Heterogeneity of glioblastoma with gliomatosis cerebri growth pattern on diffusion and perfusion MRI.

BACKGROUND AND PURPOSE: Gliomatosis cerebri (GC) is a rare growth pattern of glioblastoma whose diffuse nature is reflected by unspecific, relatively uniform findings on conventional MRI. In the present study we sought to evaluate the additional value of diffusion (DWI) and perfusion weighted (PWI) MRI for a more detailed characterization. METHODS: We analyzed the MRI findings in patients with histologically proven glioblastoma with GC growth pattern with a specific emphasis on T2 lesion pattern, volume, relative apparent diffusion coefficient (rACD), and relative cerebral blood volume (rCBV) and compared these to age-/gender-matched patients with localized glioblastoma. RESULTS: Overall, 16 patients (median age 59.5 years, 4 male) were included in the study. Of these, 8 patients had a glioblastoma with GC growth pattern, and 8 a classical localized growth pattern. While the median rADC (1.27 [IQR 1.12-1.41]) within the T2 lesion was significant lower in glioblastoma with GC growth pattern compared to localized glioblastoma (1.74 [IQR 1.45-1.96]; p = 0.003), the median T2 lesion volume and rCBV within the T2 lesion did not differ significantly. Furthermore, six patients with glioblastoma with GC growth pattern showed focal areas with significantly reduced rADC (p = 0.043), and/or increased rCBV (p = 0.028). CONCLUSIONS: Lower rADC in glioblastoma with GC growth pattern might reflect the diffuse tumor cell infiltration whereas focal areas with decreased rADC and/or increased rCBV probably indicate high tumor cell density and/or abnormal tumor vessels which may be useful for biopsy guidance.

[Treatment-associated neurological symptoms of cancer patients and their treatment]. / Therapieassoziierte neurologische Symptome onkologischer Patienten und deren Behandlung.

With the increasing understanding of the specific molecular and cellular pathogenesis of cancer, systemic cancer treatment has become much more targeted and in part substantially more effective. The increased number of long-term survivors and the number of highly specific targeted therapies have resulted in a wide range of neurological complications. Neurologists are increasingly confronted with previously unknown neurological complications of cancer treatment. A profound understanding of the molecular and cellular mechanisms of action of anticancer drugs is the key for a prompt diagnosis and appropriate treatment of these treatment-associated neurological complications.

The promises of immunotherapy in gliomas.

PURPOSE OF REVIEW: Also owing to the limited efficacy of targeted therapies, there has been a renewed interest in targeting gliomas with immunotherapy. But despite considerable efforts using sophisticated approaches, proof of efficacy beyond case studies is still lacking. The purpose of this review is to summarize and discuss current immunotherapeutic approaches and efforts to understand mechanisms of response and resistance. RECENT FINDINGS: The recent failure of large randomized clinical trials using targeted vaccines and checkpoint inhibitors to improve clinical outcome have underlined the grand challenges in this therapeutic arena and illustrated the necessity to understand the biology of immunotherapeutic interventions before conducting large randomized studies. However, these failures should not distract us from continuing to optimize immunotherapeutic concepts. The recent developments in transgenic T cell technologies and personalized vaccines but also rational combinatorial approaches offer tremendous opportunities and should be exploited carefully in early scientifically driven clinical trials. SUMMARY: A profound understanding of the cellular and molecular mechanisms of response and resistance to immunotherapy to be gained from these thoroughly designed clinical trials will be essential to carve out successful strategies in selected patient populations.

Bevacizumab for Patients with Recurrent Gliomas Presenting with a Gliomatosis Cerebri Growth Pattern.

Bevacizumab has been shown to improve progression-free survival and neurologic function, but failed to improve overall survival in newly diagnosed glioblastoma and at first recurrence. Nonetheless, bevacizumab is widely used in patients with recurrent glioma. However, its use in patients with gliomas showing a gliomatosis cerebri growth pattern is contentious. Due to the marked diffuse and infiltrative growth with less angiogenic tumor growth, it may appear questionable whether bevacizumab can have a therapeutic effect in those patients. However, the development of nodular, necrotic, and/or contrast-enhancing lesions in patients with a gliomatosis cerebri growth pattern is not uncommon and may indicate focal neo-angiogenesis. Therefore, control of growth of these lesions as well as control of edema and reduction of steroid use may be regarded as rationales for the use of bevacizumab in these patients. In this retrospective patient series, we report on 17 patients with primary brain tumors displaying a gliomatosis cerebri growth pattern (including seven glioblastomas, two anaplastic astrocytomas, one anaplastic oligodendroglioma, and seven diffuse astrocytomas). Patients have been treated with bevacizumab alone or in combination with lomustine or irinotecan. Seventeen matched patients treated with bevacizumab for gliomas with a classical growth pattern served as a control cohort. Response rate, progression-free survival, and overall survival were similar in both groups. Based on these results, anti-angiogenic therapy with bevacizumab should also be considered in patients suffering from gliomas with a mainly infiltrative phenotype.

