A Phase 1/2 Study of Disulfiram and Copper With Concurrent Radiation Therapy and Temozolomide for Patients With Newly Diagnosed Glioblastoma.

PURPOSE: This phase 1/2 study aimed to evaluate the safety and preliminary efficacy of combining disulfiram and copper (DSF/Cu) with radiation therapy (RT) and temozolomide (TMZ) in patients with newly diagnosed glioblastoma (GBM). METHODS AND MATERIALS: Patients received standard RT and TMZ with DSF (250-375 mg/d) and Cu, followed by adjuvant TMZ plus DSF (500 mg/d) and Cu. Pharmacokinetic analyses determined drug concentrations in plasma and tumors using high-performance liquid chromatography-mass spectrometry. RESULTS: Thirty-three patients, with a median follow-up of 26.0 months, were treated, including 12 IDH-mutant, 9 NF1-mutant, 3 BRAF-mutant, and 9 other IDH-wild-type cases. In the phase 1 arm, 18 patients were treated; dose-limiting toxicity probabilities were 10% (95% CI, 3%-29%) at 250 mg/d and 21% (95% CI, 7%-42%) at 375 mg/d. The phase 2 arm treated 15 additional patients at 250 mg/d. No significant difference in overall survival or progression-free survival was noted between IDH- and NF1-mutant cohorts compared with institutional counterparts treated without DSF/Cu. However, extended remission occurred in 3 BRAF-mutant patients. Diethyl-dithiocarbamate-copper, the proposed active metabolite of DSF/Cu, was detected in plasma but not in tumors. CONCLUSIONS: The maximum tolerated dose of DSF with RT and TMZ is 375 mg/d. DSF/Cu showed limited clinical efficacy for most patients. However, promising efficacy was observed in BRAF-mutant GBM, warranting further investigation.

Multi-Omic Analysis of CIC's Functional Networks Reveals Novel Interaction Partners and a Potential Role in Mitotic Fidelity.

CIC encodes a transcriptional repressor and MAPK signalling effector that is inactivated by loss-of-function mutations in several cancer types, consistent with a role as a tumour suppressor. Here, we used bioinformatic, genomic, and proteomic approaches to investigate CIC's interaction networks. We observed both previously identified and novel candidate interactions between CIC and SWI/SNF complex members, as well as novel interactions between CIC and cell cycle regulators and RNA processing factors. We found that CIC loss is associated with an increased frequency of mitotic defects in human cell lines and an in vivo mouse model and with dysregulated expression of mitotic regulators. We also observed aberrant splicing in CIC-deficient cell lines, predominantly at 3' and 5' untranslated regions of genes, including genes involved in MAPK signalling, DNA repair, and cell cycle regulation. Our study thus characterises the complexity of CIC's functional network and describes the effect of its loss on cell cycle regulation, mitotic integrity, and transcriptional splicing, thereby expanding our understanding of CIC's potential roles in cancer. In addition, our work exemplifies how multi-omic, network-based analyses can be used to uncover novel insights into the interconnected functions of pleiotropic genes/proteins across cellular contexts.

Joint Final Report of EORTC 26951 and RTOG 9402: Phase III Trials With Procarbazine, Lomustine, and Vincristine Chemotherapy for Anaplastic Oligodendroglial Tumors.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the basis of the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.Anaplastic oligodendroglial tumors (AOTs) are chemotherapy-sensitive brain tumors. We report the final very long-term survival results from European Organization for the Research and Treatment of Cancer 26951 and Radiation Therapy Oncology Group 9402 phase III trials initiated in 1990s, which both studied radiotherapy with/without neo/adjuvant procarbazine, lomustine, and vincristine (PCV) for newly diagnosed anaplastic oligodendroglial tumors. The median follow-up duration in both was 18-19 years. For European Organization for the Research and Treatment of Cancer 26951, median, 14-year, and probable 20-year overall survival rates without versus with PCV were 2.6 years, 13.4%, and 10.1% versus 3.5 years, 25.1%, and 16.8% (N = 368 overall; hazard ratio [HR] 0.78; 95% CI, 0.63 to 0.98; P = .033), with 1p19q codeletion 9.3 years, 26.2%, and 13.6% versus 14.2 years, 51.0%, and 37.1% (n = 80; HR 0.60; 95% CI, 0.35 to 1.03; P = .063), respectively. For Radiation Therapy Oncology Group 9402, analogous results were 4.8 years, 16.5%, and 11.2% versus 4.8 years, 29.1%, and 24.6% (N = 289 overall; HR 0.79; 95% CI, 0.61 to 1.03; P = .08), with codeletion 7.3 years, 25.0%, and 14.9% versus 13.2 years, 46.1%, and 37% (n = 125; HR 0.61; 95% CI, 0.40 to 0.94; P = .02), respectively. With that, the studies show similar long-term survival even without tumor recurrence in a significant proportion of patients after first-line treatment with radiotherapy/PCV.

