Photo-immune therapy with liposomally formulated phospholipid-conjugated indocyanine green induces specific antitumor responses with heat shock protein-70 expression in a glioblastoma model.

Glioblastoma (GBM) is the most common malignant brain tumor, and infiltrates into the surrounding normal brain tissue. Induction of a tumor-specific immune response is one of the best methods to obtain tumor-specific cytotoxicity. Photodynamic therapy (PDT) is known to effectively induce an antitumor immune response. We have developed a clinically translatable nanoparticle, liposomally formulated phospholipid-conjugated indocyanine green (LP-iDOPE), applicable for PDT. This nanoparticle accumulates in tumor tissues by the enhanced permeability and retention effect, and releases heat and singlet oxygen to injure cancer cells when activated by near infrared (NIR) light. We assessed the effectiveness of the LP-iDOPE system in brain using the rat 9L glioblastoma model. Treatment with LP-iDOPE and NIR irradiation resulted in significant tumor growth suppression and prolongation of survival. Histopathological examination showed induction of both apoptosis and necrosis and accumulation of CD8+ T-cells and macrophages/microglia accompanied by marked expressions of heat shock protein-70 (HSP70), which was not induced by mild hyperthermia alone at 45° C or an interleukin-2-mediated immune reaction. Notably, the efficacy was lost in immunocompromised nude rats. These results collectively show that the novel nanoparticle LP-iDOPE in combination with NIR irradiation can efficiently induce a tumor-specific immune reaction for malignant gliomas possibly by inducing HSP70 expression which is known to activate antigen-presenting cells through toll-like receptor signaling.

[Clinical Characteristics of Craniocervical Junction Arteriovenous Fistulas].

OBJECTIVE: Craniocervical junction arteriovenous fistulas(CCJ-AVFs)are extremely rare lesions that may result in both subarachnoid hemorrhage(SAH)and myelopathy. Diagnosis of CCJ-AVF is difficult and may be delayed due to variable clinical features and a spectrum of neuroradiological findings. To elucidate the clinical characteristics of CCJ-AVF, we analyzed the clinical symptoms, neuroimaging findings, and the results of surgical treatment in five patients. RESULTS: Among the five patients, four were diagnosed with dural AVFs, and the remaining patient was diagnosed with radicular AVF. Two of the five patients presented with SAH, and the rest presented with myelopathy. In both the SAH patients, the initial digital subtraction angiography(DSA)failed to reveal the AVFs, and a definitive diagnosis was made only after repeated DSAs. In two of the three myelopathy patients, the diagnosis was delayed because of nonspecific chronic neurological symptoms which resembled a thoracolumbar lesion. Four patients underwent shunt occlusion through direct surgery and demonstrated favorable outcomes. One myelopathy patient, however, demonstrated abrupt onset, associated with progressive neurological deterioration, which resulted in poor prognosis. The magnetic resonance imaging(MRI)findings, which included intramedullary high intensity on a T2 weighted image, flow void, and varix at the cervical cord, were specific for the myelopathy patients. CONCLUSION: A thorough 4-vessel DSA study, including the cervical region, is mandatory for SAH patients whose clots are predominantly in the posterior fossa, and repeated DSA must be considered in cases of unknown origin. CCJ-AVF may cause myelopathy, with symptoms such as urinary dysfunction and/or paraparesis. Screening with a cervical MRI is useful for detecting CCJ-AVF in cases of myelopathy. Emergency radical treatment must be attempted for those patients demonstrating abrupt onset associated with symptoms of progressive deterioration, such as respiratory dysfunction or bulbar palsy.

[Hemorrhagic Onset of Subependymal Giant Cell Astrocytoma Associated with Tuberous Sclerosis:A Case Report and Review of Literature].

We report on a case of subependymal giant cell astrocytoma(SEGA)in a patient with tuberous sclerosis(TSC)that presented with intratumoral hemorrhage and acute hydrocephalus. Initial treatment was external ventricular drainage to control the intracranial pressure;however, the tumor increased in size due to recurrent hemorrhage. Subsequently, the tumor was successfully removed via the transcortical-transventricular approach without neurological deterioration. Although intratumoral hemorrhage is extremely rare in patients with SEGA, subsequent acute hydrocephalus resulting from obstruction of the foramen of Monro will be fatal if prompt surgical treatment is not available. Careful and periodical radiographic examination of the central nervous system will be mandatory in patients with TSC, especially in those who have subependymal nodules(SEN)or SEGA around the foramen of Monro. Radical surgical removal should be considered before they become symptomatic.