Model-Based Iterative Reconstruction in CT Enterography.

OBJECTIVE: The purpose of this study was to analyze the performance of pure model-based iterative reconstruction (MBIR) in low-dose CT enterography. SUBJECTS AND METHODS: Forty-four patients with Crohn disease referred for CT enterography were included. Low-dose modified-protocol and conventional-protocol CT datasets were contemporaneously acquired. Conventional-protocol image formation was performed with 40% adaptive statistical iterative reconstruction (ASIR). Modified-protocol data were reconstructed with 100% MBIR and 40% ASIR. Image quality was assessed subjectively and objectively at six levels. Independent clinical interpretations by two fully blinded radiologists were compared with reference standard consensus reviews by two nonblinded readers who had access to clinical information, previous imaging studies, and medical records. RESULTS: A 74.7% average radiation dose reduction was seen: low-dose modified-protocol effective dose, 1.61 ± 1.18 mSv (size-specific-dose-estimate, 2.47 ± 1.21 mGy); conventional-protocol effective dose, 6.05 ± 2.84 mSv (size-specific-dose-estimate, 9.25 ± 2.9 mGy). Image quality assessment yielded 9372 data points. Objective noise on modified-protocol MBIR images was superior (p < 0.05) to that with the conventional protocol at three of six levels and comparable at the other three levels. Modified-protocol images were superior to conventional-protocol ASIR images (p < 0.05 in all cases) for subjective noise, spatial resolution, contrast resolution, streak artifact, and diagnostic acceptability on coronal reconstructions. Axial diagnostic acceptability was superior for conventional-protocol ASIR (p = 0.76). For both readers, modified-protocol MBIR clinical readings agreed more closely with reference standard readings than did conventional-protocol ASIR readings with regard to bowel wall disease assessment (κ = 0.589 and 0.700 vs 0.583 and 0.564). Overall Crohn disease activity grade (κ = 0.549 and 0.441 vs 0.315 and 0.596) and detection of acute complications (κ = 1.0 and 0.689 vs 0.896 and 0.896) were comparable when evaluated on conventional-protocol ASIR and modified-protocol MBIR images. CONCLUSION: Low-dose CT enterography with MBIR yields images that are comparable to or superior to conventional images.

H3K27M neoepitope vaccination in diffuse midline glioma induces B and T cell responses across diverse HLA loci of a recovered patient.

H3K27M, a driver mutation with T and B cell neoepitope characteristics, defines an aggressive subtype of diffuse glioma with poor survival. We functionally dissect the immune response of one patient treated with an H3K27M peptide vaccine who subsequently entered complete remission. The vaccine robustly expanded class II human leukocyte antigen (HLA)-restricted peripheral H3K27M-specific T cells. Using functional assays, we characterized 34 clonally unique H3K27M-reactive T cell receptors and identified critical, conserved motifs in their complementarity-determining region 3 regions. Using detailed HLA mapping, we further demonstrate that diverse HLA-DQ and HLA-DR alleles present immunogenic H3K27M epitopes. Furthermore, we identified and profiled H3K27M-reactive B cell receptors from activated B cells in the cerebrospinal fluid. Our results uncover the breadth of the adaptive immune response against a shared clonal neoantigen across multiple HLA allelotypes and support the use of class II-restricted peptide vaccines to stimulate tumor-specific T and B cells harboring receptors with therapeutic potential.

Cerebrospinal Fluid cfDNA Sequencing for Classification of Central Nervous System Glioma.