Single-cell landscapes of primary glioblastomas and matched explants and cell lines show variable retention of inter- and intratumor heterogeneity.

Glioblastomas (GBMs) are aggressive brain tumors characterized by extensive inter- and intratumor heterogeneity. Patient-derived models, such as organoids and explants, have recently emerged as useful models to study such heterogeneity, although the extent to which they can recapitulate GBM genomic features remains unclear. Here, we analyze bulk exome and single-cell genome and transcriptome profiles of 12 IDH wild-type GBMs, including two recurrent tumors, and of patient-derived explants (PDEs) and gliomasphere (GS) lines derived from these tumors. We find that PDEs are genetically similar to, and variably retain gene expression characteristics of, their parent tumors. Notably, PDEs appear to exhibit similar levels of transcriptional heterogeneity compared with their parent tumors, whereas GS lines tend to be enriched for cells in a more uniform transcriptional state. The approaches and datasets introduced here will provide a valuable resource to help guide experiments using GBM-derived models, especially in the context of studying cellular heterogeneity.

Corrigendum to: Impact of lymphopenia on survival for elderly patients with glioblastoma: A secondary analysis of the CCTG CE.6 (EORTC 26062-22061, TROG 08.02) randomized clinical trial.

[This corrects the article DOI: 10.1093/noajnl/vdab153.].

Impact of lymphopenia on survival for elderly patients with glioblastoma: A secondary analysis of the CCTG CE.6 (EORTC 26062-22061, TROG03.01) randomized clinical trial.

BACKGROUND: Lymphopenia may lead to worse outcomes for glioblastoma patients. This study is a secondary analysis of the CCTG CE.6 trial evaluating the impact of chemotherapy and radiation on lymphopenia, and effects of lymphopenia on overall survival (OS). METHODS: CCTG CE.6 randomized elderly glioblastoma patients (≥ 65 years) to short-course radiation alone (RT) or short-course radiation with temozolomide (RT + TMZ). Lymphopenia (mild-moderate: grade 1-2; severe: grade 3-4) was defined per CTCAE v3.0, and measured at baseline, 1 week and 4 weeks post-RT. Preselected key factors for analysis included age, sex, ECOG, resection extent, MGMT methylation, Mini-Mental State Examination, and steroid use. Multinomial logistic regression and multivariable Cox regression models were used to identify lymphopenia-associated factors and association with survival. RESULTS: Five hundred and sixty-two patients were analyzed (281 RT vs 281 RT+TMZ). At baseline, both arms had similar rates of mild-moderate (21.4% vs 21.4%) and severe (3.2% vs 2.9%) lymphopenia. However, at 4 weeks post-RT, RT+TMZ was more likely to develop lymphopenia (mild-moderate: 27.9% vs 18.2%; severe: 9.3% vs 1.8%; p<0.001). Developing any lymphopenia post-RT was associated with baseline lymphopenia (P < .001). Baseline lymphopenia (hazard ratio [HR] 1.3) was associated with worse OS (HR: 1.30, 95% confidence interval [CI] 1.05-1.62; P = .02), regardless of MGMT status. CONCLUSIONS: Development of post-RT lymphopenia is associated with addition of TMZ and baseline lymphopenia and not with RT alone in patients treated with short-course radiation. However, regardless of MGMT status, only baseline lymphopenia is associated with worse OS, which may be considered as a prognostic biomarker for elderly glioblastoma patients.

