Ventricular opening and cerebrospinal fluid circulation accelerate the biodegradation process of carmustine wafers suggesting their immunomodulation potential in the human brain.

PURPOSE: Opening the ventricular system during glioblastoma surgery is often necessary, but the consequent effect on the tumor microenvironment of glioblastoma remains unknown. Implantation of carmustine wafer enables direct drug delivery to the tumor site; however, the exact mechanism of the wafer's biodegradation process is unclear, and the available data is limited to in vivo non-human mammalian studies. We hypothesized that the ventricular opening affects the degradation process of the wafer and the glioblastoma tumor microenvironment. METHODS: This study included 30 glioblastoma patients. 21 patients underwent carmustine wafer implantation during initial surgery. All patients underwent repeated surgical resection upon recurrence, allowing for pathological comparison of changes associated with wafer implantation. Immunohistochemical analyses were performed using CD68, TMEM119, CD163, IBA1, BIN1, and CD31 antibodies to highlight microglia, macrophages, and tumor vascularity, and the quantitative scoring results were correlated with clinical, molecular, and surgical variables, including the effect of the ventricular opening. RESULTS: The carmustine wafer implanted group presented significantly less TMEM119-positive microglia within the tumor (P = 0.0002). Simple and multiple regression analyses revealed that the decrease in TMEM119-positive microglia was correlated with longer intervals between surgeries and opened ventricular systems. No correlation was observed between age, methylated O6-methylguanine DNA methyltransferase promoter expression, and the extent of surgical resection. CONCLUSIONS: Our study findings strongly suggest that biomaterials may possess immunomodulation capacity, which is significantly impacted by the ventricular opening procedure. Furthermore, our data highlights the pathophysiological effects of the ventricular opening within the surrounding human brain, especially after the wafer implantation.

Tumor microenvironment after biodegradable BCNU wafer implantation: special consideration of immune system.

Biomaterials to treat cancers hold therapeutic potential; however, their translation to bedside treatment requires further study. The carmustine (1,3-bis (2-chloroethyl)-1-nitrosourea; BCNU) wafer, a biodegradable polymer, currently is the only drug that is able to be placed at the surgical site to treat malignant tumors. However, how this wafer affects the surrounding tumor microenvironment is not well understood to date. We retrospectively reviewed all patients with glioblastoma treated with and without BCNU wafers who underwent repeat resection at tumor recurrence. We investigated radiological imaging; the interval between the two surgeries; and immunohistochemistry of CD3, CD4, CD8, CD20, CD68, FOXP3, and PD1. We implanted BCNU wafers in 41 newly diagnosed glioblastoma patients after approval of the wafer in Japan. Of them, 14 underwent surgery at recurrence and tissue was obtained from around the wafers. The interval between the first and second surgeries ranged from 63 to 421 days. The wafer could be observed on magnetic resonance imaging at up to 226 days, whereas intraoperatively the biodegraded material of the wafer could be found at up to 421 days after the initial surgery. Immunohistochemical analysis demonstrated that CD8+ and CD68+ cells were significantly increased, but FOXP3+ cells did not increase, after wafer implantation compared to tissue from cases without wafer implantation. MRI data and immune cells, as well as interval between surgeries and immune cells, demonstrated positive correlation. These results helped us to understand the bioactivity of bioengineered materials and to establish a new approach for immunotherapy.

PDGFRA gene rearrangements are frequent genetic events in PDGFRA-amplified glioblastomas.

