Combination of Ad-SGE-REIC and bevacizumab modulates glioma progression by suppressing tumor invasion and angiogenesis.

Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and its overexpression has been shown to exert anti-tumor effects as a therapeutic target gene in many human cancers. Recently, we demonstrated the anti-glioma effects of an adenoviral vector carrying REIC/Dkk-3 with the super gene expression system (Ad-SGE-REIC). Anti-vascular endothelial growth factor treatments such as bevacizumab have demonstrated convincing therapeutic advantage in patients with glioblastoma. However, bevacizumab did not improve overall survival in patients with newly diagnosed glioblastoma. In this study, we examined the effects of Ad-SGE-REIC on glioma treated with bevacizumab. Ad-SGE-REIC treatment resulted in a significant reduction in the number of invasion cells treated with bevacizumab. Western blot analyses revealed the increased expression of several endoplasmic reticulum stress markers in cells treated with both bevacizumab and Ad-SGE-REIC, as well as decreased ß-catenin protein levels. In malignant glioma mouse models, overall survival was extended in the combination therapy group. These results suggest that the combination therapy of Ad-SGE-REIC and bevacizumab exerts anti-glioma effects by suppressing the angiogenesis and invasion of tumors. Combined Ad-SGE-REIC and bevacizumab might be a promising strategy for the treatment of malignant glioma.

Differentiated glioblastoma cells accelerate tumor progression by shaping the tumor microenvironment via CCN1-mediated macrophage infiltration.

Glioblastoma (GBM) is the most lethal primary brain tumor characterized by significant cellular heterogeneity, namely tumor cells, including GBM stem-like cells (GSCs) and differentiated GBM cells (DGCs), and non-tumor cells such as endothelial cells, vascular pericytes, macrophages, and other types of immune cells. GSCs are essential to drive tumor progression, whereas the biological roles of DGCs are largely unknown. In this study, we focused on the roles of DGCs in the tumor microenvironment. To this end, we extracted DGC-specific signature genes from transcriptomic profiles of matched pairs of in vitro GSC and DGC models. By evaluating the DGC signature using single cell data, we confirmed the presence of cell subpopulations emulated by in vitro culture models within a primary tumor. The DGC signature was correlated with the mesenchymal subtype and a poor prognosis in large GBM cohorts such as The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project. In silico signaling pathway analysis suggested a role of DGCs in macrophage infiltration. Consistent with in silico findings, in vitro DGC models promoted macrophage migration. In vivo, coimplantation of DGCs and GSCs reduced the survival of tumor xenograft-bearing mice and increased macrophage infiltration into tumor tissue compared with transplantation of GSCs alone. DGCs exhibited a significant increase in YAP/TAZ/TEAD activity compared with GSCs. CCN1, a transcriptional target of YAP/TAZ, was selected from the DGC signature as a candidate secreted protein involved in macrophage recruitment. In fact, CCN1 was secreted abundantly from DGCs, but not GSCs. DGCs promoted macrophage migration in vitro and macrophage infiltration into tumor tissue in vivo through secretion of CCN1. Collectively, these results demonstrate that DGCs contribute to GSC-dependent tumor progression by shaping a mesenchymal microenvironment via CCN1-mediated macrophage infiltration. This study provides new insight into the complex GBM microenvironment consisting of heterogeneous cells.

Spinal Cord Diffuse Midline Glioma, H3K27M- mutant Effectively Treated with Bevacizumab: A Report of Two Cases.

"Diffuse midline glioma (DMG), H3K27M-mutant" was newly classified in the revised World Health Organization (WHO) 2016 classification of central nervous system tumors. Spinal cord DMG, H3K27M-mutant is relatively rare, with poor prognosis, and there are no effective treatment protocols. In this study, we report two cases of spinal cord DMG, H3K27M-mutant treated with bevacizumab. The two patients were women in their 40s who initially presented with sensory impairment. MRI showed spinal intramedullary tumors, and each patient underwent laminectomy/laminoplasty and biopsy of the tumors. Histological examination initially suggested low-grade astrocytoma in case 1 and glioblastoma in case 2. Upon further immunohistochemical examination in case 1 and molecular examination in case 2, however, both cases were diagnosed as DMG, H3K27M-mutant. Case 1 was treated with radiation therapy and temozolomide (TMZ) chemotherapy, which induced a transient improvement of symptoms; 3 months after surgery, however, the patient's symptoms rapidly deteriorated. MRI showed tumor enlargement with edema to the medulla. Triweekly administration of bevacizumab improved her symptoms for the following 12 months. Case 2 was treated with bevacizumab from the beginning because of acute deterioration of breathing. After bevacizumab administration, both cases showed tumor regression on MRI and drastic improvement of symptoms within a few days. Although spinal cord DMG, H3K27M-mutant has an aggressive clinical course and poor prognosis, bevacizumab administration may offer the significant clinical benefit of alleviating edema, which improves patient's capacity for activities of daily life.

