Glioblastomas shed large quantities of small, membrane-bound microvesicles into the circulation. Although these hold promise as potential biomarkers of therapeutic response, their identification and ...quantification remain challenging. Here, we describe a highly sensitive and rapid analytical technique for profiling circulating microvesicles directly from blood samples of patients with glioblastoma. Microvesicles, introduced onto a dedicated microfluidic chip, are labeled with target-specific magnetic nanoparticles and detected by a miniaturized nuclear magnetic resonance system. Compared with current methods, this integrated system has a much higher detection sensitivity and can differentiate glioblastoma multiforme (GBM) microvesicles from nontumor host cell-derived microvesicles. We also show that circulating GBM microvesicles can be used to analyze primary tumor mutations and as a predictive metric of treatment-induced changes. This platform could provide both an early indicator of drug efficacy and a potential molecular stratifier for human clinical trials.
Point mutations of the NADP⁺-dependent isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) occur early in the pathogenesis of gliomas. When mutated, IDH1 and IDH2 gain the ability to produce the ...metabolite (R)-2-hydroxyglutarate (2HG), but the downstream effects of mutant IDH1 and IDH2 proteins or of 2HG on cellular metabolism are unknown. We profiled >200 metabolites in human oligodendroglioma (HOG) cells to determine the effects of expression of IDH1 and IDH2 mutants. Levels of amino acids, glutathione metabolites, choline derivatives, and tricarboxylic acid (TCA) cycle intermediates were altered in mutant IDH1- and IDH2-expressing cells. These changes were similar to those identified after treatment of the cells with 2HG. Remarkably, N-acetyl-aspartyl-glutamate (NAAG), a common dipeptide in brain, was 50-fold reduced in cells expressing IDH1 mutants and 8.3-fold reduced in cells expressing IDH2 mutants. NAAG also was significantly lower in human glioma tissues containing IDH mutations than in gliomas without such mutations. These metabolic changes provide clues to the pathogenesis of tumors associated with IDH gene mutations.
The prognosis of patients with recurrent World Health Organization (WHO) grade IV malignant glioma is dismal, and there is currently no effective therapy. We conducted a dose-finding and toxicity ...study in this population of patients, evaluating convection-enhanced, intratumoral delivery of the recombinant nonpathogenic polio-rhinovirus chimera (PVSRIPO). PVSRIPO recognizes the poliovirus receptor CD155, which is widely expressed in neoplastic cells of solid tumors and in major components of the tumor microenvironment.
We enrolled consecutive adult patients who had recurrent supratentorial WHO grade IV malignant glioma, confirmed on histopathological testing, with measurable disease (contrast-enhancing tumor of ≥1 cm and ≤5.5 cm in the greatest dimension). The study evaluated seven doses, ranging between 10
and 10
50% tissue-culture infectious doses (TCID
), first in a dose-escalation phase and then in a dose-expansion phase.
From May 2012 through May 2017, a total of 61 patients were enrolled and received a dose of PVSRIPO. Dose level -1 (5.0×10
TCID
) was identified as the phase 2 dose. One dose-limiting toxic effect was observed; a patient in whom dose level 5 (10
TCID
) was administered had a grade 4 intracranial hemorrhage immediately after the catheter was removed. To mitigate locoregional inflammation of the infused tumor with prolonged glucocorticoid use, dose level 5 was deescalated to reach the phase 2 dose. In the dose-expansion phase, 19% of the patients had a PVSRIPO-related adverse event of grade 3 or higher. Overall survival among the patients who received PVSRIPO reached a plateau of 21% (95% confidence interval, 11 to 33) at 24 months that was sustained at 36 months.
Intratumoral infusion of PVSRIPO in patients with recurrent WHO grade IV malignant glioma confirmed the absence of neurovirulent potential. The survival rate among patients who received PVSRIPO immunotherapy was higher at 24 and 36 months than the rate among historical controls. (Funded by the Brain Tumor Research Charity and others; ClinicalTrials.gov number, NCT01491893 .).
Glioblastoma is one of the most challenging forms of cancer to treat. Here we describe a computational platform that integrates the analysis of copy number variations and somatic mutations and ...unravels the landscape of in-frame gene fusions in glioblastoma. We found mutations with loss of heterozygosity in LZTR1, encoding an adaptor of CUL3-containing E3 ligase complexes. Mutations and deletions disrupt LZTR1 function, which restrains the self renewal and growth of glioma spheres that retain stem cell features. Loss-of-function mutations in CTNND2 target a neural-specific gene and are associated with the transformation of glioma cells along the very aggressive mesenchymal phenotype. We also report recurrent translocations that fuse the coding sequence of EGFR to several partners, with EGFR-SEPT14 being the most frequent functional gene fusion in human glioblastoma. EGFR-SEPT14 fusions activate STAT3 signaling and confer mitogen independence and sensitivity to EGFR inhibition. These results provide insights into the pathogenesis of glioblastoma and highlight new targets for therapeutic intervention.