The Pluripotent Stem-Cell Marker Alkaline Phosphatase is Highly Expressed in Refractory Glioblastoma with DNA Hypomethylation.

Background: Hypomethylation of genomic DNA induces stem-cell properties in cancer cells and contributes to the treatment resistance of various malignancies. Objective: To examine the correlation between the methylation status of stem-cell-related genes and the treatment outcomes in patients with glioblastoma (GBM). Methods: The genome-wide DNA methylation status was determined using HumanMethylation450 BeadChips, and the methylation status was compared between a group of patients with good prognosis (survival > 4 yr) and a group with poor prognosis (survival < 1 yr). Immunohistochemistry for proteins translated from hypomethylated genes, including alkaline phosphatase (ALPL), CD133, and CD44, was performed in 70 GBMs and 60 oligodendroglial tumors. Results: The genomic DNA in refractory GBM was more hypomethylated than in GBM from patients with relatively long survival (P = .0111). Stem-cell-related genes including ALPL, CD133, and CD44 were also significantly hypomethylated. A validation study using immunohistochemistry showed that DNA hypomethylation was strongly correlated with high protein expression of ALPL, CD133, and CD44. GBM patients with short survival showed high expression of these stem-cell markers. Multivariate analysis confirmed that co-expression of ALPL + CD133 or ALPL + CD44 was a strong predictor of short survival. Anaplastic oligodendroglial tumors without isocitrate dehydrogenase 1 mutation were significantly correlated with high ALPL expression and poor survival. Conclusion: Accumulation of stem-cell properties due to aberrant DNA hypomethylation is associated with the refractory nature of GBM.

Transforming growth factor-ß and stem cell markers are highly expressed around necrotic areas in glioblastoma.

Invasion into surrounding normal brain and resistance to genotoxic therapies are the main devastating aspects of glioblastoma (GBM). These biological features may be associated with the stem cell phenotype, which can be induced through a dedifferentiation process known as epithelial-mesenchymal transition (EMT). We show here that tumor cells around pseudopalisading necrotic areas in human GBM tissues highly express the most important EMT inducer, transforming growth factor (TGF-ß), concurrently with the EMT-related transcriptional factor, TWIST. In addition, the stem cell markers CD133 and alkaline phosphatase (ALPL) were also highly expressed around necrotic foci in GBM tissues. The high expression of TGF-ß around necrotic regions was significantly correlated with shorter progression-free survival and overall survival in patients with GBM. High expression of stem cell markers, ALPL, CD133, and CD44 was also correlated with poor outcomes. These results collectively support the hypothesis that tissue hypoxia induces the stem cell phenotype through TGF-ß-related EMT and contributes to the poor outcome of GBM patients.

Molecular imaging of 1p/19q deletion in oligodendroglial tumours with 11C-methionine positron emission tomography.

OBJECTIVE: Chromosome 1p/19q deletion is an established prognostic and predictive marker in the WHO grade III oligodendroglial tumours (OT). To estimate the genetic status preoperatively, the authors investigated the correlation between the uptake of (11)C-methionine in positron emission tomography (PET) and the 1p/19q status in grades II and III OT. METHODS: We retrospectively reviewed 144 patients with gliomas who received (11)C-methionine PET. 66 cases with grades II-III oligodendrogliomas or oligoastrocytomas underwent fluorescence in situ hybridisation to determine the 1p/19q status. The tissue uptake of (11)C-methionine was expressed as the ratio of the maximum standardised uptake value (SUVmax) in tumour areas to the mean SUV (SUVmean) in the contralateral normal brain (tumour-to-normal tissue (T/N) ratio). RESULTS: The T/N ratio in (11)C-methionine PET was significantly higher in grade III OT than in grade II tumours. The mean T/N ratio of the grade II tumours without 1p/19q deletion was significantly higher than that of the grade II tumours with 1p/19q deletion (mean 2.67 vs 1.94, respectively; p=0.0457). In grade III tumours, the mean T/N ratio of the tumours without 1p/19q deletion was also significantly higher than that of the tumours with 1p/19q deletion (mean 4.83 vs 3.49, respectively; p=0.0261). The rate of IDH1 mutation was lower and the rate of contrast enhancement on MRIs was higher in the 1p/19q non-deleted OT than those with 1p/19q deletion, which may contribute to the high T/N ratio. CONCLUSIONS: Among suspected OT, (11)C-methionine PET may help us preoperatively discriminate tumours with and without 1p/19q deletion.

Liposomally formulated phospholipid-conjugated indocyanine green for intra-operative brain tumor detection and resection.