TERT promoter mutation confers favorable prognosis regardless of 1p/19q status in adult diffuse gliomas with IDH1/2 mutations.

TERT promoter mutations are commonly associated with 1p/19q codeletion in IDH-mutated gliomas. However, whether these mutations have an impact on patient survival independent of 1p/19q codeletion is unknown. In this study, we investigated the impact of TERT promoter mutations on survival in IDH-mutated glioma cases. Detailed clinical information and molecular status data were collected for a cohort of 560 adult patients with IDH-mutated gliomas. Among these patients, 279 had both TERT promoter mutation and 1p/19q codeletion, while 30 had either TERT promoter mutation (n = 24) or 1p/19q codeletion (n = 6) alone. A univariable Cox proportional hazard analysis for survival using clinical and genetic factors indicated that a Karnofsky performance status score (KPS) of 90 or 100, WHO grade II or III, TERT promoter mutation, 1p/19q codeletion, radiation therapy, and extent of resection (90-100%) were associated with favorable prognosis (p < 0.05). A multivariable Cox regression model revealed that TERT promoter mutation had a significantly favorable prognostic impact (hazard ratio = 0.421, p = 0.049), while 1p/19q codeletion did not have a significant impact (hazard ratio = 0.648, p = 0.349). Analyses incorporating patient clinical and genetic information were further conducted to identify subgroups showing the favorable prognostic impact of TERT promoter mutation. Among the grade II-III glioma patients with a KPS score of 90 or 100, those with IDH-TERT co-mutation and intact 1p/19q (n = 17) showed significantly longer survival than those with IDH mutation, wild-type TERT, and intact 1p/19q (n = 185) (5-year overall survival, 94% and 77%, respectively; p = 0.032). Our results demonstrate that TERT promoter mutation predicts favorable prognosis independent of 1p/19q codeletion in IDH-mutated gliomas. Combined with its adverse effect on survival among IDH-wild glioma cases, the bivalent prognostic impact of TERT promoter mutation may help further refine the molecular diagnosis and prognostication of diffuse gliomas.

PD-L1 and PD-L2 expression in the tumor microenvironment including peritumoral tissue in primary central nervous system lymphoma.

BACKGROUND: The prevalence of programmed death-ligand 1 (PD-L1) and PD-L2 expression on tumor cells and tumor-infiltrating immune cells in primary central nervous system lymphoma (PCNSL) remains unclear. In the present study, we analyzed needle biopsy and craniotomy specimens of patients with PCNSL to compare the PD-L1 and PD-L2 levels in the tumor and surrounding (peritumoral) tissue. We also assessed the correlation between biological factors and the prognostic significance of PD-L1 and PD-L2 expression. METHODS: We retrospectively analyzed the cases of 70 patients histologically diagnosed with PCNSL (diffuse large B-cell lymphoma). Immunohistochemistry for CD20, CD68, PD-L1, and PD-L2 was performed. In cases with specimens taken by craniotomy, the percentages of PD-L1- and PD-L2-positive macrophages were evaluated in both tumor and peritumoral tissue. The Kaplan-Meier method with log-rank test and Cox proportional hazard model were used for survival analysis. RESULTS: The tumor cells expressed little or no PD-L1 and PD-L2, but macrophages expressed PD-L1 and PD-L2 in most of the patients. The median percentage of PD-L2-positive cells was significantly higher among peritumoral macrophages (32.5%; 95% CI: 0-94.6) than intratumoral macrophages (27.5%; 95% CI: 0-81.1, p = 0.0014). There was a significant correlation between the percentages of PD-L2-positive intratumoral macrophages and PD-L2-positive peritumoral macrophages (p = 0.0429), with very low coefficient correlation (ρ = 0.098535). PD-L1 expression on macrophages was significantly associated with biological factors (intratumoral macrophages: better KPS, p = 0.0008; better MSKCC score, p = 0.0103; peritumoral macrophages: low proportion of LDH elevation, p = 0.0064) and longer OS (for intratumoral macrophages: high PD-L1 = 60 months, 95% CI = 30-132.6; low PD-L1 = 24 months, 95% CI = 11-48; p = 0.032; for peritumoral macrophages: high PD-L1 = 60 months, 95% CI = 30.7-NR; low PD-L1 = 14 months, 95% CI = 3-26). PD-L1 expression on peritumoral macrophages was strongly predictive of a favorable outcome (HR = 0.30, 95% CI = 0.12-0.77, p = 0.0129). CONCLUSIONS: Macrophages in intratumoral and peritumoral tissue expressed PD-L1 and PD-L2 at a higher rate than tumor cells. PD-L1 expression, especially on peritumoral macrophages, seems to be an important prognostic factor in PCNSL. Future comprehensive analysis of checkpoint molecules in the tumor microenvironment, including the peritumoral tissue, is warranted.