Tumors thrive in an immunosuppressive microenvironment that impedes antitumor innate and adaptive immune responses. Thus, approaches that can overcome immunosuppression and engage antitumor immunity ...are needed. This study defines the adjuvant and cancer immunotherapy potential of the recombinant poliovirus/rhinovirus chimera PVSRIPO. PVSRIPO is currently in clinical trials against recurrent World Health Organization grade IV malignant glioma, a notoriously treatment-refractory cancer. Cytopathogenic infection of neoplastic cells releases the proteome and exposes pathogen- and damage-associated molecular patterns. At the same time, sublethal infection of antigen-presenting cells, such as dendritic cells and macrophages, yields potent, sustained type I interferon-dominant activation in an immunosuppressed microenvironment and promotes the development of tumor antigen-specific T cell responses in vitro and antitumor immunity in vivo. PVSRIPO's immune adjuvancy stimulates canonical innate anti-pathogen inflammatory responses within the tumor microenvironment that culminate in dendritic cell and T cell infiltration. Our findings provide mechanistic evidence that PVSRIPO functions as a potent intratumor immune adjuvant that generates tumor antigen-specific cytotoxic T lymphocyte responses.
Immunologic targeting of tumor-specific gene mutations may allow precise eradication of neoplastic cells without toxicity. Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively ...activated and immunogenic mutation not expressed in normal tissues but widely expressed in glioblastoma multiforme (GBM) and other neoplasms.
A phase II, multicenter trial was undertaken to assess the immunogenicity of an EGFRvIII-targeted peptide vaccine and to estimate the progression-free survival (PFS) and overall survival (OS) of vaccinated patients with newly diagnosed EGFRvIII-expressing GBM with minimal residual disease. Intradermal vaccinations were given until toxicity or tumor progression was observed. Sample size was calculated to differentiate between PFS rates of 20% and 40% 6 months after vaccination.
There were no symptomatic autoimmune reactions. The 6-month PFS rate after vaccination was 67% (95% CI, 40% to 83%) and after diagnosis was 94% (95% CI, 67% to 99%; n = 18). The median OS was 26.0 months (95% CI, 21.0 to 47.7 months). After adjustment for age and Karnofsky performance status, the OS of vaccinated patients was greater than that observed in a control group matched for eligibility criteria, prognostic factors, and temozolomide treatment (hazard ratio, 5.3; P = .0013; n = 17). The development of specific antibody (P = .025) or delayed-type hypersensitivity (P = .03) responses to EGFRvIII had a significant effect on OS. At recurrence, 82% (95% CI, 48% to 97%) of patients had lost EGFRvIII expression (P < .001).
EGFRvIII-targeted vaccination in patients with GBM warrants investigation in a phase III, randomized trial.
Several immunotherapy clinical trials in recurrent glioblastoma have reported long-term survival benefits in 10-20% of patients. Here we perform genomic analysis of tumor tissue from recurrent WHO ...grade IV glioblastoma patients acquired prior to immunotherapy intervention. We report that very low tumor mutation burden is associated with longer survival after recombinant polio virotherapy or after immune checkpoint blockade in recurrent glioblastoma patients. A relationship between tumor mutation burden and survival is not observed in cohorts of immunotherapy naïve newly diagnosed or recurrent glioblastoma patients. Transcriptomic analyses reveal an inverse relationship between tumor mutation burden and enrichment of inflammatory gene signatures in cohorts of recurrent, but not newly diagnosed glioblastoma tumors, implying that a relationship between tumor mutation burden and tumor-intrinsic inflammation evolves upon recurrence.
Malignant gliomas are highly lethal cancers dependent on angiogenesis. Critical tumor subpopulations within gliomas share characteristics with neural stem cells. We examined the potential of stem ...cell-like glioma cells (SCLGC) to support tumor angiogenesis. SCLGC isolated from human glioblastoma biopsy specimens and xenografts potently generated tumors when implanted into the brains of immunocompromised mice, whereas non-SCLGC tumor cells isolated from only a few tumors formed secondary tumors when xenotransplanted. Tumors derived from SCLGC were morphologically distinguishable from non-SCLGC tumor populations by widespread tumor angiogenesis, necrosis, and hemorrhage. To determine a potential molecular mechanism for SCLGC in angiogenesis, we measured the expression of a panel of angiogenic factors secreted by SCLGC. In comparison with matched non-SCLGC populations, SCLGC consistently secreted markedly elevated levels of vascular endothelial growth factor (VEGF), which were further induced by hypoxia. In an in vitro model of angiogenesis, SCLGC-conditioned medium significantly increased endothelial cell migration and tube formation compared with non-SCLGC tumor cell-conditioned medium. The proangiogenic effects of glioma SCLGC on endothelial cells were specifically abolished by the anti-VEGF neutralizing antibody bevacizumab, which is in clinical use for cancer therapy. Furthermore, bevacizumab displayed potent antiangiogenic efficacy in vivo and suppressed growth of xenografts derived from SCLGC but limited efficacy against xenografts derived from a matched non-SCLGC population. Together these data indicate that stem cell-like tumor cells can be a crucial source of key angiogenic factors in cancers and that targeting proangiogenic factors from stem cell-like tumor populations may be critical for patient therapy.
Mutations of the isocitrate dehydrogenase (IDH) metabolic enzymes IDH1 and IDH2 have been found to be frequent and early genetic alterations in astrocytomas and oligodendrogliomas. All mutations ...identified to date affect a single amino acid located within the isocitrate binding site (R132 of IDH1 and the analogous R172 residue of IDH2). IDH1 and IDH2 mutations define a specific subtype of gliomas and may have significant utility for the diagnosis, prognosis, and treatment of patients with these tumors.
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