Gene rearrangement in the form of an intragenic deletion is the primary mechanism of oncogenic mutation of the epidermal growth factor receptor (EGFR) gene in gliomas. However, the incidence of platelet-derived growth factor receptor-α (PDGFRA) gene rearrangement in these tumors is unknown. We investigated the PDGFRA locus in PDGFRA-amplified gliomas and identified two rearrangements, including the first case of a gene fusion between kinase insert domain receptor (KDR) (VEGFRII) and the PDGFRA gene, and six cases of PDGFRA(Δ8, 9), an intragenic deletion rearrangement. The PDGFRA(Δ8, 9) mutant was common, being present in 40% of the glioblastoma multiformes (GBMs) with PDGFRA amplification. Tumors with these two types of PDGFRA rearrangement displayed histologic features of oligodendroglioma, and the gene products of both rearrangements showed constitutively elevated tyrosine kinase activity and transforming potential that was reversed by PDGFR blockade. These results suggest the possibility that these PDGFRA mutants behave as oncogenes in this subset of gliomas, and that the prevalence of such rearrangements may have been considerably underestimated.

Long-term follow-up after BCNU wafer implantation in patients with newly diagnosed glioblastoma.

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU, or Carmustine) wafers are intraoperatively implantable wafers used to achieve local tumor control. There is scarce data about the behavior of wafers in the long-term follow-up of implanted cases. We reviewed the data of 64 patients with newly diagnosed glioblastoma treated by surgery, BCNU wafers, radiation therapy, and temozolomide administration. This cohort included 55 patients who presented first recurrence, and 49 of them showed tumor progression to death. The MR imaging of each patient at the terminal stage and an autopsy case were used to elucidate the tumor progression pattern after the wafer implantation. We subdivided the first recurrence pattern into local, distant, and multifocal based on MR imaging or into infield, outfield, and marginal based on the radiation field. The first recurrence pattern was 33 patients (60%) with local, 13 (24%) with distant, and nine (16%) with multifocal recurrence, or 38 patients (69%) with infield, 13 (24%) with outfield, and four (7%) with marginal. The median and mean time intervals between MR imaging at the terminal stage and death were 2.0 and 2.3 months, respectively. Of note, 13 patients with first distant recurrence had no obvious radiological local tumor progression even at the terminal stage. Long-term follow-up after BCNU wafer implantation revealed that patients with first distant recurrence had long-lasting local tumor control until the terminal stage.

A De Novo Mouse Model of C11orf95-RELA Fusion-Driven Ependymoma Identifies Driver Functions in Addition to NF-κB.

The majority of supratentorial ependymomas (ST-ependymomas) have few mutations but frequently display chromothripsis of chromosome 11q that generates a fusion between C11orf95 and RELA (RELAFUS). Neural stem cells transduced with RELAFUSex vivo form ependymomas when implanted in the brain. These tumors display enhanced NF-κB signaling, suggesting that this aberrant signal is the principal mechanism of oncogenesis. However, it is not known whether RELAFUS is sufficient to drive de novo ependymoma tumorigenesis in the brain and, if so, whether these tumors also arise from neural stem cells. We show that RELAFUS drives ST-ependymoma formation from periventricular neural stem cells in mice and that RELAFUS-induced tumorigenesis is likely dependent on a series of cell signaling pathways in addition to NF-κB.

Down-regulation of O6-methylguanine-DNA methyltransferase gene expression in gliomas by platinum compounds.

Forty-two patients with malignant gliomas that had received two courses of chemotherapy more than 2 months apart were examined. Among these 42 patients, 31 were treated with 1-(4-amino-2-methyl-5-pyrimidynyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), and 11 were treated with platinum compounds such as cis-platinum (CDDP) or carboplatin (CBDCA), as the first-line chemotherapy. The response rate of the second chemotherapy in the 31 patients treated first with ACNU was significantly lower than that in the 11 patients treated with platinum compounds, regardless of the type of the second chemotherapy (P=0.0292 by Fisher's exact probability test). O(6)-methylguanine-DNA methyltransferase (MGMT) mRNA expression was measured twice by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) using Sybr-Green I in 16 of 42 patients. The relative quantitation value (RQV) of MGMT mRNA normalized to the level of 2-microglobulin decreased after chemotherapy in all 5 patients treated with platinum compound. U373MG and A172 human glioma cells were cultured for 5 days with 1 microM of CDDP or 4 microM of CBDCA. The RQV of MGMT in these cells treated with platinum compounds obviously decreased, and these cells were more sensitive to ACNU than the control cells based on colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Both the clinical findings and laboratory results suggest that platinum compounds may play a role in the down-regulation of MGMT mRNA expression and up-regulation of the sensitivity to ACNU. Platinum compounds may be strong candidates for use as first-line chemotherapeutic agents against malignant gliomas.

