Hiroshi Aoki

Mild Heat Shock Induces Autophagic Growth Arrest, But Not Apoptosis in U251-MG and U87-MG Human Malignant Glioma Cells

Although hyperthermia has been used as a treatment of malignant brain tumors, it is not yet clear what is the mechanism of the cell growth inhibition by heat shock, especially by the temperature which has clinically been applied to tumor–brain border-zone, 42–43°C. Therefore, we evaluated the change of U251-MG and U87-MG human malignant glioma cells after 43°C-heat shock comparing with that of 45°C. First, we observed that cell growth was transiently inhibited after 43°C-heat shock for 3 or 5 days, in U251-MG or U87-MG cells, respectively, which was followed by regrowth. During the period of transient growth inhibition, mild G2/M arrest was observed. However, apoptosis was observed in only 2.7% or 1.5%, of 43°C-heated cells, in U251-MG or U87-MG cells, respectively. Instead, transmission electron micrography showed the formation of vacuoles, degeneration of mitochondria, and autophagosomes. Moreover, in the both cell lines, flow-cytometric analysis with acridine orange revealed the induction of acidic vesicle organelles, which was blocked by 3-methyladenine (3-MA), suggesting the involvement of autophagy. Furthermore, while 3-MA did not increase the anti-tumor effect of 43°C-heat shock, bafilomycin A1, another autophagy inhibitor, did significantly enhance the effect in U251-MG cells. Taken together, mild heat shock (43°C for 2 h) causes autophagy and mild G2/M arrest, but does not induce apparent apoptosis in U251-MG and U87-MG glioma cells. Inhibition of autophagy with bafilomycin A1 may increase the anti-tumor efficacy of mild heat shock against some malignant glioma cells.

Therapeutic efficacy of targeting chemotherapy using local hyperthermia and thermosensitive liposome: evaluation of drug distribution in a rat glioma model

A method was developed of targeting chemotherapy using thermosensitive liposomes to treat malignant gliomas. Using the brain heating system, when the tumour core is heated to >43°C, the tumour infiltrating zone is exposed to mild hyperthermia (40–43°C). Thermosensitive liposomes were designed to release their contents at 40°C to target both the tumour core and tumour infiltrating zone. The present study investigated the anti-tumour effect on rat glioma models in tumour drug uptake and tumour growth delay studies. Elevated accumulation of ADR in the rat C6 glioma after treatment was obtained in the area heated to >40°C. However, there was no significant difference between the areas heated to 40–42°C and >43°C. Furthermore, it was found that ADR concentrations in the mildly hyperthermic areas were significantly higher following treatment with liposomal ADR than with free ADR. The animals treated with the new combination therapy had significantly longer overall survival time in comparison to those receiving other treatments. Thus, thermosensitive liposomes release their contents in response to mild hyperthermia and this combination therapy has a greater therapeutic efficacy for malignant brain tumours. This method is a promising approach for the treatment of malignant glioma patients.

Induction of autophagy in temozolomide treated malignant gliomas

Autophagy is a dynamic process of protein degradation. Induction of autophagy by temozolomide (TMZ) has been noted in glioma cell lines. Twenty‐eight specimens, obtained from 14 patients before and after TMZ treatment, were analyzed to investigate whether induction of autophagy could be detected in surgical specimens by immunohistochemical analysis. Macroautophagy was monitored by immunohistochemical analysis employing anti‐light chain 3 isoform B (LC3B) and anti‐lysosome‐associated membrane protein 1 (LAMP1) antibodies; chaperone‐mediated autophagy was monitored by anti‐LAMP2A antibody immunostaining. Furthermore, detection of LC3B protein by Western blotting was performed on six specimens obtained from the preserved frozen tissues of three patients. All specimens showed dot‐like staining for each immunostain in the cytoplasm of glioma cells, indicating induction of autophagy. LC3B, LAMP1 and LAMP2A immunostains were semiquantitatively scored from 1 to 3 points. Combination of the three scores after TMZ treatment (6.4 ± 1.2) showed a significant increase (P = 0.020) compared to pre‐treatment scores (5.2 ± 1.5). Western blotting for LC3B showed increased LC3B‐I and LC3B‐II expression after TMZ treatment. The present study proved that autophagy monitoring by immunohistochemical staining of surgical specimens was feasible. These results suggest that autophagy is induced by TMZ.