Annexin A2-STAT3-Oncostatin M receptor axis drives phenotypic and mesenchymal changes in glioblastoma.

Glioblastoma (GBM) is characterized by extensive tumor cell invasion, angiogenesis, and proliferation. We previously established subclones of GBM cells with distinct invasive phenotypes and identified annexin A2 (ANXA2) as an activator of angiogenesis and perivascular invasion. Here, we further explored the role of ANXA2 in regulating phenotypic transition in GBM. We identified oncostatin M receptor (OSMR) as a key ANXA2 target gene in GBM utilizing microarray analysis and hierarchical clustering analysis of the Ivy Glioblastoma Atlas Project and The Cancer Genome Atlas datasets. Overexpression of ANXA2 in GBM cells increased the expression of OSMR and phosphorylated signal transducer and activator of transcription 3 (STAT3) and enhanced cell invasion, angiogenesis, proliferation, and mesenchymal transition. Silencing of OSMR reversed the ANXA2-induced phenotype, and STAT3 knockdown reduced OSMR protein expression. Exposure of GBM cells to hypoxic conditions activated the ANXA2-STAT3-OSMR signaling axis. Mice bearing ANXA2-overexpressing GBM exhibited shorter survival times compared with control tumor-bearing mice, whereas OSMR knockdown increased the survival time and diminished ANXA2-mediated tumor invasion, angiogenesis, and growth. Further, we uncovered a significant relationship between ANXA2 and OSMR expression in clinical GBM specimens, and demonstrated their correlation with tumor histopathology and patient prognosis. Our results indicate that the ANXA2-STAT3-OSMR axis regulates malignant phenotypic changes and mesenchymal transition in GBM, suggesting that this axis is a promising therapeutic target to treat GBM aggressiveness.

Oncolytic Herpes Virus Armed with Vasculostatin in Combination with Bevacizumab Abrogates Glioma Invasion via the CCN1 and AKT Signaling Pathways.

Anti-VEGF treatments such as bevacizumab have demonstrated convincing therapeutic advantage in patients with glioblastoma. However, bevacizumab has also been reported to induce invasiveness of glioma. In this study, we examined the effects of rapid antiangiogenesis mediated by oncolytic virus (RAMBO), an oncolytic herpes simplex virus-1 expressing vasculostatin, on bevacizumab-induced glioma invasion. The effect of the combination of RAMBO and bevacizumab in vitro was assessed by cytotoxicity, migration, and invasion assays. For in vivo experiments, glioma cells were stereotactically inoculated into the brain of mice. RAMBO was intratumorally injected 7 days after tumor inoculation, and bevacizumab was administered intraperitoneally twice a week. RAMBO significantly decreased both the migration and invasion of glioma cells treated with bevacizumab. In mice treated with bevacizumab and RAMBO combination, the survival time was significantly longer and the depth of tumor invasion was significantly smaller than those treated with bevacizumab monotherapy. Interestingly, RAMBO decreased the expression of cysteine-rich protein 61 and phosphorylation of AKT, which were increased by bevacizumab. These results suggest that RAMBO suppresses bevacizumab-induced glioma invasion, which could be a promising approach to glioma therapy.

δ-Catenin Promotes Bevacizumab-Induced Glioma Invasion.

The combination of bevacizumab with temozolomide and radiotherapy was shown to prolong progression-free survival in newly diagnosed patients with glioblastoma, and this emphasizes the potential of bevacizumab as a glioma treatment. However, although bevacizumab effectively inhibits angiogenesis, it has also been reported to induce invasive proliferation. This study examined gene expression in glioma cells to investigate the mechanisms of bevacizumab-induced invasion. We made a human glioma U87ΔEGFR cell xenograft model by stereotactically injecting these cells into the brain of animals. We administered bevacizumab intraperitoneally three times per week. At 18 days after tumor implantation, the brains were removed for histopathology and mRNA was extracted. In vivo, bevacizumab treatment increased glioma cell invasion. qRT-PCR array analysis revealed upregulation of δ-catenin (CTNND2) and several other factors. In vitro, bevacizumab treatment upregulated δ-catenin expression. A low concentration of bevacizumab was not cytotoxic, but tumor cell motility was increased in scratch wound assays and two-chamber assays. Overexpression of δ-catenin increased the tumor invasion in vitro and in vivo However, δ-catenin knockdown decreased glioma cell invasiveness. The depth of tumor invasion in the U87ΔEGFR cells expressing δ-catenin was significantly increased compared with empty vector-transfected cells. The increase in invasive capacity induced by bevacizumab therapy was associated with upregulation of δ-catenin expression in invasive tumor cells. This finding suggests that δ-catenin is related to tumor invasion and migration.