Copy-scAT: Deconvoluting single-cell chromatin accessibility of genetic subclones in cancer.

Single-cell epigenomic assays have tremendous potential to illuminate mechanisms of transcriptional control in functionally diverse cancer cell populations. However, application of these techniques to clinical tumor specimens has been hampered by the current inability to distinguish malignant from nonmalignant cells, which potently confounds data analysis and interpretation. Here, we describe Copy-scAT, an R package that uses single-cell epigenomic data to infer copy number variants (CNVs) that define cancer cells. Copy-scAT enables studies of subclonal chromatin dynamics in complex tumors like glioblastoma. By deploying Copy-scAT, we uncovered potent influences of genetics on chromatin accessibility profiles in individual subclones. Consequently, some genetic subclones were predisposed to acquire stem-like or more differentiated molecular phenotypes, reminiscent of developmental paradigms. Copy-scAT is ideal for studies of the relationships between genetics and epigenetics in malignancies with high levels of intratumoral heterogeneity and to investigate how cancer cells interface with their microenvironment.

CODEL: phase III study of RT, RT + TMZ, or TMZ for newly diagnosed 1p/19q codeleted oligodendroglioma. Analysis from the initial study design.

BACKGROUND: We report the analysis involving patients treated on the initial CODEL design. METHODS: Adults (>18) with newly diagnosed 1p/19q World Health Organization (WHO) grade III oligodendroglioma were randomized to radiotherapy (RT; 5940 centigray ) alone (arm A); RT with concomitant and adjuvant temozolomide (TMZ) (arm B); or TMZ alone (arm C). Primary endpoint was overall survival (OS), arm A versus B. Secondary comparisons were performed for OS and progression-free survival (PFS), comparing pooled RT arms versus TMZ-alone arm. RESULTS: Thirty-six patients were randomized equally. At median follow-up of 7.5 years, 83.3% (10/12) TMZ-alone patients progressed, versus 37.5% (9/24) on the RT arms. PFS was significantly shorter in TMZ-alone patients compared with RT patients (hazard ratio [HR] = 3.12; 95% CI: 1.26, 7.69; P = 0.014). Death from disease progression occurred in 3/12 (25%) of TMZ-alone patients and 4/24 (16.7%) on the RT arms. OS did not statistically differ between arms (comparison underpowered). After adjustment for isocitrate dehydrogenase (IDH) status (mutated/wildtype) in a Cox regression model utilizing IDH and RT treatment status as covariables (arm C vs pooled arms A + B), PFS remained shorter for patients not receiving RT (HR = 3.33; 95% CI: 1.31, 8.45; P = 0.011), but not OS ((HR = 2.78; 95% CI: 0.58, 13.22, P = 0.20). Grade 3+ adverse events occurred in 25%, 42%, and 33% of patients (arms A, B, and C). There were no differences between arms in neurocognitive decline comparing baseline to 3 months. CONCLUSIONS: TMZ-alone patients experienced significantly shorter PFS than patients treated on the RT arms. The ongoing CODEL trial has been redesigned to compare RT + PCV versus RT + TMZ.

Proceedings of the Comprehensive Oncology Network Evaluating Rare CNS Tumors (NCI-CONNECT) Oligodendroglioma Workshop.

BACKGROUND: Oligodendroglioma is a rare primary central nervous system (CNS) tumor with highly variable outcome and for which therapy is usually not curative. At present, little is known regarding the pathways involved with progression of oligodendrogliomas or optimal biomarkers for stratifying risk. Developing new therapies for this rare cancer is especially challenging. To overcome these challenges, the neuro-oncology community must be particularly innovative, seeking multi-institutional and international collaborations, and establishing partnerships with patients and advocacy groups thereby ensuring that each patient enrolled in a study is as informative as possible. METHODS: The mission of the National Cancer Institute's NCI-CONNECT program is to address the challenges and unmet needs in rare CNS cancer research and treatment by connecting patients, health care providers, researchers, and advocacy organizations to work in partnership. On November 19, 2018, the program convened a workshop on oligodendroglioma, one of the 12 rare CNS cancers included in its initial portfolio. The purpose of this workshop was to discuss scientific progress and regulatory challenges in oligodendroglioma research and develop a call to action to advance research and treatment for this cancer. RESULTS: The recommendations of the workshop include a multifaceted and interrelated approach covering: biology and preclinical models, data sharing and advanced molecular diagnosis and imaging; clinical trial design; and patient outreach and engagement. CONCLUSIONS: The NCI-CONNECT program is well positioned to address challenges in oligodendroglioma care and research in collaboration with other stakeholders and is developing a list of action items for future initiatives.