Accumulation of 2-hydroxyglutarate in gliomas correlates with survival: a study by 3.0-tesla magnetic resonance spectroscopy

IntroductionPrevious magnetic resonance spectroscopy (MRS) and mass spectroscopy studies have shown accumulation of 2-hydroxyglutarate (2HG) in mutant isocitrate dehydrogenase (IDH) gliomas. IDH mutation is known to be a powerful positive prognostic marker in malignant gliomas. Hence, 2HG accumulation in gliomas was assumed to be a positive prognostic factor in gliomas, but this has not yet been proven. Here, we analyzed 52 patients harboring World Health Organization (WHO) grade II and III gliomas utilizing 3.0-tesla MRS.ResultsMutant IDH gliomas showed significantly higher accumulation of 2HG (median 5.077 vs. 0.000, p =0.0002, Mann-Whitney test). 2HG was detectable in all mutant IDH gliomas, whereas in 10 out of 27 (37.0%) wild-type IDH gliomas, 2HG was below the detectable range (2HG =0) (p =0.0003, chi-squared test). Screening for IDH mutation by 2HG analysis was highly sensitive (cutoff 2HG =1.489 mM, sensitivity 100.0%, specificity 72.2%). Gliomas with high 2HG accumulation had better overall survival than gliomas with low 2HG accumulation (p =0.0401, Kaplan-Meier analysis).Discussion2HG accumulation detected by 3.0-tesla MRS not only correlates well with IDH status, but also positively correlates with survival in WHO grade II and III gliomas.

Immunohistochemical profiles of IDH1, MGMT and P53: Practical significance for prognostication of patients with diffuse gliomas

Genetic and epigenetic status, including mutations of isocitrate dehydrogenase (IDH) and TP53 and methylation of O6‐methylguanine‐DNA methyltransferase (MGMT), are associated with the development of various types of glioma and are useful for prognostication. Here, using routinely available histology sections from 312 patients with diffuse gliomas, we performed immunohistochemistry using antibodies specific for IDH1 mutation, MGMT methylation status, and aberrant p53 expression to evaluate the possible prognostic significance of these features. With regard to overall survival (OS), univariate analysis indicated that an IDH1‐positive profile in patients with glioblastoma (GBM), anaplastic astrocytoma (AA), anaplastic oligoastrocytoma and oligodendroglioma, or a MGMT‐negative profile in patients with GBM and AA were significantly associated with a favorable outcome. Multivariate analysis revealed that both profiles were independent factors influencing prognosis. The OS of patients with IDH1‐positive/MGMT‐negative profiles was significantly longer than that of patients with negative/negative and negative/positive profiles. A p53 profile was not an independent prognostic factor. However, for GBM/AA patients with IDH1‐negative/MGMT‐negative profiles, p53 overexpression was significantly associated with an unfavorable outcome. Thus, the immunohistochemical profiles of IDH1 and MGMT are of considerable significance in gliomas, and a combination of IDH1, MGMT and p53 profiles may be useful for prognostication of GBM/AA.

Epstein‐Barr virus‐associated primary central nervous system cytotoxic T‐cell lymphoma

Primary central nervous system lymphoma (PCNSL) expressing T‐cell markers is rare, among which nasal‐type extranodal NK/T‐cell lymphoma is an extremely rare subtype associated with Epstein‐Barr virus (EBV) infection. Here we report the clinicopathologic features of a case of EBV‐associated PCNSL showing a cytotoxic T‐cell phenotype. The patient, a 73‐year‐old woman, presented with rapidly progressive mental deterioration. Brain MRI revealed multiple lesions with swelling in the bilateral cerebral hemispheres, which were hypointense on T1‐weighted images, hyperintense on T2‐weighted and fluid‐attenuated inversion recovery images, and slightly hyperintense on diffusion‐weighted images. Biopsy specimens from the temporal region showed many medium‐sized anaplastic lymphocytic cells with perivascular and angio‐invasive patterns in the cortex. Immunohistochemically, the cells were positive for CD3, CD8, T‐cell‐restricted intracellular antigen‐1 (TIA‐1), granzyme B and perforin, but negative for CD56 and CD20. In situ hybridization revealed EBV‐encoded RNAs in the tumor cell nuclei. A rearrangement study showed T‐cell receptor γ–chain gene rearrangement with a clonal appearance. The patient died 6 months after surgery, and a general autopsy revealed no lymphoma cells outside the brain. These cellular profiles are inconsistent with those of extranodal NK/T‐cell lymphoma, and have not been previously described. This case appears to represent an unusual CNS manifestation of EBV‐associated T‐cell lymphoma.

Gene expression signature-based prognostic risk score in patients with primary central nervous system lymphoma.

