DNA damage response in brain tumors: A Society for Neuro-Oncology consensus review on mechanisms and translational efforts in neuro-oncology.

DNA damage response (DDR) mechanisms are critical to maintenance of overall genomic stability, and their dysfunction can contribute to oncogenesis. Significant advances in our understanding of DDR pathways have raised the possibility of developing therapies that exploit these processes. In this expert-driven consensus review, we examine mechanisms of response to DNA damage, progress in development of DDR inhibitors in IDH-wild-type glioblastoma and IDH-mutant gliomas, and other important considerations such as biomarker development, preclinical models, combination therapies, mechanisms of resistance and clinical trial design considerations.

The Virtual Child.

SUMMARY: We are building the world's first Virtual Child-a computer model of normal and cancerous human development at the level of each individual cell. The Virtual Child will "develop cancer" that we will subject to unlimited virtual clinical trials that pinpoint, predict, and prioritize potential new treatments, bringing forward the day when no child dies of cancer, giving each one the opportunity to lead a full and healthy life.

Adult brain tumour research in 2024: Status, challenges and recommendations.

In 2015, a groundswell of brain tumour patient, carer and charity activism compelled the UK Minister for Life Sciences to form a brain tumour research task and finish group. This resulted, in 2018, with the UK government pledging £20m of funding, to be paralleled with £25m from Cancer Research UK, specifically for neuro-oncology research over the subsequent 5 years. Herein, we review if and how the adult brain tumour research landscape in the United Kingdom has changed over that time and what challenges and bottlenecks remain. We have identified seven universal brain tumour research priorities and three cross-cutting themes, which span the research spectrum from bench to bedside and back again. We discuss the status, challenges and recommendations for each one, specific to the United Kingdom.

Aberrant ATM signaling and homology-directed DNA repair as a vulnerability of p53-mutant GBM to AZD1390-mediated radiosensitization.

ATM is a key mediator of radiation response, and pharmacological inhibition of ATM is a rational strategy to radiosensitize tumors. AZD1390 is a brain-penetrant ATM inhibitor and a potent radiosensitizer. This study evaluated the spectrum of radiosensitizing effects and the impact of TP53 mutation status in a panel of IDH1 wild-type (WT) glioblastoma (GBM) patient-derived xenografts (PDXs). AZD1390 suppressed radiation-induced ATM signaling, abrogated G0-G1 arrest, and promoted a proapoptotic response specifically in p53-mutant GBM in vitro. In a preclinical trial using 10 orthotopic GBM models, AZD1390/RT afforded benefit in a cohort of TP53-mutant tumors but not in TP53-WT PDXs. In mechanistic studies, increased endogenous DNA damage and constitutive ATM signaling were observed in TP53-mutant, but not in TP53-WT, PDXs. In plasmid-based reporter assays, GBM43 (TP53-mutant) showed elevated DNA repair capacity compared with that in GBM14 (p53-WT), whereas treatment with AZD1390 specifically suppressed homologous recombination (HR) efficiency, in part, by stalling RAD51 unloading. Furthermore, overexpression of a dominant-negative TP53 (p53DD) construct resulted in enhanced basal ATM signaling, HR activity, and AZD1390-mediated radiosensitization in GBM14. Analyzing RNA-seq data from TCGA showed up-regulation of HR pathway genes in TP53-mutant human GBM. Together, our results imply that increased basal ATM signaling and enhanced dependence on HR represent a unique susceptibility of TP53-mutant cells to ATM inhibitor-mediated radiosensitization.

Preclinical Characterization of AZD9574, a Blood-Brain Barrier Penetrant Inhibitor of PARP1.