PIK3R1Met326Ile germline mutation correlates with cysteine-rich protein 61 expression and poor prognosis in glioblastoma.

Despite therapeutic advances, glioblastoma represents a lethal brain tumor. Recently, research to identify prognostic markers for glioblastoma has intensified. Our previous study demonstrated that median progression-free survival (PFS) and overall survival (OS) of patients with high cysteine-rich protein 61 (CCN1) expression was significantly shorter than that of patients with low CCN1 expression. To understand the molecular mechanisms that regulate CCN1 expression, we examined 147 tumour samples from 80 patients with glioblastoma and 67 patients with lower grade glioma. Next-generation and Sanger sequencing showed that PIK3R1Met326Ile was more frequent in the CCN1 high expression group (10/37 cases, 27.0%) than the CCN1 low expression group (3/38 cases, 7.9%) in glioblastoma. This mutation was also detected in corresponding blood samples. In multivariate analysis, high CCN1 expression and PIK3R1Met326Ile in glioblastoma patients were prognostic factors for OS [HR = 2.488 (1.298-4.769), p = 0.006] and [HR = 2.089 (1.020-4.277), p = 0.0439], respectively. Thus, the PIK3R1Met326Ile germline appears to be correlated with CCN1 expression and poor prognosis in glioblastoma.

[Stent-Assisted Coil Embolization of a Dissecting Aneurysm of the Posterior Cerebral Artery: A Case Report].

Dissecting aneurysms of the posterior cerebral artery (PCA) are rare, especially those at the P1 segment. Here, we describe the case of a 57-year-old woman with a subarachnoid hemorrhage (SAH). Computed tomography angiography (CTA) and digital subtraction angiography (DSA) revealed a small (3 mm) dissecting aneurysm with the typical pearl-and-string sign at the right P1 segment. Fourteen days after onset, the patient developed aphasia. DSA revealed vasospasm of the right middle cerebral artery, and we performed endovascular treatment by the intra-arterial injection of 1-(5-isoquinolinesulfonyl) homopiperazine. After this treatment, the patient's symptoms recovered immediately. Vertebral angiography revealed enlargement of the dissecting aneurysm (up to 7 mm diameter). We started a loading dose of 300 mg aspirin and 400 mg clopidogrel after observing growth of the aneurysm. Fifteen days after onset, we performed a stent-assisted coil embolization, and obtained nearly complete obliteration of the aneurysm with preserved patency of the parent artery. Six-month follow-up DSA demonstrated complete occlusion of the aneurysm with good patency of the stented PCA; the patient was at modified Rankin Scale 1. In the treatment of ruptured dissecting aneurysms, parent vessel occlusion (PVO) with aneurysm is common. However, PVO may cause both cerebral infarction of the distal area and perforator occlusion of the occluded vessel. Stent-assisted coil embolization can preserve parent vessel flow and obliterate the aneurysm. Stents offer a therapeutic alternative for PCA dissecting aneurysms, especially when PVO cannot be tolerated.

[Endovascular treatment for cervical carotid artery aneurysm: a case report].

We report here a case of cervical carotid artery aneurysm. This 37-year-old male suffered from acute neck swelling when he was taking lunch after physical work. Ultrasonography demonstrated a cervical pseudoaneurysm and a jet flow, which was blowing into the dome from the carotid artery. Angiogram revealed an aneurysm with a diameter of 3 cm at the bifurcation of the common carotid artery. Coil embolization using double-catheter technique was performed and complete occlusion of the aneurysm was obtained without any complications. The patient returned to his normal life. Cervical carotid artery aneurysms are rare and they induce pain by swelling in the neck, hoarseness, swallowing disturbance, hemorrhage, and cerebral ischemia due to embolism. In case of a cervical carotid artery aneurysm, safe and effective treatments are required and endovascular treatment should be considered. Although stent-assisted coil embolization or covered-stent placement were reported as an effective treatment for cervical aneurysms, coil embolization without using a stent was performed in this particular patient who is a young, blue-collar worker because the avoidance of long-term anti-platelet therapy was desirable. Preoperative evaluation is important to select adequate treatment.