Hypoxia-mediated cancer stem cells in pseudopalisades with activation of hypoxia-inducible factor-1α/Akt axis in glioblastoma.

Pseudopalisades (Ps) around necrotic foci are severely hypoxic and overexpress hypoxia-inducible factor (HIF) in glioblastoma (GBM). Hypoxic regions have been proposed as one of several distinct niches for cancer stem cells (CSCs) in GBM, but little is known about the association between Ps features and CSC properties. Herein, we focused on the biological role of Ps lesions. In clinical cases of GBM, expression of hypoxia-related molecules including HIF-1α, Glut-1, p27(Kip1), and pAkt was significantly increased in perinecrotic Ps lesions compared with nonnecrotic areas and perinecrotic lesions lacking Ps features. Significantly higher expression levels of several CSC-related markers, including CD133, Sox2, CD44s, and aldehyde dehydrogenase (ALDH) 1, were also observed in Ps lesions, which were positively correlated with expression of hypoxia-related molecules and pAkt. Ps lesions also showed increased number of apoptotic cells and decreased bcl-2 and survivin expression compared with the surrounding tissue. Short-term exposure of astrocytoma cell lines to cobalt chloride, which is known to mimic the effect of hypoxia, caused an increase in expression of both hypoxia- and CSC-related markers, in line with increases in the ALDH(high) cell population and number of spheroids. Inhibition of endogenous Akt by LY294002 resulted in decreased expression of Sox2, ALDH1, and CD133, leading to enhancement of cobalt chloride-mediated apoptotic events due to altered ratio of bcl-2 to bax expression. These findings suggest that Ps lesions within GBM may serve as a specialized hypoxic niche, in which the HIF-1α/pAkt axis is activated, in response to severe hypoxia.

Light microscopic demonstration of the microlumen of ependymoma: a study of the usefulness of antigen retrieval for epithelial membrane antigen (EMA) immunostaining.

To determine the origin of dotlike epithelial membrane antigen (EMA) immunoreactivity of ependymoma, which is consistent with the eosinophilic globular body in hematoxylin and eosin (H&E) stain, an immunoelectron microscopic study was undertaken. The usefulness of antigen retrieval pretreatment in detecting the dotlike EMA immunoreactivity in ependymomas was also studied. The materials were 29 ependymomas, 7 autopsy brains as a normal control, and 50 brain tumors of various types. The study confirmed that most of the brown dots in EMA immunostain in ependymoma represented microlumina of tumor cells. In ependymomas, plain EMA immunostaining showed dotlike positivity in only six cases (21%), and antigen retrieval pretreatment increased the number of positives up to 26 cases (90%). Antigen retrieved CD99 detected 23 positive cases (80%) in ependymomas. On the basis of the results, although some false positive findings were raised by antigen retrieval pretreatment, the authors positively recommend adoption of the technique, especially when ependymoma remains as one of the differential diagnoses of the tumor.

Granulofilamentous meningioma.

A 55-year old female was referred to the Department of Neurosurgery, Kitasato Hospital, because of a hearing impairment. Neuroimaging revealed a typical meningioma attached to the falx in the right frontal region. During surgery, an encapsulated, circumscribed, reddish-gray, slightly hard tumor attached to the falx was completely removed by an interhemispheric approach. On light microscopy, many of the tumor cells contained eosinophilic inclusions with single or multiple vacuoles that displaced the cytoplasm. The nuclei of the tumor cells were eccentric. There were no signs of malignancy in the specimen. Electron microscopy revealed that most of the eosinophilic inclusions were composed of filaments measuring 12 nm in diameter. There have been several reports of benign meningiomas with eosinophilic inclusions composed of intermediate filaments. The microscopic differences between these types of tumor and rhabdoid meningiomas are very subtle, and it is important the two types of tumors are not confused. Benign meningiomas with eosinophilic inclusions comprising intermediate filaments, for example the tumor described in this report, have been diagnosed as granulofilamentous meningiomas, which is a subtype of benign meningioma.