Some tumor-specific near-infrared (NIR) fluorescent dyes such as indocyanine green (ICG), IDRye800CW, and 5-aminolevulinic acid have been used clinically for detecting tumor margins or micro-cancer lesions. In this study, we evaluated the physicochemical properties of liposomally formulated phospholipid-conjugated ICG, denoted by LP-iDOPE, as a clinically translatable NIR imaging nanoparticle for brain tumors. We also confirmed its brain-tumor-specific biodistribution and its characteristics as the intra-operative NIR imaging nanoparticles for brain tumor surgery. These properties of LP-iDOPE may enable neurosurgeons to achieve more accurate identification and more complete resection of brain tumor.

IDH1 mutation is prognostic for diffuse astrocytoma but not low-grade oligodendrogliomas in patients not treated with early radiotherapy.

Despite accumulating knowledge regarding molecular backgrounds, the optimal management strategy for low-grade gliomas remains controversial. One reason is the marked heterogeneity in the clinical course. To establish an accurate subclassification of low-grade gliomas, we retrospectively evaluated isocitrate dehydrogenase-1 (IDH1) mutation in clinical specimens of diffuse astrocytomas (DA) and oligodendroglial tumors separately. No patients were treated with early radiotherapy, and modified PCV chemotherapy was used for postoperative residual tumors or recurrence in oligodendroglial tumors. Immunohistochemical evaluation of IDH status, p53 status, O(6)-methylguanine methyltransferase expression, and the MIB-1 index were performed. The 1p and 19q status was analyzed with fluorescence in situ hybridization. Ninety-four patients were followed for a median period of 8.5 years. For DAs, p53 was prognostic for progression- free survival (PFS) and IDH1 was significant for overall survival (OS) with multivariate analysis. In contrast, for oligodendroglial tumors, none of the parameters was significant for PFS or OS. Thus, the significance of IDH1 mutation is not clear in oligodendroglial tumors that are homogeneously indolent and chemosensitive. In contrast, DAs are heterogeneous tumors including some potentially malignant tumors that can be predicted by examining the IDH1 mutation status.

Aqueous microwave-assisted solid-phase peptide synthesis using Fmoc strategy. III: racemization studies and water-based synthesis of histidine-containing peptides.

In this study, we describe the first aqueous microwave-assisted synthesis of histidine-containing peptides in high purity and with low racemization. We have previously shown the effectiveness of our synthesis methodology for peptides including difficult sequences using water-dispersible 9-fluorenylmethoxycarbonyl-amino acid nanoparticles. It is an organic solvent-free, environmentally friendly method for chemical peptide synthesis. Here, we studied the racemization of histidine during an aqueous-based coupling reaction with microwave irradiation. Under our microwave-assisted protocol using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, the coupling reaction can be efficiently performed with low levels of racemization of histidine. Application of this water-based microwave-assisted protocol with water-dispersible 9-fluorenylmethoxycarbonyl-amino acid nanoparticles led to the successful synthesis of the histidine-containing hexapeptide neuropeptide W-30 (10-15), Tyr-His-Thr-Val-Gly-Arg-NH2, in high yield and with greatly reduced histidine racemization.

Circulating anti-filamin C autoantibody as a potential serum biomarker for low-grade gliomas.

BACKGROUND: Glioma is the most common primary malignant central nervous system tumor in adult, and is usually not curable due to its invasive nature. Establishment of serum biomarkers for glioma would be beneficial both for early diagnosis and adequate therapeutic intervention. Filamins are an actin cross-linker and filamin C (FLNC), normally restricted in muscle tissues, offers many signaling molecules an essential communication fields. Recently, filamins have been considered important for tumorigenesis in cancers. METHODS: We searched for novel glioma-associated antigens by serological identification of antigens utilizing recombinant cDNA expression cloning (SEREX), and found FLNC as a candidate protein. Tissue expressions of FLNC (both in normal and tumor tissues) were examined by immunohistochemistry and quantitative RT-PCR analyses. Serum anti-FLNC autoantibody level was measured by ELISA in normal volunteers and in the patients with various grade gliomas. RESULTS: FLNC was expressed in glioma tissues and its level got higher as tumor grade advanced. Anti-FLNC autoantibody was also detected in the serum of glioma patients, but its levels were inversely correlated with the tissue expression. Serum anti-FLNC autoantibody level was significantly higher in low-grade glioma patients than in high-grade glioma patients or in normal volunteers, which was confirmed in an independent validation set of patients' sera. The autoantibody levels in the patients with meningioma or cerebral infarction were at the same level of normal volunteers, and they were significantly lower than that of low-grade gliomas. Total IgG and anti-glutatione S-transferase (GST) antibody level were not altered among the patient groups, which suggest that the autoantibody response was specific for FLNC. CONCLUSIONS: The present results suggest that serum anti-FLNC autoantibody can be a potential serum biomarker for early diagnosis of low-grade gliomas while it needs a large-scale clinical study.