PURPOSE: Primary central nervous system (CNS) gliomas can be classified by characteristic genetic alterations. In addition to solid tissue obtained via surgery or biopsy, cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) is an alternative source of material for genomic analyses. EXPERIMENTAL DESIGN: We performed targeted next-generation sequencing of CSF cfDNA in a representative cohort of 85 patients presenting at two neurooncological centers with suspicion of primary or recurrent glioma. Copy-number variation (CNV) profiles, single-nucleotide variants (SNV), and small insertions/deletions (indel) were combined into a molecular-guided tumor classification. Comparison with the solid tumor was performed for 38 cases with matching solid tissue available. RESULTS: Cases were stratified into four groups: glioblastoma (n = 32), other glioma (n = 19), nonmalignant (n = 17), and nondiagnostic (n = 17). We introduced a molecular-guided tumor classification, which enabled identification of tumor entities and/or cancer-specific alterations in 75.0% (n = 24) of glioblastoma and 52.6% (n = 10) of other glioma cases. The overlap between CSF and matching solid tissue was highest for CNVs (26%-48%) and SNVs at predefined gene loci (44%), followed by SNVs/indels identified via uninformed variant calling (8%-14%). A molecular-guided tumor classification was possible for 23.5% (n = 4) of nondiagnostic cases. CONCLUSIONS: We developed a targeted sequencing workflow for CSF cfDNA as well as a strategy for interpretation and reporting of sequencing results based on a molecular-guided tumor classification in glioma. See related commentary by Abdullah, p. 2860.

Concurrent gliomas in patients with multiple sclerosis.

BACKGROUND: Concurrent malignant brain tumors in patients with multiple sclerosis (MS) constitute a rare but paradigmatic phenomenon for studying neuroimmunological mechanisms from both molecular and clinical perspectives. METHODS: A multicenter cohort of 26 patients diagnosed with both primary brain tumors and multiple sclerosis was studied for disease localization, tumor treatment-related MS activity, and molecular characteristics specific for diffuse glioma in MS patients. RESULTS: MS neither predisposes nor protects from the development of gliomas. Patients with glioblastoma WHO grade 4 without isocitratdehydrogenase (IDH) mutations have a longstanding history of MS, whereas patients diagnosed with IDH-mutant astrocytoma WHO grade 2 receive multiple sclerosis diagnosis mostly at the same time or later. Concurrent MS is associated with a lesser extent of tumor resection and a worse prognosis in IDH-mutant glioma patients (PFS 32 vs. 64 months, p = 0.0206). When assessing tumor-intrinsic differences no distinct subgroup-defining methylation pattern is identified in gliomas of MS patients compared to other glioma samples. However, differential methylation of immune-related genetic loci including human leukocyte antigen locus on 6p21 and interleukin locus on 5q31 is found in MS patients vs. matched non-MS patients. In line, inflammatory disease activity increases in 42% of multiple sclerosis patients after brain tumor radiotherapy suggesting a susceptibility of multiple sclerosis brain tissue to pro-inflammatory stimuli such as ionizing radiation. CONCLUSIONS: Concurrent low-grade gliomas should be considered in multiple sclerosis patients with slowly progressive, expansive T2/FLAIR lesions. Our findings of typically reduced extent of resection in MS patients and increased MS activity after radiation may inform future treatment decisions.

Brain tumors such as gliomas can evade attacks by the immune system. In contrast, some diseases of the central nervous system such as multiple sclerosis (MS) are caused by an overactive immune system. Our study looks at a cohort of rare patients with both malignant glioma and concurrent MS and examines how each disease and their treatments affect each other. Our data suggest that even in patients with known MS, if medical imaging findings are unusual, a concurrent brain tumor should be excluded at an early stage. Radiotherapy, as is the standard of care for malignant brain tumors, may worsen the inflammatory disease activity in MS patients, which may be associated with certain genetic risk factors. Our findings may help to inform treatment of patients with brain tumors and MS.

A H3K27M-targeted vaccine in adults with diffuse midline glioma.

Substitution of lysine 27 to methionine in histone H3 (H3K27M) defines an aggressive subtype of diffuse glioma. Previous studies have shown that a H3K27M-specific long peptide vaccine (H3K27M-vac) induces mutation-specific immune responses that control H3K27M+ tumors in major histocompatibility complex-humanized mice. Here we describe a first-in-human treatment with H3K27M-vac of eight adult patients with progressive H3K27M+ diffuse midline glioma on a compassionate use basis. Five patients received H3K27M-vac combined with anti-PD-1 treatment based on physician's discretion. Repeat vaccinations with H3K27M-vac were safe and induced CD4+ T cell-dominated, mutation-specific immune responses in five of eight patients across multiple human leukocyte antigen types. Median progression-free survival after vaccination was 6.2 months and median overall survival was 12.8 months. One patient with a strong mutation-specific T cell response after H3K27M-vac showed pseudoprogression followed by sustained complete remission for >31 months. Our data demonstrate safety and immunogenicity of H3K27M-vac in patients with progressive H3K27M+ diffuse midline glioma.