Glioma-derived IL-33 orchestrates an inflammatory brain tumor microenvironment that accelerates glioma progression.

Despite a deeper molecular understanding, human glioblastoma remains one of the most treatment refractory and fatal cancers. It is known that the presence of macrophages and microglia impact glioblastoma tumorigenesis and prevent durable response. Herein we identify the dual function cytokine IL-33 as an orchestrator of the glioblastoma microenvironment that contributes to tumorigenesis. We find that IL-33 expression in a large subset of human glioma specimens and murine models correlates with increased tumor-associated macrophages/monocytes/microglia. In addition, nuclear and secreted functions of IL-33 regulate chemokines that collectively recruit and activate circulating and resident innate immune cells creating a pro-tumorigenic environment. Conversely, loss of nuclear IL-33 cripples recruitment, dramatically suppresses glioma growth, and increases survival. Our data supports the paradigm that recruitment and activation of immune cells, when instructed appropriately, offer a therapeutic strategy that switches the focus from the cancer cell alone to one that includes the normal host environment.

Temozolomide and seizure outcomes in a randomized clinical trial of elderly glioblastoma patients.

INTRODUCTION: Tumor-related epilepsy may respond to chemotherapy. In a previously-published multi-centre randomized clinical trial of 562 elderly glioblastoma patients, temozolomide plus short-course radiotherapy conferred a survival benefit over radiotherapy alone. Seizure outcomes were not reported. METHODS: We performed an unplanned secondary analysis of this trial's data. The trial design has been previously reported. Seizures were recorded by clinicians as adverse events and by patients in quality of life questionnaires. A Chi-square test of seizure rates between the two groups (α = 0.05) and a Kaplan-Meier estimator of time-to-first self-reported seizure were planned. RESULTS: Almost all patients were followed until they died. In the radiotherapy alone group, 68 patients (24%) had a documented or self-reported seizure versus 83 patients (30%) in the temozolomide plus radiotherapy group, Chi-square analysis showed no difference (p = 0.15). Patients receiving radiotherapy alone tended to develop seizures earlier than those receiving temozolomide plus radiotherapy (p = 0.054). Patients with seizures had shorter overall survival than those without seizures (hazard ratio 1.24, p = 0.02). CONCLUSIONS: This study was not powered to detect differences in seizure outcomes, but temozolomide seemed to have minimal impact on seizure control in elderly patients with glioblastoma. CLINICAL TRIAL REGISTRATION: NCT00482677 2007-06-05.

PARP inhibition suppresses the emergence of temozolomide resistance in a model system.

INTRODUCTION: Temozolomide (TMZ) is a life prolonging DNA alkylating agent active against glioblastomas (GBM) in which the O6-methylguanine-DNA methyltransferase (MGMT) gene is silenced by promoter methylation. Unfortunately acquired TMZ resistance severely undermines its clinical efficacy. Using an in vitro model, we tested whether poly (ADP-ribose) polymerase-1 and -2 (PARP) inhibition could suppress the emergence of resistance to enhance the effectiveness of TMZ. METHODS: Using the MGMT-methylated GBM line U251N, in which TMZ resistance can be induced, we developed a method to rapidly recreate mechanisms of TMZ resistance seen in GBMs, including MMR mutations and MGMT re-expression. We then assessed whether TMZ resistant U251N sub-clones could be re-sensitized to TMZ by co-treatment with the PARP inhibitor ABT-888, and also whether the emergence of resistance could be suppressed by PARP inhibition. RESULTS: U251N cultures chronically exposed to TMZ developed discrete colonies that expanded during TMZ treatment. These colonies were isolated, expanded further as sub-clones, and assessed for mechanisms of TMZ resistance. Most resistant sub-clones had detectable mutations in one or more mismatch repair (MMR) genes, frequently MSH6, and displayed infrequent re-expression of MGMT. TMZ resistance was associated with isolated poly(ADP-ribose) (pADPr) up-regulation in one sub-clone and was unexplained in several others. TMZ resistant sub-clones regressed during co-treatment with TMZ and ABT-888, and early co-treatment of U251N parental cultures suppressed the emergence of TMZ resistant colonies. CONCLUSION: In a model of acquired resistance, co-treatment with TMZ and a PARP inhibitor had two important benefits: re-sensitization of TMZ resistant cells and suppression of TMZ resistance.

