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Glioblastoma is the most aggressive tumor in the central nervous system, with a survival rate of less than 15 months despite multimodal therapy. Tumor recurrence frequently occurs after removal. Tumoral angiogenesis, the formation of neovessels, has a positive impact on tumor progression and invasion, although there are controversial results in the specialized literature regarding its impact on survival. This study aims to correlate the immunoexpression of angiogenesis markers (CD34, CD105) with the proliferation index Ki67 and p53 in primary and secondary glioblastomas. This retrospective study included 54 patients diagnosed with glioblastoma at the Pathology Department of County Emergency Clinical Hospital Târgu Mureș. Microvascular density was determined using CD34 and CD105 antibodies, and the results were correlated with the immunoexpression of p53, IDH1, ATRX and Ki67. The number of neoformed blood vessels varied among cases, characterized by different shapes and calibers, with endothelial cells showing modified morphology and moderate to marked pleomorphism. Neovessels with a glomeruloid aspect, associated with intense positivity for CD34 or CD105 in endothelial cells, were observed, characteristic of glioblastomas. Mean microvascular density values were higher for the CD34 marker in all cases, though there were no statistically significant differences compared to CD105. Mutant IDH1 and ATRX glioblastomas, wild-type p53 glioblastomas, and those with a Ki67 index above 20% showed a more abundant microvascular density, with statistical correlations not reaching significance. This study highlighted a variety of percentage intervals of microvascular density in primary and secondary glioblastomas using immunohistochemical markers CD34 and CD105, respectively, with no statistically significant correlation between evaluated microvascular density and p53 or Ki67.

Mutations in isocitrate dehydrogenase isoform 1 (IDH1) are primarily found in secondary glioblastoma (GBM) and low-grade glioma but are rare in primary GBM. The standard treatment for GBM includes radiation combined with temozolomide, an alkylating agent. Fortunately, IDH1 mutant gliomas are sensitive to this treatment, resulting in a more favorable prognosis. However, it\'s estimated that up to 75 % of IDH1 mutant gliomas will progress to WHO grade IV over time and develop resistance to alkylating agents. Therefore, understanding the mechanism(s) by which IDH1 mutant gliomas confer sensitivity to alkylating agents is crucial for developing targeted chemotherapeutic approaches. The base excision repair (BER) pathway is responsible for repairing most base damage induced by alkylating agents. Defects in this pathway can lead to hypersensitivity to these agents due to unresolved DNA damage. The coordinated assembly and disassembly of BER protein complexes are essential for cell survival and for maintaining genomic integrity following alkylating agent exposure. These complexes rely on poly-ADP-ribose formation, an NAD+-dependent post-translational modification synthesized by PARP1 and PARP2 during the BER process. At the lesion site, poly-ADP-ribose facilitates the recruitment of XRCC1. This scaffold protein helps assemble BER proteins like DNA polymerase beta (Polβ), a bifunctional DNA polymerase containing both DNA synthesis and 5\'-deoxyribose-phosphate lyase (5\'dRP lyase) activity. Here, we confirm that IDH1 mutant glioma cells have defective NAD+ metabolism, but still produce sufficient nuclear NAD+ for robust PARP1 activation and BER complex formation in response to DNA damage. However, the overproduction of 2-hydroxyglutarate, an oncometabolite produced by the IDH1 R132H mutant protein, suppresses BER capacity by reducing Polβ protein levels. This defines a novel mechanism by which the IDH1 mutation in gliomas confers cellular sensitivity to alkylating agents and to inhibitors of the poly-ADP-ribose glycohydrolase, PARG.

IDH1 and IDH2 mutational status is a critical biomarker with diagnostic, prognostic, and treatment implications in glioma. Although IDH1 p.R132H-specific immunohistochemistry is available, it is unable to identify other mutations in IDH1/2. Next-generation sequencing can accurately determine IDH1/2 mutational status but suffers from long turnaround time when urgent treatment planning and initiation is medically necessary. The Idylla assay can detect IDH1/2 mutational status from unstained formalin-fixed paraffin-embedded (FFPE) slides in as little as a few hours. In a clinical validation, we demonstrate clinical accuracy of 97% compared to next-generation sequencing. Sensitivity studies demonstrated a limit of detection of 2.5-5% variant allele frequency, even at DNA inputs below the manufacturer\'s recommended threshold. Overall, the assay is an effective and accurate method for rapid determination of IDH1/2 mutational status.