INTERCEPT H3: a multicenter phase I peptide vaccine trial for the treatment of H3-mutated diffuse midline gliomas.

INTRODUCTION: Diffuse midline gliomas (DMG) are universally lethal central nervous system tumors that carry almost unanimously the clonal driver mutation histone-3 K27M (H3K27M). The single amino acid substitution of lysine to methionine harbors a neoantigen that is presented in tumor tissue. The long peptide vaccine H3K27M-vac targeting this major histocompatibility complex class II (MHC class II)-restricted neoantigen induces mutation-specific immune responses that suppress the growth of H3K27M+ flank tumors in an MHC-humanized rodent model. METHODS: INTERCEPT H3 is a non-controlled open label, single arm, multicenter national phase 1 trial to assess safety, tolerability and immunogenicity of H3K27M-vac in combination with standard radiotherapy and the immune checkpoint inhibitor atezolizumab (ATE). 15 adult patients with newly diagnosed K27M-mutant histone-3.1 (H3.1K27M) or histone-3.3 (H3.3K27M) DMG will be enrolled in this trial. The 27mer peptide vaccine H3K27M-vac will be administered concomitantly to standard radiotherapy (RT) followed by combinatorial treatment with the programmed death-ligand 1 (PD-L1) targeting antibody ATE. The first three vaccines will be administered bi-weekly (q2w) followed by a dose at the beginning of recovery after RT and six-weekly administrations of doses 5 to 11 thereafter. In a safety lead-in, the first three patients (pts. 1-3) will be enrolled sequentially. PERSPECTIVE: H3K27M-vac is a neoepitope targeting long peptide vaccine derived from the clonal driver mutation H3K27M in DMG. The INTERCEPT H3 trial aims at demonstrating (1) safety and (2) immunogenicity of repeated fixed dose vaccinations of H3K27M-vac administered with RT and ATE in adult patients with newly diagnosed H3K27M-mutant DMG. TRIAL REGISTRATION: NCT04808245.

Intracranial injection of natural killer cells engineered with a HER2-targeted chimeric antigen receptor in patients with recurrent glioblastoma.

BACKGROUND: Glioblastoma (GB) is incurable at present without established treatment options for recurrent disease. In this phase I first-in-human clinical trial we investigated safety and feasibility of adoptive transfer of clonal chimeric antigen receptor (CAR)-NK cells (NK-92/5.28.z) targeting HER2, which is expressed at elevated levels by a subset of glioblastomas. METHODS: Nine patients with recurrent HER2-positive GB were treated with single doses of 1 × 107, 3 × 107, or 1 × 108 irradiated CAR-NK cells injected into the margins of the surgical cavity during relapse surgery. Imaging at baseline and follow-up, peripheral blood lymphocyte phenotyping and analyses of the immune architecture by multiplex immunohistochemistry and spatial digital profiling were performed. RESULTS: There were no dose-limiting toxicities, and none of the patients developed a cytokine release syndrome or immune effector cell-associated neurotoxicity syndrome. Five patients showed stable disease after relapse surgery and CAR-NK injection that lasted 7 to 37 weeks. Four patients had progressive disease. Pseudoprogression was found at injection sites in 2 patients, suggestive of a treatment-induced immune response. For all patients, median progression-free survival was 7 weeks, and median overall survival was 31 weeks. Furthermore, the level of CD8+ T-cell infiltration in recurrent tumor tissue prior to CAR-NK cell injection positively correlated with time to progression. CONCLUSIONS: Intracranial injection of HER2-targeted CAR-NK cells is feasible and safe in patients with recurrent GB. 1 × 108 NK-92/5.28.z cells was determined as the maximum feasible dose for a subsequent expansion cohort with repetitive local injections of CAR-NK cells.

Intrathecal activation of CD8+ memory T cells in IgG4-related disease of the brain parenchyma.