PURPOSE: We evaluated the properties and activity of AZD9574, a blood-brain barrier (BBB) penetrant selective inhibitor of PARP1, and assessed its efficacy and safety alone and in combination with temozolomide (TMZ) in preclinical models. EXPERIMENTAL DESIGN: AZD9574 was interrogated in vitro for selectivity, PARylation inhibition, PARP-DNA trapping, the ability to cross the BBB, and the potential to inhibit cancer cell proliferation. In vivo efficacy was determined using subcutaneous as well as intracranial mouse xenograft models. Mouse, rat, and monkey were used to assess AZD9574 BBB penetration and rat models were used to evaluate potential hematotoxicity for AZD9574 monotherapy and the TMZ combination. RESULTS: AZD9574 demonstrated PARP1-selectivity in fluorescence anisotropy, PARylation, and PARP-DNA trapping assays and in vivo experiments demonstrated BBB penetration. AZD9574 showed potent single agent efficacy in preclinical models with homologous recombination repair deficiency in vitro and in vivo. In an O6-methylguanine-DNA methyltransferase (MGMT)-methylated orthotopic glioma model, AZD9574 in combination with TMZ was superior in extending the survival of tumor-bearing mice compared with TMZ alone. CONCLUSIONS: The combination of three key features-PARP1 selectivity, PARP1 trapping profile, and high central nervous system penetration in a single molecule-supports the development of AZD9574 as the best-in-class PARP inhibitor for the treatment of primary and secondary brain tumors. As documented by in vitro and in vivo studies, AZD9574 shows robust anticancer efficacy as a single agent as well as in combination with TMZ. AZD9574 is currently in a phase I trial (NCT05417594). See related commentary by Lynce and Lin, p. 1217.

ATM-Inhibitor AZD1390 Is a Radiosensitizer for Breast Cancer CNS Metastasis.

PURPOSE: Limited effective treatments are currently available for central nervous system (CNS) metastasis (CM). This is largely driven by the inability of current therapeutics to penetrate the blood brain barrier (BBB) and the lack of preclinical models for testing new therapies. Here we study the efficacy of AZD1390, a BBB penetrating ataxia-telangiectasia mutated inhibitor, as a radiosensitizer for breast cancer CM treatment. EXPERIMENTAL DESIGN: Three patient-derived xenograft (PDX) tumors including 2 HER2+ and 1 triple-negative breast cancer harboring DNA damage response (DDR) gene mutations, were implanted subcutaneously in the flank of mice to assess tumor growth inhibition by AZD1390 combined with radiation. Animal survival was further assessed by implanting the best responding PDX model orthotopically in the brain. RESULTS: Pretreatment with AZD1390 followed by radiation therapy inhibited growth of PDX tumors implanted in the flank, and improved survival in orthotopic models with average survival of 222 days compared with 123 days in controls. Administration of AZD1390 posttreatment for 21 days had no further benefits. While the combination therapy resulted in sustained tumor inhibition, sporadic regrowth was observed in some mice 50 to 100 days posttreatment in all models. Gene expression comparing these tumors with complete responders demonstrated changes in upregulation of oncogenic proteins, which are potential drivers of tumor growth after treatment. CONCLUSIONS: Our results demonstrate that AZD1390 effectively sensitizes breast cancer CM to radiation therapy in DDR mutant tumors. This study demonstrates the potential of using AZD1390 as a novel therapeutic agent for patients with breast cancer CM.

Immunostaining protocol for infiltrating brain cancer spheroids for light-sheet imaging.

Glioblastoma tumors form in brains' white matter and are fast-growing and aggressive. Poor prognosis is the result of therapeutic resistance and infiltrating growth into the surrounding brain. Here we present a protocol for the detection of the cytoskeleton intermediate filament, vimentin, in cells at the proliferating spheroid surface. By combining a classical invasion assay with immunofluorescence and light-sheet imaging, we find that it is exactly these cytoskeleton-reinforcing cells on the spheroid's surface that will start the infiltration. We anticipate our results to be the starting point of more sophisticated investigation of anti-cancer drug effects on cytoskeleton reorganisation.

Non-metabolic functions of phosphofructokinase-1 orchestrate tumor cellular invasion and genome maintenance under bevacizumab therapy.