Adult cerebellar glioblastoma cases have different characteristics from supratentorial glioblastoma.

This study is a histological and clinical investigation of four cases of cerebellar glioblastoma, a rare tumor. The cases included three males and one female, from 33 to 67 years in age (mean 49 years). Tumor resection, postoperative irradiation and chemotherapy were performed in all cases. Two patients died of local tumor recurrence after 14 and 27 months. Another patient relapsed after 10 months; however, after additional tumor resection and second line chemotherapy, she remains disease-free 41 months after the initial treatment. The fourth patient has not relapsed in the 6 months since initial treatment. The histopathology of all cases was glioblastoma with pseudopalisading necrosis. However, low-grade glioma histopathology was found in three patients. All glioblastomas were immunopositive for p53 and immunonegative for epidermal growth factor receptor (EGFR) and isocitrate dehydrogenase 1 (IDH1). These adult cerebellar glioblastoma cases had similar clinical and pathological characteristics, and had different characteristics compared with supratentorial glioblastomas.

Pilocytic astrocytoma with abundant oligodendroglioma-like component.

An 18-year-old girl presented with a history of visual disturbance without headache, nausea, or vomiting in May 2010. In July 2010, the patient visited our hospital because of visual disturbance. Head magnetic resonance images revealed hydrocephalus caused by a ring-enhancing mass lesion located in the vermis. Total tumor removal was performed. Histological findings revealed that honeycomb cells resembling oligodendrocytes accounted for most parts of the tumor. Rosenthal fibers and hyaline droplets were seen in a small portion. The tumor cells were immunoreactive for GFAP and Olig2, but none of the tumor cells were immunoreactive for Symaptophysin, EMA, or IDH 1. according to these findings, the tumor was diagnosed as pilocytic astrocytoma with an abundant oligodendroglioma-like component. Pilocytic astrocytoma is known to be associated with an oligodendroglioma-like component; however, the differential diagnosis for oligodendroglioma may be difficult when an oligodendroglioma-like component occupies most of the tumor.

Pathophysiological evaluation of cerecyte coil embolization for experimental broad neck aneurysms.

Cerecyte second-generation coils feature inner surfaces coated with an absorbable polyglycolic acid (PGA) polymer. Their use is expected to accelerate aneurysm organization, but time course data are limited. The present experimental study was therefore conducted to clarify the processes by pathological examination. Methods. Two types of experimental aneurysms were initially generated in adult mongrel dogs, one bifurcation and another of lateral wall type. Long-term persistence of each was defined by follow-up angiography for more than 1 year. Embolization of the aneurysms was then performed using only cerecyte coils, and follow-up angiography was conducted after 2 and 4 weeks followed by pathological examination. Results. Organization of both types of broad neck aneurysm was apparent 4 weeks after embolization, which is earlier as compared with already reported data for bare coils.

Epstein-Barr virus (EBV)-associated primary central nervous system lymphoma: is incidence of EBV expression associated with median survival time?

The frequency and clinical features of Epstein-Barr virus (EBV)-associated primary central nervous system lymphoma (PCNSL) in elderly patients were investigated in this study. Thirty-three PCNSL cases were enrolled in the retrospective study. Biopsies were performed, and tissue was embedded in paraffin and sectioned. In-situ hybridization of EBV-encoded small RNAs was then conducted. Specimens were scored as having one of three possible results: negative (no EBV-positive cells), slightly positive (<50% EBV-positive cells), and strongly positive (>50% EBV-positive cells). Fifteen cases were negative for EBV expression. Sixteen cases were slightly positive, and two cases (68 and 79 years of age) were strongly positive. The incidence of strongly positive EBV expression in PCNSL was 6.1%. The incidence of strongly positive EBV expression in PCNSL patients ≥65 years of age was 13%. Median survival time differed significantly among PCNSL patients treated with high-dose methotrexate and radiotherapy. Importantly, the strongly EBV-positive PCNSL cases had the worst outcomes, and the EBV-negative PCNSL cases had the best outcomes. These results suggest that EBV infection may affect the treatment outcome of PCNSL. In the future, examination of EBV expression in PCNSL patients who receive individualized treatment may be useful.

