generation sequencing and functionally evaluated using high-throughput in vitro drug screening to characterize pharmacological treatment sensitivities. RESULTS: Next-generation sequencing identified matched genetic alterations in brain metastasis tissue samples and corresponding short-term cultures, suggesting that short-term cultures of brain metastases are suitable models for recapitulating the genetic profile of brain metastases that may determine their sensitivity to anti-cancer drugs. Employing a high-throughput in vitro drug screening platform, we successfully screened the cultures of five brain metastases for response to 267 anticancer compounds and related drug response to genetic data. Among others, we found that targeted treatment with JAK3, HER2, or FGFR3 inhibitors showed anti-cancer effects in individual brain metastasis cultures. CONCLUSION: Our preclinical study provides a proof-of-concept for combining molecular profiling with in vitro drug screening for predictive evaluation of therapeutic vulnerabilities in brain metastasis patients. This approach could advance the use of patient-derived cancer cells in clinical practice and might eventually facilitate decision-making for personalized drug treatment.

Epigenetic modification and characterization of the MGMT promoter region using CRISPRoff in glioblastoma cells.

The methylation status of the O6-methylguanine DNA methyltransferase (MGMT) promoter region is a critical predictor of response to alkylating agents in glioblastoma. However, current approaches to study the MGMT status focus on analyzing models with non-identical backgrounds. Here, we present an epigenetic editing approach using CRISPRoff to introduce site-specific CpG methylation in the MGMT promoter region of glioma cell lines. Sanger sequencing revealed successful introduction of methylation, effectively generating differently methylated glioma cell lines with an isogenic background. The introduced methylation resulted in reduced MGMT mRNA and protein levels. Furthermore, the cell lines with MGMT promoter region methylation exhibited increased sensitivity to temozolomide, consistent with the impact of methylation on treatment outcomes in patients with glioblastoma. This precise epigenome-editing approach provides valuable insights into the functional relevance of MGMT promoter regional methylation and its potential for prognostic and predictive assessments, as well as epigenetic-targeted therapies.

Glioma.

Gliomas are primary brain tumours that are thought to develop from neural stem or progenitor cells that carry tumour-initiating genetic alterations. Based on microscopic appearance and molecular characteristics, they are classified according to the WHO classification of central nervous system (CNS) tumours and graded into CNS WHO grades 1-4 from a low to high grade of malignancy. Diffusely infiltrating gliomas in adults comprise three tumour types with distinct natural course of disease, response to treatment and outcome: isocitrate dehydrogenase (IDH)-mutant and 1p/19q-codeleted oligodendrogliomas with the best prognosis; IDH-mutant astrocytomas with intermediate outcome; and IDH-wild-type glioblastomas with poor prognosis. Pilocytic astrocytoma is the most common glioma in children and is characterized by circumscribed growth, frequent BRAF alterations and favourable prognosis. Diffuse gliomas in children are divided into clinically indolent low-grade tumours and high-grade tumours with aggressive behaviour, with histone 3 K27-altered diffuse midline glioma being the leading cause of glioma-related death in children. Ependymal tumours are subdivided into biologically and prognostically distinct types on the basis of histology, molecular biomarkers and location. Although surgery, radiotherapy and alkylating agent chemotherapy are the mainstay of glioma treatment, individually tailored strategies based on tumour-intrinsic dominant signalling pathways have improved outcome in subsets of patients.

Zotiraciclib (TG02) for newly diagnosed glioblastoma in the elderly or for recurrent glioblastoma: The EORTC 1608 STEAM trial.

BACKGROUND: Zotiraciclib (TG02) is an oral multi-cyclin dependent kinase (CDK) inhibitor thought to inhibit tumor growth via CDK-9-dependent depletion of survival proteins such as c-MYC and MCL-1 which are frequently overexpressed in glioblastoma. METHODS: EORTC 1608 (NCT03224104) (STEAM) had a three parallel group (A,B,C) phase Ib, open-label, non-randomized, multicenter design in IDH wild-type newly diagnosed glioblastoma or anaplastic astrocytoma. Groups A and B explored the maximum tolerated dose (MTD) of TG02 in elderly patients, in combination with hypofractionated radiotherapy alone (group A) or temozolomide alone (group B), according to O6-methylguanine DNA methyltransferase promoter methylation status determined centrally. Group C explored single agent activity of TG02 at first relapse after temozolomide chemoradiotherapy with a primary endpoint of progression-free survival at 6 months (PFS-6). Tumor expression of CDK-9, c-MYC and MCL-1 was determined by immunohistochemistry. RESULTS: The MTD was 150 mg twice weekly in combination with radiotherapy alone (group A) or temozolomide alone (group B). Two dose-limiting toxicities were observed at 150 mg: one in group A (grade 3 seizure), one in group B (multiple grade 1 events). Main toxicities included neutropenia, gastrointestinal disorders and hepatotoxicity. PFS-6 in group C was 6.7%. CDK-9, c-MYC and MCL-1 were confirmed to be expressed and their expression was moderately cross-correlated. High protein levels of MCL-1 were associated with inferior survival. CONCLUSIONS: TG02 exhibits overlapping toxicity with alkylating agents and low single agent clinical activity in recurrent glioblastoma. The role of CDK-9 and its down-stream effectors as prognostic factors and therapeutic targets in glioblastoma warrants further study.