BACKGROUND: Glioblastoma (GBM) is a highly lethal malignancy for which neoangiogenesis serves as a defining hallmark. The anti-VEGF antibody, bevacizumab, has been approved for the treatment of recurrent GBM, but resistance is universal. METHODS: We analyzed expression data of GBM patients treated with bevacizumab to discover potential resistance mechanisms. Patient-derived xenografts (PDXs) and cultures were interrogated for effects of phosphofructokinase-1, muscle isoform (PFKM) loss on tumor cell motility, migration, and invasion through genetic and pharmacologic targeting. RESULTS: We identified PFKM as a driver of bevacizumab resistance. PFKM functions dichotomize based on subcellular location: cytosolic PFKM interacted with KIF11, a tubular motor protein, to promote tumor invasion, whereas nuclear PFKM safeguarded genomic stability of tumor cells through interaction with NBS1. Leveraging differential transcriptional profiling, bupivacaine phenocopied genetic targeting of PFKM, and enhanced efficacy of bevacizumab in preclinical GBM models in vivo. CONCLUSION: PFKM drives novel molecular pathways in GBM, offering a translational path to a novel therapeutic paradigm.

Therapeutic Opportunities of Disrupting Genome Integrity in Adult Diffuse Glioma.

Adult diffuse glioma, particularly glioblastoma (GBM), is a devastating tumor of the central nervous system. The existential threat of this disease requires on-going treatment to counteract tumor progression. The present outcome is discouraging as most patients will succumb to this disease. The low cure rate is consistent with the failure of first-line therapy, radiation and temozolomide (TMZ). Even with their therapeutic mechanism of action to incur lethal DNA lesions, tumor growth remains undeterred. Delivering additional treatments only delays the inescapable development of therapeutic tolerance and disease recurrence. The urgency of establishing lifelong tumor control needs to be re-examined with a greater focus on eliminating resistance. Early genomic and transcriptome studies suggest each tumor subtype possesses a unique molecular network to safeguard genome integrity. Subsequent seminal work on post-therapy tumor progression sheds light on the involvement of DNA repair as the causative contributor for hypermutation and therapeutic failure. In this review, we will provide an overview of known molecular factors that influence the engagement of different DNA repair pathways, including targetable vulnerabilities, which can be exploited for clinical benefit with the use of specific inhibitors.

Tumor mutational burden and purity adjustment before and after treatment with temozolomide in 27 paired samples of glioblastoma: a prospective study.

Treatment of glioblastoma (GBM) remains a challenging task, with limited treatment options, none offering a cure. Immune therapy has proven effective across different cancers with remarkable response rates. Tumor mutational burden (TMB) is a marker of response, but technical and methodological differences in TMB estimates have made a proper assessment and comparison challenging. Here, we analyzed a prospective collection of paired samples from 35 patients with newly diagnosed GBM, all of whom were wild-type (WT) for isocitrate dehydrogenase, before and after treatment with radiotherapy and temozolomide. Seven patients (20%) had O6-methylguanine-DNA methyltransferase-methylated tumors. Six patients (17%) had two relapse surgeries, and tissue from all three surgeries was collected. We found that accurate evaluation of TMB was confounded by high variability in the cancer cell fraction of relapse samples. To ameliorate this, we developed a model to adjust for tumor purity based on the relative density distribution of variant allele frequencies in each primary-relapse pair. Additionally, we examined the mutation spectra of shared and private mutations. After tumor purity adjustment, we found TMB comparison reliable in tumors with tumor purity between 15% and 40%, resulting in 27/35 patients (77.1%). TMB remained unchanged from 0.65 mutations per megabase (Mb) to 0.67/Mb before and after treatment, respectively. Examination of the mutation spectra revealed a dominance of C > T transitions at CpG sites in both shared and relapse-private mutations, consistent with cytosine deamination and the clock-like mutational signature 1. We present and apply a cellularity correction approach that enables more accurate assessment of TMB in paired tumor samples. We did not find a significant increase in TMB after correcting for cancer cell fraction. Our study raises significant concerns when determining TMB. Although a small sample size, corrected TMB can have a clinical significance when stratifying patients to experimental treatment, for example, immune checkpoint therapy.

Cell medium-dependent dynamic modulation of size and structural transformations of binary phospholipid/ω-3 fatty acid liquid crystalline nano-self-assemblies: Implications in interpretation of cell uptake studies.