Purpose: Better understanding of the underlying biology of primary central nervous system lymphomas (PCNSL) is critical for the development of early detection strategies, molecular markers, and new therapeutics. This study aimed to define genes associated with survival of patients with PCNSL. Experimental Design: Expression profiling was conducted on 32 PCNSLs. A gene classifier was developed using the random survival forests model. On the basis of this, prognosis prediction score (PPS) using immunohistochemical analysis is also developed and validated in another data set with 43 PCNSLs. Results: We identified 23 genes in which expressions were strongly and consistently related to patient survival. A PPS was developed for overall survival (OS) using a univariate Cox model. Survival analyses using the selected 23-gene classifiers revealed a prognostic value for high-dose methotrexate (HD-MTX) and HD-MTX–containing polychemotherapy regimen–treated patients. Patients predicted to have good outcomes by the PPS showed significantly longer survival than those with poor predicted outcomes (P < 0.0001). PPS using immunohistochemical analysis is also significant in test (P = 0.0004) and validation data set (P = 0.0281). The gene-based predictor was an independent prognostic factor in a multivariate model that included clinical risk stratification (P < 0.0001). Among the genes, BRCA1 protein expressions were most strongly associated with patient survival. Conclusion: We have identified gene expression signatures that can accurately predict survival in patients with PCNSL. These predictive genes should be useful as molecular biomarkers and they could provide novel targets for therapeutic interventions. Clin Cancer Res; 18(20); 5672–81. ©2012 AACR.

Suppressed Expression of Autophagosomal Protein LC3 in Cortical Tubers of Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is characterized by benign tumors and hamartomas, including cortical tubers. Hamartin and tuberin, encoded by the TSC 1 and 2 genes, respectively, constitute a functional complex that negatively regulates the mammalian target of rapamycin (mTOR) signaling pathway, eventually promoting the induction of autophagy. In the present study, we assessed the induction of autophagy in cortical tubers surgically removed from seven patients with TSC in comparison with five controls of cortical tissue taken from non‐TSC patients with epilepsy. Immunoblotting demonstrated a marked reduction of LC3B‐I and LC3B‐II in tubers relative to the controls. In tubers, strong, diffuse and dot‐like immunoreactivity (IR) for LC3B was observed in dysmorphic neurons and balloon cells, but LC3B‐IR in other neurons with normal morphology was significantly weaker than that in neurons in the controls. Immunoelectron microscopy revealed diffuse distribution of LC3B‐IR within the cytoplasm of balloon cells. The dot‐like pattern may correspond to abnormal aggregation bodies involving LC3. In an autopsy patient with TSC, we observed that LC3B‐IR in neurons located outside of the tubers was preserved. Thus, autophagy is suppressed in tubers presumably through the mTOR pathway, and possibly a pathological autophagy reaction occurs in the dysmorphic neurons and balloon cells.

Near-infrared spectroscopic study and the Wada test for presurgical evaluation of expressive and receptive language functions in glioma patients: With a case report of dissociated language functions

Near-infrared spectroscopy (NIRS) has proven to be useful for the evaluation of language lateralization in healthy subjects, infants, and epileptic patients. This study for the first time investigated the expressive and receptive language functions separately, using NIRS in presurgical glioma patients. We also describe a special case with dissociated pattern of language functions. Ten glioma patients were examined. Using NIRS, the hemodynamic changes during a verb generation task or story listening task were measured in the cerebral hemisphere on either side covering the language areas. Following the NIRS study, the Wada test was performed in all the patients. The NIRS study revealed increases of oxyhemoglobin and decreases of deoxyhemoglobin in the language areas elicited by both tasks. In 9 patients, who were all right-handed, the expressive and receptive language functions were lateralized to the left hemisphere. The results of the NIRS study were completely consistent with those of the Wada test. In the remaining 1 patient with a right sided insular glioma, who was right-handed, the NIRS study revealed stronger activation of the right inferior frontal region during the verb generation task, and stronger activation of the left superior temporal region during the story listening task. This dissociated language function was validated by the Wada test and the postoperative neurological course. These results demonstrate that a NIRS study using our technique is extremely valuable for preoperative assessment of the language functions and exemplifies how a preoperative NIRS study can allow detection of unforeseen language lateralization.

Mild hyperthermia plus adenoviral p53 over-expression additively inhibits the viability of human malignant glioma cells

Adenoviral replacement of the p53 gene has already been proved effective for the treatment of various tumours, including malignant gliomas. However, it is difficult to treat malignant glioma with p53 gene therapy alone because of problems with resistance or a less-than-satisfactory response to the treatment. This study investigated whether heat shock at 43°C (mild hyperthermia) augments the cytotoxic effect of p53 gene transfer on malignant glioma cells expressing wild-type p53 (D54) or mutant p53 (U373-MG and U251-MG). The combination of mild hyperthermia and adenoviral p53 over-expression had an additive inhibitory effect on cellular proliferation in all three cell lines studied. Further, both cell cycle analysis and a DNA fragmentation assay showed that apoptosis was induced by p53 over-expression alone but not by heat shock at 43°C alone. However, p53 over-expression followed by mild hyperthermia additively increased the proportion of cells in which apoptosis was induced, regardless of the endogenous p53 status of the tumour cells. Interestingly, a caspase-independent mechanism was observed to be involved in the p53-induced apoptosis in U251-MG and D54 cells. Taken together, the findings showed that combining adenoviral p53 transfer with mild hyperthermia inhibits the proliferation of malignant glioma cells in an additive manner, irrespective of their endogenous p53 status, suggesting a novel treatment strategy for this malignancy.