IgG4-related disease (IgG4-RD) is a fibroinflammatory disorder signified by aberrant infiltration of IgG4-restricted plasma cells into a variety of organs. Clinical presentation is heterogeneous, and pathophysiological mechanisms of IgG4-RD remain elusive. There are very few cases of IgG4-RD with isolated central nervous system manifestation. By leveraging single-cell sequencing of the cerebrospinal fluid (CSF) of a patient with an inflammatory intracranial pseudotumor, we provide novel insights into the immunopathophysiology of IgG4-RD. Our data illustrate an IgG4-RD-associated polyclonal T-cell response in the CSF and an oligoclonal T-cell response in the parenchymal lesions, the latter being the result of a multifaceted cell-cell interaction between immune cell subsets and pathogenic B cells. We demonstrate that CD8+ T effector memory cells might drive and sustain autoimmunity via macrophage migration inhibitory factor (MIF)-CD74 signaling to immature B cells and CC-chemokine ligand 5 (CCL5)-mediated recruitment of cytotoxic CD4+ T cells. These findings highlight the central role of T cells in sustaining IgG4-RD and open novel avenues for targeted therapies.

Coronary Artery Disease in Patients Presenting With Acute Ischemic Stroke or Transient Ischemic Attack and Elevated Troponin Levels.

Background: There is little information concerning the invasive coronary angiography (ICA) findings of patients with acute ischemic stroke (AIS) or transient ischemic attack (TIA) with elevated troponin levels and suspected myocardial infarction (MI). This study analyzed patient characteristics associated with ICA outcomes. Methods: A total of 8,322 patients with AIS or TIA, treated between March 2010 and May 2020, were retrospectively screened for elevated serum troponin I at hospital admission. Patients in whom ICA was performed, due to suspected type 1 MI based on symptoms, echocardiography, and ECG, were categorized according to ICA results (non-obstructive coronary artery disease (CAD): ≥1 stenosis ≥50% but no stenosis ≥80%; obstructive CAD: any stenosis ≥80% or hemodynamically relevant stenosis assessed by FFR/iwFR). Results: Elevated troponin levels were detected in 2,205 (22.5%) patients, of whom 123 (5.6%) underwent ICA (mean age 71 ± 12 years; 67% male). CAD was present in 98 (80%) patients, of whom 51 (41%) were diagnosed with obstructive CAD. Thus, ICA findings of obstructive CAD accounted for 2.3% of patients with troponin elevation and 0.6% of all stroke patients. The clinical hallmarks of myocardial ischemia, including angina pectoris (31 vs. 15%, p < 0.05) and regional wall motion abnormalities (49 vs. 32%, p = 0.07), and increased cardiovascular risk indicated obstructive CAD. While there was no association between lesion site or stroke severity and ICA findings, causal large-artery atherosclerosis was significantly more common in patients with obstructive coronary disease (p < 0.05). Conclusion: The rate of obstructive CAD in patients with stroke or TIA and elevated troponin levels with suspected concomitant type I MI is low. The cumulation of several cardiovascular risk factors and clinical signs of MI were predictive. AIS patients with large-artery atherosclerosis and elevated troponin may represent an especially vulnerable subgroup of stroke patients with risk for obstructive CAD.

AMPK activation protects astrocytes from hypoxia­induced cell death.

Adenosine monophosphate (AMP)­activated protein kinase (AMPK) is a major cellular energy sensor that is activated by an increase in the AMP/adenosine triphosphate (ATP) ratio. This causes the initiation of adaptive cellular programs, leading to the inhibition of anabolic pathways and increasing ATP synthesis. AMPK indirectly inhibits mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a serine/threonine kinase and central regulator of cell growth and metabolism, which integrates various growth inhibitory signals, such as the depletion of glucose, amino acids, ATP and oxygen. While neuroprotective approaches by definition focus on neurons, that are more sensitive under cell stress conditions, astrocytes play an important role in the cerebral energy homeostasis during ischemia. Therefore, the protection of astrocytic cells or other glial cells may contribute to the preservation of neuronal integrity and function. In the present study, it was thus hypothesized that a preventive induction of energy deprivation­activated signaling pathways via AMPK may protect astrocytes from hypoxia and glucose deprivation. Hypoxia­induced cell death was measured in a paradigm of hypoxia and partial glucose deprivation in vitro in the immortalized human astrocytic cell line SVG. Both the glycolysis inhibitor 2­deoxy­d­glucose (2DG) and the AMPK activator A­769662 induced the phosphorylation of AMPK, resulting in mTORC1 inhibition, as evidenced by a decrease in the phosphorylation of the target ribosomal protein S6 (RPS6). Treatment with both 2DG and A­769662 also decreased glucose consumption and lactate production. Furthermore, A­769662, but not 2DG induced an increase in oxygen consumption, possibly indicating a more efficient glucose utilization through oxidative phosphorylation. Hypoxia­induced cell death was profoundly reduced by treatment with 2DG or A­769662. On the whole, the findings of the present study demonstrate, that AMPK activation via 2DG or A­769662 protects astrocytes under hypoxic and glucose­depleted conditions.