The biological significance of tumor grade, age, enhancement and extent of resection in IDH mutant gliomas: how should they inform treatment decision in the era of IDH inhibitors? Invited review.

The 2016 and 2021 World Health Organization (WHO) 2021 Classification of Central Nervous System (CNS) tumors have resulted in a major improvement of the classification of IDH-mutant gliomas. With more effective treatments many patients experience prolonged survival . However, treatment guidelines are often still based on information from historical series comprising both patients with IDHwt and IDH mutant tumors. They provide recommendations for radiotherapy and chemotherapy for so-called high-risk patients, usually based on residual tumor after surgery and age over 40. More up-to-date studies give a better insight into clinical, radiological and molecular factors associated with outcome of patients with IDH-mutant glioma. These insights should be used today for risk stratification and for treatment decisions. In many patients with an IDH-mutant grade 2 and grade 3 glioma, if carefully monitored postponing radiotherapy and chemotherapy is safe, and will not jeopardize overall outcome of patients. With the INDIGO trial showing patient benefit from the IDH inhibitor vorasidenib, there is a sizable population in which it seems reasonable to try this class of agents before recommending radio-chemotherapy with its delayed adverse event profile affecting quality of survival. Ongoing trials should help to further identify the patients that are benefiting from this treatment.

mTORC1 regulates cell survival under glucose starvation through 4EBP1/2-mediated translational reprogramming of fatty acid metabolism.

Energetic stress compels cells to evolve adaptive mechanisms to adjust their metabolism. Inhibition of mTOR kinase complex 1 (mTORC1) is essential for cell survival during glucose starvation. How mTORC1 controls cell viability during glucose starvation is not well understood. Here we show that the mTORC1 effectors eukaryotic initiation factor 4E binding proteins 1/2 (4EBP1/2) confer protection to mammalian cells and budding yeast under glucose starvation. Mechanistically, 4EBP1/2 promote NADPH homeostasis by preventing NADPH-consuming fatty acid synthesis via translational repression of Acetyl-CoA Carboxylase 1 (ACC1), thereby mitigating oxidative stress. This has important relevance for cancer, as oncogene-transformed cells and glioma cells exploit the 4EBP1/2 regulation of ACC1 expression and redox balance to combat energetic stress, thereby supporting transformation and tumorigenicity in vitro and in vivo. Clinically, high EIF4EBP1 expression is associated with poor outcomes in several cancer types. Our data reveal that the mTORC1-4EBP1/2 axis provokes a metabolic switch essential for survival during glucose starvation which is exploited by transformed and tumor cells.

The Epigenetic Evolution of Glioma Is Determined by the IDH1 Mutation Status and Treatment Regimen.

Tumor adaptation or selection is thought to underlie therapy resistance in glioma. To investigate longitudinal epigenetic evolution of gliomas in response to therapeutic pressure, we performed an epigenomic analysis of 132 matched initial and recurrent tumors from patients with IDH-wildtype (IDHwt) and IDH-mutant (IDHmut) glioma. IDHwt gliomas showed a stable epigenome over time with relatively low levels of global methylation. The epigenome of IDHmut gliomas showed initial high levels of genome-wide DNA methylation that was progressively reduced to levels similar to those of IDHwt tumors. Integration of epigenomics, gene expression, and functional genomics identified HOXD13 as a master regulator of IDHmut astrocytoma evolution. Furthermore, relapse of IDHmut tumors was accompanied by histologic progression that was associated with survival, as validated in an independent cohort. Finally, the initial cell composition of the tumor microenvironment varied between IDHwt and IDHmut tumors and changed differentially following treatment, suggesting increased neoangiogenesis and T-cell infiltration upon treatment of IDHmut gliomas. This study provides one of the largest cohorts of paired longitudinal glioma samples with epigenomic, transcriptomic, and genomic profiling and suggests that treatment of IDHmut glioma is associated with epigenomic evolution toward an IDHwt-like phenotype. SIGNIFICANCE: Standard treatments are related to loss of DNA methylation in IDHmut glioma, resulting in epigenetic activation of genes associated with tumor progression and alterations in the microenvironment that resemble treatment-naïve IDHwt glioma.