Development of a peptide-based delivery platform for targeting malignant brain tumors.

Despite extensive molecular characterization, human glioblastoma remains a fatal disease with survival rates measured in months. Little improvement is seen with standard surgery, radiotherapy and chemotherapy. Clinical progress is hampered by the inability to detect and target glioblastoma disease reservoirs based on a diffuse invasive pattern and the presence of molecular and phenotypic heterogeneity. The goal of this study was to target the invasive and stem-like glioblastoma cells that evade first-line treatments using agents capable of delivering imaging enhancers or biotherapeutic cargo. To accomplish this, a combinatorial phage display library was biopanned against glioblastoma cell model systems that accurately recapitulate the intra- and inter-tumor heterogeneity and infiltrative nature of the disease. Candidate peptides were screened for specificity and ability to target glioblastoma cells in vivo. Cargo-conjugated peptides delivered contrast-enhancing agents to highly infiltrative tumor populations in intracranial xenograft models without the obvious need for blood brain barrier disruption. Simultaneous use of five independent targeting peptides provided greater coverage of this complex tumor and selected peptides have the capacity to deliver a therapeutic cargo (oncolytic virus VSVΔM51) to the tumor cells in vivo. Herein, we have identified a series of peptides with utility as an innovative platform to assist in targeting glioblastoma for the purpose of diagnostic or prognostic imaging, image-guided surgery, and/or improved delivery of therapeutic agents to glioblastoma cells implicated in disease relapse.

Comprehensive genomic profiling of glioblastoma tumors, BTICs, and xenografts reveals stability and adaptation to growth environments.

Glioblastoma multiforme (GBM) is the most deadly brain tumor, and currently lacks effective treatment options. Brain tumor-initiating cells (BTICs) and orthotopic xenografts are widely used in investigating GBM biology and new therapies for this aggressive disease. However, the genomic characteristics and molecular resemblance of these models to GBM tumors remain undetermined. We used massively parallel sequencing technology to decode the genomes and transcriptomes of BTICs and xenografts and their matched tumors in order to delineate the potential impacts of the distinct growth environments. Using data generated from whole-genome sequencing of 201 samples and RNA sequencing of 118 samples, we show that BTICs and xenografts resemble their parental tumor at the genomic level but differ at the mRNA expression and epigenomic levels, likely due to the different growth environment for each sample type. These findings suggest that a comprehensive genomic understanding of in vitro and in vivo GBM model systems is crucial for interpreting data from drug screens, and can help control for biases introduced by cell-culture conditions and the microenvironment in mouse models. We also found that lack of MGMT expression in pretreated GBM is linked to hypermutation, which in turn contributes to increased genomic heterogeneity and requires new strategies for GBM treatment.

Capicua regulates neural stem cell proliferation and lineage specification through control of Ets factors.

Capicua (Cic) is a transcriptional repressor mutated in the brain cancer oligodendroglioma. Despite its cancer link, little is known of Cic's function in the brain. We show that nuclear Cic expression is strongest in astrocytes and neurons but weaker in stem cells and oligodendroglial lineage cells. Using a new conditional Cic knockout mouse, we demonstrate that forebrain-specific Cic deletion increases proliferation and self-renewal of neural stem cells. Furthermore, Cic loss biases neural stem cells toward glial lineage selection, expanding the pool of oligodendrocyte precursor cells (OPCs). These proliferation and lineage effects are dependent on de-repression of Ets transcription factors. In patient-derived oligodendroglioma cells, CIC re-expression or ETV5 blockade decreases lineage bias, proliferation, self-renewal, and tumorigenicity. Our results identify Cic as an important regulator of cell fate in neurodevelopment and oligodendroglioma, and suggest that its loss contributes to oligodendroglioma by promoting proliferation and an OPC-like identity via Ets overactivity.