Lyotropic non-lamellar liquid crystalline (LLC) nanoparticles, with their tunable structural features and capability of loading a wide range of drugs and reporter probes, are emerging as versatile injectable nanopharmaceuticals. Secondary emulsifiers, such as Pluronic block copolymers, are commonly used for colloidal stabilization of LLC nanoparticles, but their inclusion often compromises the biological safety (e.g., poor hemocompatibility and enhanced cytotoxicity) of the formulation. Here, we introduce a library of colloidally stable, structurally tunable, and pH-responsive lamellar and non-lamellar liquid crystalline nanoparticles from binary mixtures of a phospholipid (phosphatidylglycerol) and three types of omega-3 fatty acids (ω-3 PUFAs), prepared in the absence of a secondary emulsifier and organic solvents. We study formulation size distribution, morphological heterogeneity, and the arrangement of their internal self-assembled architectures by nanoparticle tracking analysis, synchrotron small-angle X-ray scattering, and cryo-transmission electron microscopy. The results show the influence of type and concentration of ω-3 PUFAs in nanoparticle structural transitions spanning from a lamellar (Lα) phase to inverse discontinuous (micellar) cubic Fd3m and hexagonal phase (H2) phases, respectively. We further report on cell-culture medium-dependent dynamic fluctuations in nanoparticle size, number and morphology, and simultaneously monitor uptake kinetics in two human cell lines. We discuss the role of these multiparametric biophysical transformations on nanoparticle-cell interaction kinetics and internalization mechanisms. Collectively, our findings contribute to the understanding of fundamental steps that are imperative for improved engineering of LLC nanoparticles with necessary attributes for pharmaceutical development.

Targeting EYA2 tyrosine phosphatase activity in glioblastoma stem cells induces mitotic catastrophe.

Glioblastoma ranks among the most lethal of primary brain malignancies, with glioblastoma stem cells (GSCs) at the apex of tumor cellular hierarchies. Here, to discover novel therapeutic GSC targets, we interrogated gene expression profiles from GSCs, differentiated glioblastoma cells (DGCs), and neural stem cells (NSCs), revealing EYA2 as preferentially expressed by GSCs. Targeting EYA2 impaired GSC maintenance and induced cell cycle arrest, apoptosis, and loss of self-renewal. EYA2 displayed novel localization to centrosomes in GSCs, and EYA2 tyrosine (Tyr) phosphatase activity was essential for proper mitotic spindle assembly and survival of GSCs. Inhibition of the EYA2 Tyr phosphatase activity, via genetic or pharmacological means, mimicked EYA2 loss in GSCs in vitro and extended the survival of tumor-bearing mice. Supporting the clinical relevance of these findings, EYA2 portends poor patient prognosis in glioblastoma. Collectively, our data indicate that EYA2 phosphatase function plays selective critical roles in the growth and survival of GSCs, potentially offering a high therapeutic index for EYA2 inhibitors.

Optical tissue clearing and machine learning can precisely characterize extravasation and blood vessel architecture in brain tumors.

Precise methods for quantifying drug accumulation in brain tissue are currently very limited, challenging the development of new therapeutics for brain disorders. Transcardial perfusion is instrumental for removing the intravascular fraction of an injected compound, thereby allowing for ex vivo assessment of extravasation into the brain. However, pathological remodeling of tissue microenvironment can affect the efficiency of transcardial perfusion, which has been largely overlooked. We show that, in contrast to healthy vasculature, transcardial perfusion cannot remove an injected compound from the tumor vasculature to a sufficient extent leading to considerable overestimation of compound extravasation. We demonstrate that 3D deep imaging of optically cleared tumor samples overcomes this limitation. We developed two machine learning-based semi-automated image analysis workflows, which provide detailed quantitative characterization of compound extravasation patterns as well as tumor angioarchitecture in large three-dimensional datasets from optically cleared samples. This methodology provides a precise and comprehensive analysis of extravasation in brain tumors and allows for correlation of extravasation patterns with specific features of the heterogeneous brain tumor vasculature.

Plasma IL-8 and ICOSLG as prognostic biomarkers in glioblastoma.