Non-deep-seated primary CNS lymphoma: therapeutic responses and a molecular signature.

The survival of patients with primary CNS lymphoma (PCNSL) has been improved by high-dose methotrexate (HD-MTX). Since the combination therapy of HD-MTX and whole-brain radiotherapy (WBRT) carries a significant risk for delayed neurotoxicity, it is important to know the therapeutic potential and prognostic factors for HD-MTX without WBRT. We retrospectively reviewed 46 consecutive patients with PCNSL treated with a HD-MTX (3.5 g/m(2)) and deferred WBRT. Patients who achieved complete response or partial response after three courses of HD-MTX were cautiously followed-up without additional treatment. Patients who had either stable disease, progressive disease, or disease relapse were offered salvage therapy. The median progression-free survival period was 10 months and the median overall survival period was 52 months, with a 5-year survival rate of 39 %. Nineteen patients (49 % of the evaluable patients) achieved a complete response at the initial response assessment. Involvement of deep structures of the brain (corpus callosum, basal ganglia and brainstem) was significantly associated with the worse progression-free survivals (p = 0.0058) and overall survivals (p = 0.0177). Gene expression profiling analysis by microarray was compared in eight patients between PCNSLs located in the deep structures of the brain and non-deep-seated tumors. The result showed that up-regulation of signal transduction-related genes and down-regulation of catalytic activity-related genes in the non-deep-seated PCNSL compared with the deep-seated tumors. The present study shows that PCNSL located in non-deep structures of the brain responds better to HD-MTX alone than those involved deep-structures.

Aqueous microwave-assisted solid-phase peptide synthesis using fmoc strategy. II. Racemization studies and water based synthesis of cysteine-containing peptides.

We have developed a microwave (MW)-assisted peptide synthesis using Fmoc-amino acid nanoparticles in water previously. It is an organic solvent-free, environmentally friendly method for peptide synthesis. In this study, we have investigated the racemization of cysteine during an aqueous based coupling reaction with MW irradiation. Under our MW-assisted protocol using WSCI and DMTMM, the coupling reaction can be performed with low levels of racemization of cysteine. We also demonstrated the synthesis of the nonapeptide oxytocin analogue, Cys(Acm)-Tyr-Ile-Gln-Asn- Cys(Acm)-Pro-Leu-Gly-NH2 using our water based MW-assisted protocol with Fmoc-amino acid nanoparticles.

Anaplastic oligodendroglial tumors harboring 1p/19q deletion can be successfully treated without radiotherapy.

Although anaplastic oligodendroglial tumors are known to be chemosensitive, patients under this diagnosis have been traditionally treated with radiotherapy. To avoid possible neurotoxicity, we prospectively treated patients with anaplastic oligodendroglial tumors harboring 1p/19q deletion, with exclusive procarbazine, ACNU, and vincristine chemotherapy without radiotherapy. Twenty-five patients were enrolled in the study (12 with 1p/19q co-deletion, 2 with 1p mono-deletion, 2 with 19q mono-deletion, and 9 without 1p/19q deletion). The median progression-free survival (PFS) was 50 months for all the patients, and those with tumors harboring 1p/19q deletion were progression free for a significantly longer period than those without the deletion (p=0.0391). The median overall survival (OS) time was not reached in both patient groups with and without 1p/19q deletion (p=0.230), and the 5-year OS rate was 62.2% for all patients. The excellent treatment results warrant a large-scale clinical study to confirm the efficacy of upfront chemotherapy omitting radiotherapy as initial therapy for anaplastic oligodendroglial tumors with 1p/19q deletion.

Discrimination between low-grade oligodendrogliomas and diffuse astrocytoma with the aid of 11C-methionine positron emission tomography.

