Kuniaki Tanahashi

Mutational landscape and clonal architecture in grade II and III gliomas

Grade II and III gliomas are generally slowly progressing brain cancers, many of which eventually transform into more aggressive tumors. Despite recent findings of frequent mutations in IDH1 and other genes, knowledge about their pathogenesis is still incomplete. Here, combining two large sets of high-throughput sequencing data, we delineate the entire picture of genetic alterations and affected pathways in these glioma types, with sensitive detection of driver genes. Grade II and III gliomas comprise three distinct subtypes characterized by discrete sets of mutations and distinct clinical behaviors. Mutations showed significant positive and negative correlations and a chronological hierarchy, as inferred from different allelic burdens among coexisting mutations, suggesting that there is functional interplay between the mutations that drive clonal selection. Extensive serial and multi-regional sampling analyses further supported this finding and also identified a high degree of temporal and spatial heterogeneity generated during tumor expansion and relapse, which is likely shaped by the complex but ordered processes of multiple clonal selection and evolutionary events.

Expression of miR-17-92 enhances anti-tumor activity of T-cells transduced with the anti-EGFRvIII chimeric antigen receptor in mice bearing human GBM xenografts

BackgroundExpression of miR-17-92 enhances T-cell survival and interferon (IFN)-γ production. We previously reported that miR-17-92 is down-regulated in T-cells derived from glioblastoma (GBM) patients. We hypothesized that transgene-derived co-expression of miR17-92 and chimeric antigen receptor (CAR) in T-cells would improve the efficacy of adoptive transfer therapy against GBM.MethodsWe constructed novel lentiviral vectors for miR-17-92 (FG12-EF1a-miR-17/92) and a CAR consisting of an epidermal growth factor receptor variant III (EGFRvIII)-specific, single-chain variable fragment (scFv) coupled to the T-cell receptor CD3ζ chain signaling module and co-stimulatory motifs of CD137 (4-1BB) and CD28 in tandem (pELNS-3C10-CAR). Human T-cells were transduced with these lentiviral vectors, and their anti-tumor effects were evaluated both in vitro and in vivo.ResultsCAR-transduced T-cells (CAR-T-cells) exhibited potent, antigen-specific, cytotoxic activity against U87 GBM cells that stably express EGFRvIII (U87-EGFRvIII) and, when co-transduced with miR-17-92, exhibited improved survival in the presence of temozolomide (TMZ) compared with CAR-T-cells without miR-17-92 co-transduction. In mice bearing intracranial U87-EGFRvIII xenografts, CAR-T-cells with or without transgene-derived miR-17-92 expression demonstrated similar levels of therapeutic effect without demonstrating any uncontrolled growth of CAR-T-cells. However, when these mice were re-challenged with U87-EGFRvIII cells in their brains, mice receiving co-transduced CAR-T-cells exhibited improved protection compared with mice treated with CAR-T-cells without miR-17-92 co-transduction.ConclusionThese results warrant the development of novel CAR-T-cell strategies that incorporate miR-17-92 to improve therapeutic potency, especially in patients with GBM.

CAR T Cells Targeting Podoplanin Reduce Orthotopic Glioblastomas in Mouse Brains.

The glioblastoma subtypes with the worst prognoses often bear podoplanin. T cells expressing a chimeric antigen receptor that targets podoplanin were specific and effective against PDPN-positive glioblastoma cells in vitro and increased survival time in a mouse model. Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor in adults with a 5-year overall survival rate of less than 10%. Podoplanin (PDPN) is a type I transmembrane mucin-like glycoprotein, expressed in the lymphatic endothelium. Several solid tumors overexpress PDPN, including the mesenchymal type of GBM, which has been reported to present the worst prognosis among GBM subtypes. Chimeric antigen receptor (CAR)–transduced T cells can recognize predefined tumor surface antigens independent of MHC restriction, which is often downregulated in gliomas. We constructed a lentiviral vector expressing a third-generation CAR comprising a PDPN-specific antibody (NZ-1–based single-chain variable fragment) with CD28, 4-1BB, and CD3ζ intracellular domains. CAR-transduced peripheral blood monocytes were immunologically evaluated by calcein-mediated cytotoxic assay, ELISA, tumor size, and overall survival. The generated CAR T cells were specific and effective against PDPN-positive GBM cells in vitro. Systemic injection of the CAR T cells into an immunodeficient mouse model inhibited the growth of intracranial glioma xenografts in vivo. CAR T-cell therapy that targets PDPN would be a promising adoptive immunotherapy to treat mesenchymal GBM. Cancer Immunol Res; 4(3); 259–68. ©2016 AACR.