Subependymal giant cell astrocytoma with oncocytic change.

A 49-year-old woman presented with a history of periodic episodes of nausea and vomiting starting in 2006. In June 2009, the patient lost consciousness and was transported to our hospital. Head computed tomography (CT) revealed hydrocephalus caused by an enhancing mass lesion with calcification located in the right lateral ventricle around the foramen of Monro. Total tumor removal was performed. Histologic findings revealed fibrillated spindle tumor cells and giant tumor cells with abundant cytoplasm. The spindle tumor cells were immunoreactive for GFAP and S-100 protein, but none of the giant tumor cells were immunoreactive for GFAP or S-100 protein. Electron microscopic examination revealed abundant mitochondria in the tumor cell cytoplasm. According to these findings, this tumor was diagnosed as subependymal giant cell astrocytoma (SEGA) with oncocytic change, which is extremely rare.

Primary central nervous system lymphoma in acquired immune deficiency syndrome mimicking toxoplasmosis.

A 37-year-old man, a hepatitis B virus carrier due to mother-to-child transmission, had a medical examination in September 2008 in nearby hospitals due to anorexia and weight loss. He was transported to our hospital because computed tomography (CT) detected intracranial lesions, and he had a positive human immunodeficiency virus (HIV) antibody test. Head computed tomography (CT) revealed multiple hemorrhagic lesions and enhancement effect, suggesting a thin wall. Also, an enhancement effect was present in the ventricle walls and the subarachnoid space. No accumulation was found in the thallium-201 scintigraphy. The enhancement effect of the ventricle walls and the subarachnoid space disappeared after oral administration of pyrimethamine, sulfadiazine, and calcium folinate, contributing to the diagnosis of an abscess and meningitis due to toxoplasma. However, mass lesions did not reduce. A biopsy was performed on 30 October, and the pathological diagnosis was malignant lymphoma. He died from respiratory function deterioration on 8 November. Lymphoma cells were found in ventricle wall tissue and the subarachnoid space at the autopsy. Toxoplasmosis will typically occur as a brain lesion most commonly in acquired immune deficiency syndrome (AIDS), whereas malignant lymphoma commonly manifests as a brain neoplastic lesion. However, differentiating between images of these lesions is difficult, so diagnosis by early biopsy is recommended.

Primary central nervous system large B-cell lymphoma with prolific, mixed T-cell and macrophage infiltrates, mimicking multiple sclerosis.

Although tissue confirmation is essential for a diagnosis of primary central nervous system large B-cell lymphoma (PCNSBL), accurate assessment may still be difficult, even when tissue is obtained. We report a 59-year-old man, first diagnosed as multiple sclerosis by open biopsy at another institution, who was then correctly diagnosed as PCNSBL after stereotactic biopsy at our hospital. The initial biopsy showed heavy lymphoid and macrophage influx with visible demyelination. On rebiopsy, a diffuse infiltrate of small to medium-sized lymphocytes was prominent and largely stained as T cells (CD3) by immunohistochemistry. There was also an admixture of macrophages, but this time, relatively low numbers of large malignant cells were also identified. The latter stained as B cells (CD20), enabling a diagnosis of B-cell lymphoma, and the condition responded fully to high-dose methotrexate. It is thus possible for PCNSBL to be histologically misinterpreted as a result of ancillary inflammation, characterized here as a profusion of T cells and macrophages.