Single-Cell High-Throughput Technologies in Cerebrospinal Fluid Research and Diagnostics.

High-throughput single-cell technologies have recently emerged as essential tools in biomedical research with great potential for clinical pathology when studying liquid and solid biopsies. We provide an update on current single-cell methods in cerebrospinal fluid research and diagnostics, focusing on high-throughput cell-type specific proteomic and genomic technologies. Proteomic methods comprising flow cytometry and mass cytometry as well as genomic approaches including immune cell repertoire and single-cell transcriptomic studies are critically reviewed and future directions discussed.

Interference with the HSF1/HSP70/BAG3 Pathway Primes Glioma Cells to Matrix Detachment and BH3 Mimetic-Induced Apoptosis.

Malignant gliomas exhibit a high intrinsic resistance against stimuli triggering apoptotic cell death. HSF1 acts as transcription factor upstream of HSP70 and the HSP70 co-chaperone BAG3 that is overexpressed in glioblastoma. To specifically target this resistance mechanism, we applied the selective HSF1 inhibitor KRIBB11 and the HSP70/BAG3 interaction inhibitor YM-1 in combination with the pan-Bcl-2 inhibitor AT-101. Here, we demonstrate that lentiviral BAG3 silencing significantly enhances AT-101-induced cell death and reactivates effector caspase-mediated apoptosis in U251 glioma cells with high BAG3 expression, whereas these sensitizing effects were less pronounced in U343 cells expressing lower BAG3 levels. KRIBB11 decreased protein levels of HSP70, BAG3, and the antiapoptotic Bcl-2 protein Mcl-1, and both KRIBB11 and YM-1 elicited significantly increased mitochondrial dysfunction, effector caspase activity, and apoptotic cell death after combined treatment with AT-101 and ABT-737. Depletion of BAG3 also led to a pronounced loss of cell-matrix adhesion, FAK phosphorylation, and in vivo tumor growth in an orthotopic mouse glioma model. Furthermore, it reduced the plating efficiency of U251 cells in three-dimensional clonogenic assays and limited clonogenic survival after short-term treatment with AT-101. Collectively, our data suggest that the HSF1/HSP70/BAG3 pathway plays a pivotal role for overexpression of prosurvival Bcl-2 proteins and cell death resistance of glioma. They also support the hypothesis that interference with BAG3 function is an effective novel approach to prime glioma cells to anoikis. Mol Cancer Ther; 16(1); 156-68. ©2016 AACR.

Dual targeting of glioblastoma with chimeric antigen receptor-engineered natural killer cells overcomes heterogeneity of target antigen expression and enhances antitumor activity and survival.

Epidermal growth factor receptor (EGFR) and its mutant form EGFRvIII are overexpressed in a large proportion of glioblastomas (GBM). Immunotherapy with an EGFRvIII-specific vaccine has shown efficacy against GBM in clinical studies. However, immune escape by antigen-loss variants and lack of control of EGFR wild-type positive clones limit the usefulness of this approach. Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells may represent an alternative immunotherapeutic strategy. For targeting to GBM, we generated variants of the clinically applicable human NK cell line NK-92 that express CARs carrying a composite CD28-CD3ζ domain for signaling, and scFv antibody fragments for cell binding either recognizing EGFR, EGFRvIII, or an epitope common to both antigens. In vitro analysis revealed high and specific cytotoxicity of EGFR-targeted NK-92 against established and primary human GBM cells, which was dependent on EGFR expression and CAR signaling. EGFRvIII-targeted NK-92 only lysed EGFRvIII-positive GBM cells, while dual-specific NK cells expressing a cetuximab-based CAR were active against both types of tumor cells. In immunodeficient mice carrying intracranial GBM xenografts either expressing EGFR, EGFRvIII or both receptors, local treatment with dual-specific NK cells was superior to treatment with the corresponding monospecific CAR NK cells. This resulted in a marked extension of survival without inducing rapid immune escape as observed upon therapy with monospecific effectors. Our results demonstrate that dual targeting of CAR NK cells reduces the risk of immune escape and suggest that EGFR/EGFRvIII-targeted dual-specific CAR NK cells may have potential for adoptive immunotherapy of glioblastoma.