Lenalidomide enhances the function of chimeric antigen receptor T cells against the epidermal growth factor receptor variant III by enhancing immune synapses.

The epidermal growth factor receptor variant III (EGFRvIII) is exclusively expressed on the cell surface in ~50% of glioblastoma multiforme (GBM). This variant strongly and persistently activates the phosphatidylinositol 3-kinase-Akt signaling pathway in a ligand-independent manner resulting in enhanced tumorigenicity, cellular motility and resistance to chemoradiotherapy. Our group generated a recombinant single-chain variable fragment (scFv) antibody specific to the EGFRvIII, referred to as 3C10-scFv. In the current study, we constructed a lentiviral vector transducing the chimeric antigen receptor (CAR) that consisted of 3C10-scFv, CD3ζ, CD28 and 4-1BB (3C10-CAR). The 3C10-CAR-transduced peripheral blood mononuclear cells (PBMCs) and CD3+ T cells specifically lysed the glioma cells that express EGFRvIII. Moreover, we demonstrated that CAR CD3+ T cells migrated to the intracranial xenograft of GBM in the mice treated with 3C10-CAR PBMCs. An important and novel finding of our study was that a thalidomide derivative lenalidomide induced 3C10-CAR PBMC proliferation and enhanced the persistent antitumor effect of the cells in vivo. Lenalidomide also exhibited enhanced immunological synapses between the effector cells and the target cells as determined by CD11a and F-actin polymerization. Collectively, lentiviral-mediated transduction of CAR effectors targeting the EGFRvIII showed specific efficacy, and lenalidomide even intensified CAR cell therapy by enhanced formation of immunological synapses.

Immunohistochemical ATRX expression is not a surrogate for 1p19q codeletion

The IDH-mutant and 1p/19q co-deletion (1p19q codel) provides significant diagnostic and prognostic value in lower-grade gliomas. As ATRX mutation and 1p19q codel are mutually exclusive, ATRX immunohistochemistry (IHC) may substitute for 1p19q codel, but this has not been comprehensively examined. In the current study, we performed ATRX-IHC in 78 gliomas whose ATRX statuses were comprehensively determined by whole exome sequencing. Among the 60 IHC-positive and 18 IHC-negative cases, 86.7 and 77.8% were ATRX-wildtype and ATRX-mutant, respectively. ATRX mutational patterns were not consistent with ATRX-IHC. If our cohort had only used IDH status and IHC-based ATRX expression for diagnosis, 78 tumors would have been subtyped as 48 oligodendroglial tumors, 16 IDH-mutant astrocytic tumors, and 14 IDH-wildtype astrocytic tumors. However, when the 1p19q codel test was performed following ATRX-IHC, 8 of 48 ATRX-IHC-positive tumors were classified as “1p19q non-codel” and 3 of 16 ATRX-IHC-negative tumors were classified as “1p19q codel”; a total of 11 tumors (14%) were incorrectly classified. In summary, we observed dissociation between ATRX-IHC and actual 1p19q codel in 11 of 64 IDH-mutant LGGs. In describing the complex IHC expression of ATRX somatic mutations, our results indicate the need for caution when using ATRX-IHC as a surrogate of 1p19q status.