OBJECT: The diagnostic usefulness of (11)C-methionine PET scans in gliomas is still controversial. The authors investigated the clinical significance of (11)C-methionine PET findings in preoperative diagnosis of histological type and grade. METHODS: The tissue uptake of (11)C-methionine was assessed using PET in 70 patients with histologically confirmed intracerebral gliomas. The ratio of maximum standard uptake values in tumor areas to the mean standard uptake values in the contralateral normal brain tissue (tumor/normal tissue [T/N] ratio) was calculated and correlated with tumor type, histological grade, contrast enhancement on MR imaging, Ki 67 labeling index, and 1p/19q status. RESULTS: The T/N ratio was significantly increased as tumor grade advanced in astrocytic tumors (WHO Grade II vs Grade III, p = 0.0011; Grade III vs Grade IV, p = 0.0007). Among Grade II gliomas, the mean T/N ratio was significantly higher in oligodendroglial tumors than in diffuse astrocytomas (DAs) (p < 0.0001). All T/N ratios for oligodendroglial tumors were ≥ 1.46, and those for DA were consistently < 1.46, with the exception of 2 cases of gemistocytic astrocytoma. The Ki 67 labeling index significantly correlated with T/N ratio in astrocytic tumors, but not in oligodendrogliomas. Oligodendroglial tumors without 1p/19q deletion had a significantly higher T/N ratio than those with the codeletion. In combination with Gd-enhanced MR imaging, 67% of nonenhanced tumors with a T/N ratio of ≥ 1.46 were proved to be Grade II oligodendrogliomas. CONCLUSIONS: These results clearly show that (11)C-methionine PET T/N ratios in Grade II oligodendrogliomas were higher than those in DAs independently of their proliferative activity. This information contributes to preoperative differential diagnoses of histological type, especially in suspected low-grade gliomas.

Favorable long-term outcome of low-grade oligodendrogliomas irrespective of 1p/19q status when treated without radiotherapy.

Despite the accumulating evidences of high chemosensitivity especially in anaplastic oligodendrogliomas with loss of chromosomes 1p and 19q, the optimal management strategy for low-grade tumors using the 1p/19q information remains controversial. We have treated all low-grade oligodendrogliomas by a chemotherapy-preceding strategy without radiotherapy, and here we analyzed the survival outcomes of 36 consecutive patients in relation to 1p/19q status. The treatment protocol was as follows: (1) simple observation after gross total resection, and (2) modified PCV chemotherapy for postoperative residual tumors or recurrence after total resection. The 1p and 19q status were analyzed by fluorescence in situ hybridization. The median follow-up period was 7.5 years and no patient was lost during the follow-up periods. 1p/19q co-deletion was observed in 72% of the patients, and there was no significant association between 1p/19q co-deletion and chemotherapy response rate. The 5- and 10-year progression-free survival (PFS) rate was 75.1 and 46.9%, respectively, and the median PFS was 121 months for 1p/19q-deleted tumors and 101 months for non-deleted tumors (log-rank test: P = 0.894). Extent of surgery did not affect PFS (P = 0.685). In contrast, the elder patients (>50) had significantly shorter PFS (P = 0.0458). Recurrent tumors were well controlled by chemotherapy irrespective of 1p/19q status, and 35 out of 36 patients survived without receiving radiotherapy. The 5- and 10-year overall survival rates were 100 and 93.8%, respectively. Two of the patients in their sixties (29%) suffered from severe cognitive dysfunctions and marked brain atrophy following chemotherapy alone. These results show that low-grade oligodendrogliomas could be successfully treated by surgical resection and nitrosourea-based chemotherapy alone without radiotherapy irrespective of 1p/19q status.

Selection of chemotherapy for glioblastoma expressing O(6)-methylguanine-DNA methyltransferase.

The therapeutic benefit of nitrosoureas or temozolomide for glioblastoma is limited mainly by O(6)-methylguanine-DNA methyltransferase (MGMT) expression. The aim of this study was to evaluate the effectiveness of various anticancer drugs for MGMT-positive glioblastoma. Seventy-four glioblastoma patients were administered various anticancer drugs according to drug sensitivity testing. For the individualization, drug-induced apoptosis was quantified by flow cytometry in the primary culture of surgically resected tumor cells. The MGMT protein expression was analyzed by immunohistochemistry. The median survival of the patients receiving the individualized chemotherapy was 19.4 months (95% CI, 15.9-22.1). The patients with negative MGMT immunostaining had significantly longer survival than those with positive MGMT immunostaining [median survival, 22.3 months (95% CI, 17.6-27.0) vs. 15.1 months (95% CI, 13.4-16.8); p=0.0188]. For MGMT-positive tumors, the platinum agents and the taxanes were more frequently selected for administration than the other categories of anticancer agents. The patient survival period of MGMT-positive glioblastomas treated with the platinum agents or the taxanes [median survival, 20.1 months (95% CI, 18.0-22.7)] was significantly longer than that of MGMT-positive tumors treated with nitrosoureas (p=0.0026), and was equivalent to that of MGMT-negative glioblastomas (p=0.3047). These results suggest that the platinum agents and the taxanes offer the best probability to be effective against immunohistochemically MGMT-positive glioblastomas.