BACKGROUND: CNS immune privilege has been challenged in recent years. Glioblastoma (GBM) immune dysfunction includes complex interactions with the immune system outside the CNS. The aim of this study was to determine diagnostic and prognostic potential of immune-related proteins in plasma in GBM and interrogate biomarker presence in the brain tumor microenvironment (TME). METHODS: One hundred and fifty-eight patients with glioma WHO grade II-IV were included. Plasma collected at surgery was screened for 92 proteins using proximity extension assay technology and related to clinical outcome. Secretion and expression of candidate prognostic biomarkers were subsequently analyzed in 8 GBM cell lines and public RNAseq data. RESULTS: Plasma levels of 20 out of 92 screened proteins were significantly different in patients with GBM compared to patients with astrocytoma WHO grade II-III. High plasma interleukin-8 (IL-8) (hazard ratio [HR] = 1.52; P = .0077) and low CD244 (HR = 0.36; P = .0004) were associated with short progression-free survival and high plasma IL-8 (HR = 1.40; P = .044) and low ICOS ligand (ICOSLG) (HR = 0.17; P = .0003) were associated with short overall survival (OS) in newly diagnosed patients with GBM. A similar trend was found for ICOSLG (HR = 0.34; P = .053) in recurrent GBM. IL-8 was mostly secreted and expressed by mesenchymal GBM cell lines and expressed by vascular cells and immune cells in the TME. This was also the case for ICOSLG, although less consistent, and with additional expression in tumor-associated oligodendrocytes. CONCLUSIONS: High plasma IL-8 and low ICOSLG at surgery are associated with short OS in newly diagnosed GBM. Source of plasma ICOSLG may be found outside the TME.

AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity.

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.

Perspective: targeting VEGF-A and YKL-40 in glioblastoma - matter matters.

Glioblastomas (GBM) are heterogeneous highly vascular brain tumors exploiting the unique microenvironment in the brain to resist treatment and anti-tumor responses. Anti-angiogenic agents, immunotherapy, and targeted therapy have been studied extensively in GBM patients over a number of decades with minimal success. Despite maximal efforts, prognosis remains dismal with an overall survival of approximately 15 months.Bevacizumab, a humanized anti-vascular endothelial growth factor (VEGF) antibody, underwent accelerated approval by the U.S. Food and Drug Administration in 2009 for the treatment of recurrent GBM based on promising preclinical and early clinical studies. Unfortunately, subsequent clinical trials did not find overall survival benefit. Pursuing pleiotropic targets and leaning toward multitarget strategies may be a key to more effective therapeutic intervention in GBM, but preclinical evaluation requires careful consideration of model choices. In this study, we discuss bevacizumab resistance, dual targeting of pro-angiogenic modulators VEGF and YKL-40 in the context of brain tumor microenvironment, and how model choice impacts study conclusions and its translational significance.

A High-Affinity Peptide Ligand Targeting Syntenin Inhibits Glioblastoma.

Despite the recent advances in cancer therapeutics, highly aggressive cancer forms, such as glioblastoma (GBM), still have very low survival rates. The intracellular scaffold protein syntenin, comprising two postsynaptic density protein-95/discs-large/zona occludens-1 (PDZ) domains, has emerged as a novel therapeutic target in highly malignant phenotypes including GBM. Here, we report the development of a novel, highly potent, and metabolically stable peptide inhibitor of syntenin, KSL-128114, which binds the PDZ1 domain of syntenin with nanomolar affinity. KSL-128114 is resistant toward degradation in human plasma and mouse hepatic microsomes and displays a global PDZ domain selectivity for syntenin. An X-ray crystal structure reveals that KSL-128114 interacts with syntenin PDZ1 in an extended noncanonical binding mode. Treatment with KSL-128114 shows an inhibitory effect on primary GBM cell viability and significantly extends survival time in a patient-derived xenograft mouse model. Thus, KSL-128114 is a novel promising candidate with therapeutic potential for highly aggressive tumors, such as GBM.

The RNA m6A Reader YTHDF2 Maintains Oncogene Expression and Is a Targetable Dependency in Glioblastoma Stem Cells.