ABT-888 restores sensitivity in temozolomide resistant glioma cells and xenografts.

BACKGROUND: Temozolomide (TMZ) is active against glioblastomas (GBM) in which the O6-methylguanine-DNA methyltransferase (MGMT) gene is silenced. However, even in responsive cases, its beneficial effect is undermined by the emergence of drug resistance. Here, we tested whether inhibition of poly (ADP-ribose) polymerase-1 and -2 (PARP) enhanced the effectiveness of TMZ. METHODS: Using patient derived brain tumor initiating cells (BTICs) and orthotopic xenografts as models of newly diagnosed and recurrent high-grade glioma, we assessed the effects of TMZ, ABT-888, and the combination of TMZ and ABT-888 on the viability of BTICs and survival of tumor-bearing mice. We also studied DNA damage repair, checkpoint protein phosphorylation, and DNA replication in mismatch repair (MMR) deficient cells treated with TMZ and TMZ plus ABT-888. RESULTS: Cells and xenografts derived from newly diagnosed MGMT methylated high-grade gliomas were sensitive to TMZ while those derived from unmethylated and recurrent gliomas were typically resistant. ABT-888 had no effect on the viability of BTICs or tumor bearing mice, but co-treatment with TMZ restored sensitivity in resistant cells and xenografts from newly diagnosed unmethylated gliomas and recurrent gliomas with MSH6 mutations. In contrast, the addition of ABT-888 to TMZ had little sensitizing effect on cells and xenografts derived from newly diagnosed methylated gliomas. In a model of acquired TMZ resistance mediated by loss of MMR gene MSH6, re-sensitization to TMZ by ABT-888 was accompanied by persistent DNA strand breaks, re-engagement of checkpoint kinase signaling, and interruption of DNA synthesis. CONCLUSION: In laboratory models, the addition of ABT-888 to TMZ overcame resistance to TMZ.

2-Hydroxyglutarate-Mediated Autophagy of the Endoplasmic Reticulum Leads to an Unusual Downregulation of Phospholipid Biosynthesis in Mutant IDH1 Gliomas.

Tumor metabolism is reprogrammed to meet the demands of proliferating cancer cells. In particular, cancer cells upregulate synthesis of the membrane phospholipids phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdE) in order to allow for rapid membrane turnover. Nonetheless, we show here that, in mutant isocitrate dehydrogenase 1 (IDHmut) gliomas, which produce the oncometabolite 2-hydroxyglutarate (2-HG), PtdCho and PtdE biosynthesis is downregulated and results in lower levels of both phospholipids when compared with wild-type IDH1 cells. 2-HG inhibited collagen-4-prolyl hydroxylase activity, leading to accumulation of misfolded procollagen-IV in the endoplasmic reticulum (ER) of both genetically engineered and patient-derived IDHmut glioma models. The resulting ER stress triggered increased expression of FAM134b, which mediated autophagic degradation of the ER (ER-phagy) and a reduction in the ER area. Because the ER is the site of phospholipid synthesis, ER-phagy led to reduced PtdCho and PtdE biosynthesis. Inhibition of ER-phagy via pharmacological or molecular approaches restored phospholipid biosynthesis in IDHmut glioma cells, triggered apoptotic cell death, inhibited tumor growth, and prolonged the survival of orthotopic IDHmut glioma-bearing mice, pointing to a potential therapeutic opportunity. Glioma patient biopsies also exhibited increased ER-phagy and downregulation of PtdCho and PtdE levels in IDHmut samples compared with wild-type, clinically validating our observations. Collectively, this study provides detailed and clinically relevant insights into the functional link between oncometabolite-driven ER-phagy and phospholipid biosynthesis in IDHmut gliomas.Significance: Downregulation of phospholipid biosynthesis via ER-phagy is essential for proliferation and clonogenicity of mutant IDH1 gliomas, a finding with immediate therapeutic implications. Cancer Res; 78(9); 2290-304. ©2018 AACR.