Context  Glioblastoma multiforme (GBM) is a malignant and aggressive primary brain tumor with a poor prognosis. This adverse prognosis is due to the tumor\'s tendency for advancement and recurrence caused by highly intrusive nature of the persisting GBM cells that actively escape from the main tumor mass into the surrounding normal brain tissue. On the basis of biomarker illustration, it can be classified into molecular subgroups. Aims  (1) To determine the expression of IDH1, ATRX, p53, and Ki67 by immunohistochemistry, in a cohort of GBMs. (2) To determine whether altered protein expression of any of these growth-control genes in GBM will show association with patient survival. (3) To establish prognostically distinct molecular subgroups of GBM, irrespective of histopathological diagnosis. Results  In this prospective observational study, 35 histologically diagnosed cases of glioblastoma were enrolled. The mean age at the time of presentation was 43.46 ± 17.25 years with a male:female ratio of 1.3:1. Of the 35 cases, microvascular proliferation was seen in 23 cases. Large foci of necrosis (>50%) were seen in 10 cases and 27 cases had mitotic count ≥ 5/high power field (HPF). Of 35 cases, 5 (14.3%) cases showed IDH1 immunopositivity and 30 (85.7%) cases were negative for IDH1. ATRX was retained in 24 (68.6%) cases, while it was lost in 11 (31.4%) cases. The p53 immunoexpression was seen in 31 (88.6%) cases, whereas p53 was negative in 4 (11.4%) cases. The overall median survival (OS) was 6 months. In two protein pairs, the three compositions were IDH1-/p53+ (74.3%), ATRX +/IDH1- (62.9%), and ATRX +/p53+ (57.1%). Combined three-protein immunohistochemical analysis revealed five different molecular variants. Also, 8.6% (3/35) of the samples had aberrant protein expression of all three proteins, i.e., ATRX-/p53 +/IDH1 + , while 11.4% (4/35) were wild-type protein expression group, i.e., ATRX +/p53-/IDH1-. Conclusion  In patients with single protein expression, Kaplan-Meier survival analysis showed statistically better OS in IDH1 mutant glioblastomas. In cases with double protein pairs, IDH1/p53 revealed statistically significant association with better median OS. The survival analysis of patients with IDH1/ATRX/p53 protein combinations also denoted a better OS. Hence, GBM can be grouped into prognostically relevant subgroups using these protein expression signatures individually, as well as the combined protein expression signatures.

A subset of patients with IDH-mutant glioma respond to inhibitors of mutant IDH (IDHi), yet the molecular underpinnings of such responses are not understood. Here, we profiled by single-cell or single-nucleus RNA-sequencing three IDH-mutant oligodendrogliomas from patients who derived clinical benefit from IDHi. Importantly, the tissues were sampled on-drug, four weeks from treatment initiation. We further integrate our findings with analysis of single-cell and bulk transcriptomes from independent cohorts and experimental models. We find that IDHi treatment induces a robust differentiation toward the astrocytic lineage, accompanied by a depletion of stem-like cells and a reduction of cell proliferation. Furthermore, mutations in NOTCH1 are associated with decreased astrocytic differentiation and may limit the response to IDHi. Our study highlights the differentiating potential of IDHi on the cellular hierarchies that drive oligodendrogliomas and suggests a genetic modifier that may improve patient stratification.

In this study, we aimed to comprehensively characterize the potential relationships among the frequently mutated genes, well-known homologous recombination repair (HRR) proteins, and immune proteins in glioma from a clinical perspective. A total of 126 surgical tissues from patients initially diagnosed with glioma were included. The genetic alterations were tested using the targeted next-generation sequencing technique. The expression of HRR proteins, immune proteins, and genetic alteration-related proteins were detected using immunostaining. Integrated analysis showed that ATRX is positively correlated with STING in high-grade glioma (HGG) with wild-type ATRX and IDH1. Then, a relapse predictive risk-scoring model was established using the least absolute shrinkage and selection operator regression algorithms. The scores based on the expression of ATRX and STING significantly predict the recurrence for glioma patients, which further predict the survival for specific subgroups, characterized with high expression of RAD51 and wild-type TERT. Moreover, STING is significantly higher in patients with high relapse risk. Interestingly, STING inhibitors and agonists both suppress the growth of HGG cells, regardless of their STING levels and STING pathway activity, whereas RAD51 inhibitor B02 is found to exclusively sensitize HGG cells with high expression of STING to temozolomide in vitro and in vivo. Overall, findings in the study not only reveal that ATRX is closely correlated with STING to drive the relapse of HGG, but also provide a STING-guided combined strategy to treat patients with aggressive gliomas. Translation of these findings will ultimately improve the outcomes for ATRX and IDH1 genomically stratified subgroups in HGG.

Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant uniquely preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employed static and dynamic structural methods and found that, compared to R132H, the R132Q active site adopted a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling revealed a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.

The 2021 WHO classification of gliomas has separated gliomas based on their IDH mutation status, reflecting differences in their pathogenesis and clinical characteristics. There is a paucity of data on the prevalence of IDH mutations in gliomas in this region. This study aimed to determine the frequency of the IDH1 mutation in adult-type diffuse astrocytic gliomas in a tertiary hospital in Kenya. Approximately half of the gliomas were positive for the IDH1 mutation, with a slight male predominance. Our study provides crucial insights into the frequency of IDH1 mutations in gliomas in Kenya.

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