Glioblastoma is a universally lethal cancer driven by glioblastoma stem cells (GSC). Here, we interrogated N 6-methyladenosine (m6A) mRNA modifications in GSCs by methyl RNA immunoprecipitation followed by sequencing and transcriptome analysis, finding transcripts marked by m6A often upregulated compared with normal neural stem cells (NSC). Interrogating m6A regulators, GSCs displayed preferential expression, as well as in vitro and in vivo dependency, of the m6A reader YTHDF2, in contrast to NSCs. Although YTHDF2 has been reported to destabilize mRNAs, YTHDF2 stabilized MYC and VEGFA transcripts in GSCs in an m6A-dependent manner. We identified IGFBP3 as a downstream effector of the YTHDF2-MYC axis in GSCs. The IGF1/IGF1R inhibitor linsitinib preferentially targeted YTHDF2-expressing cells, inhibiting GSC viability without affecting NSCs and impairing in vivo glioblastoma growth. Thus, YTHDF2 links RNA epitranscriptomic modifications and GSC growth, laying the foundation for the YTHDF2-MYC-IGFBP3 axis as a specific and novel therapeutic target in glioblastoma. SIGNIFICANCE: Epitranscriptomics promotes cellular heterogeneity in cancer. RNA m6A landscapes of cancer and NSCs identified cell type-specific dependencies and therapeutic vulnerabilities. The m6A reader YTHDF2 stabilized MYC mRNA specifically in cancer stem cells. Given the challenge of targeting MYC, YTHDF2 presents a therapeutic target to perturb MYC signaling in glioblastoma.This article is highlighted in the In This Issue feature, p. 211.

Radio-Resistance and DNA Repair in Pediatric Diffuse Midline Gliomas.

Malignant gliomas (MG) are among the most prevalent and lethal primary intrinsic brain tumors. Although radiotherapy (RT) is the most effective nonsurgical therapy, recurrence is universal. Dysregulated DNA damage response pathway (DDR) signaling, rampant genomic instability, and radio-resistance are among the hallmarks of MGs, with current therapies only offering palliation. A subgroup of pediatric high-grade gliomas (pHGG) is characterized by H3K27M mutation, which drives global loss of di- and trimethylation of histone H3K27. Here, we review the most recent literature and discuss the key studies dissecting the molecular biology of H3K27M-mutated gliomas in children. We speculate that the aberrant activation and/or deactivation of some of the key components of DDR may be synthetically lethal to H3K27M mutation and thus can open novel avenues for effective therapeutic interventions for patients suffering from this deadly disease.

Genomic profiling of newly diagnosed glioblastoma patients and its potential for clinical utility - a prospective, translational study.

Glioblastoma (GBM) is an incurable brain tumor for which new treatment strategies are urgently needed. Next-generation sequencing of GBM has most often been performed retrospectively and on archival tissue from both diagnostic and relapse surgeries with limited knowledge of clinical information, including treatment given. We sought to investigate the genomic composition prospectively in treatment-naïve patients, searched for possible targetable aberrations, and investigated for prognostic and/or predictive factors. A total of 108 newly diagnosed GBM patients were included. Clinical information, progression-free survival, and overall survival (OS) were noted. Tissues were analyzed by whole-exome sequencing, single nucleotide polymorphism (SNP) and transcriptome arrays, and RNA sequencing; assessed for mutations, fusions, tumor mutational burden (TMB), and chromosomal instability (CI); and classified into GBM subgroups. Each genomic report was discussed at a multidisciplinary tumor board meeting to evaluate for matching trials. From 111 consecutive patients, 97.3% accepted inclusion in this study. Eighty-six (77%) were treated with radiation therapy/temozolomide (TMZ) and adjuvant TMZ. One NTRK2 and three FGFR3-TACC3 fusions were identified. Copy number alterations in GRB2 and SMYD4 were significantly correlated with worse median OS together with known clinical variables like age, performance status, steroid dose, and O6-methyl-guanine-DNA-methyl-transferase status. Patients with CI-median or TMB-high had significantly worse median OS compared to CI-low/high or TMB-low/median. In conclusion, performing genomic profiling at diagnosis enables evaluation of genomic-driven therapy at the first progression. Furthermore, TMB-high or CI-median patients had worse median OS, which can support the possibility of offering experimental treatment already at the first line for this group.