Activation of Yes-Associated Protein in Low-Grade Meningiomas Is Regulated by Merlin, Cell Density, and Extracellular Matrix Stiffness

Abstract The NF2 gene product Merlin is a protein containing ezrin, radixin, and moesin domains; it is a member of the 4.1 protein superfamily associated with the membrane cytoskeleton and also interacts with cell surface molecules. The mammalian Hippo cascade, a downstream signaling cascade of merlin, inactivates the Yes-associated protein (YAP). Yes-associated protein is activated by loss of the NF2 gene and functions as an oncogene in meningioma cells; however, the factors controlling YAP expression, phosphorylation, and subcellular localization in meningiomas have not been fully elucidated. Here, we demonstrate that merlin expression is heterogeneous in 1 NF2 gene-negative and 3 NF2 gene-positive World Health Organization grade I meningiomas. In the NF2 gene-positive meningiomas, regions with low levels of merlin (tumor rims) had greater numbers of cells with nuclear YAP versus regions with high merlin levels (tumor cores). Merlin expression and YAP phosphorylation were also affected by cell density in the IOMM-Lee and HKBMM human meningioma cell lines; nuclear localization of YAP was regulated by cell density and extracellular matrix (ECM) stiffness in IOMM-Lee cells. These results suggest that cell density and ECM stiffness may contribute to the heterogeneous loss of merlin and increased nuclear YAP expression in human meningiomas.

Efficacy of early carotid endarterectomy for vulnerable plaque in the common carotid artery.

Dear Editor Steno-occlusive disease in the common carotid artery (CCA) is relatively rare. Clinical and plaque features of CCA stenosis have thus seldom been investigated [6, 7]. We report here a case of refractory ischemia due to mild common carotid artery stenosis treated with carotid endarterectomy (CEA). A 59-year-old Japanese male developed mild right hemiparesis. Ischemic lesions in the left hemisphere due to mild left distal CCA stenosis with vulnerable plaque were revealed on diffusion-weighted imaging (DWI), time-of-flight imaging (TOF), and three-dimensional computed tomography angiography (3DCTA). Oral clopidgrel and rosuvastatin were initiated. Ten months later, symptoms recurred, and DWI revealed new ischemic lesions (Fig. 1a). Oral aspirin was added. TOF showed an increased plaque volume with retention of the lumen (Fig. 1b, c), indicating expansive remodeling. A fibrous cap (Fig. 1b) was disrupted, and the plaque was directly exposed to the lumen (Fig. 1c). The stenosis remained mild on 3DCTA (Fig. 1d, e upper). However, an intraluminal filling defect implied a mobile plaque (Fig. 1e lower). T1-weighted black-blood imaging showed protrusion of a high-intensity plaque into the lumen (Fig. 1f). Intravenous heparin was therefore started. Nevertheless, the third ischemic event occurred 4 days after the second onset. Six days after the third event, CEAwas performed without major perioperative complications. A large amount of fragile plaque with inner fibrous tissues was resected. A portion of the plaque was exposed directly to the lumen (Fig. 1g). No intraluminal thrombus was identified. Histopathological study showed fragile atheroma containing intraplaque hemorrhage. A portion of the inner fibrous tissues was disrupted (Fig. 1h). The patient has not developed further events after 12months of follow-up. Recently, plaque characteristics have been accurately evaluated using MRI [8, 10]. Intraplaque hemorrhage [1, 9], fibrous cap rupture [5, 9], expansive arterial remodeling [4], and a mobile plaque [2, 3] indicate plaque fragility and a higher risk of ischemic events. Steno-occlusive disease of the CCA is rare (1 %–5 % of stroke patients) because extracranial carotid atherosclerotic disease usually arises from the ICA [6, 7]. CCA lesions are supposed to frequently cause amaurosis fugax due to hemodynamic insufficiency [6]. In this case, the patient suffered repeated cerebral embolisms from mild CCA stenosis. A previous study reported that all CCA lesions showed less than 75 % stenosis [7]. However, from this case, we learned a lesson that an extent of stenosis in the CCA is not necessarily correlated with a risk of stroke as seen in the ICA. Although the vessel retains its lumen, the outer circumference might be increasing, which implies increasing plaque volume and vulnerability [4]. In this case, plaque imaging studies suggested plaque fragility and a higher * Yoshio Araki y.araki@med.nagoya-u.ac.jp