Manabu Natsumeda

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.

Clinicopathological factors related to regrowth of vestibular schwannoma after incomplete resection

OBJECT The authors retrospectively analyzed various clinicopathological factors to determine which are related to regrowth during a long-term follow-up period in patients who underwent incomplete vestibular schwannoma (VS) resection. METHODS This study involved 74 patients (25 men and 49 women) in whom a VS was treated surgically via the lateral suboccipital approach, and who had postoperative follow-up periods exceeding 5 years. The mean follow-up was 104.1 months (range 60-241 months), and the mean patient age at surgery was 48.1 years (range 19-75 years). The tumors ranged in size from 0 mm (localized within the internal auditory canal) to 56 mm (28.3 ± 12.2 mm [mean ± SD]). RESULTS Gross-total resection (GTR) was performed in 41 (55%) of the 74 patients; subtotal resection ([STR]; 90-99%) in 25 (34%); and partial resection ([PR]; < 90%) in 8 (11%). Regrowth rates in the GTR, STR, and PR groups were 2.4% (1 of 41 cases), 52% (13 of 25), and 62.5% (5 of 8), respectively, and the times to regrowth ranged from 6 to 76 months (median 31.9 months). The regrowth-free survival curves differed significantly between the complete (GTR) and incomplete (STR and PR) resection groups. Eighteen (54.5%) of the 33 patients who underwent incomplete resection showed evidence of regrowth during follow-up. Univariate and multivariate analyses of various factors revealed that both the thickness of the residual tumor, based on MR imaging after surgery, and the MIB-1 index were positively related to residual tumor regrowth. The receiver operating characteristic curves, plotted for both the thickness of the residual tumor and the MIB-1 index, identified the optimal cutoff points for these values as 7.4 mm (sensitivity 83.3%, specificity 86.7%) and 1.6 (sensitivity 83.3%, specificity 66.7%), respectively. CONCLUSIONS Greater residual tumor thickness, based on MR imaging after the initial surgery, and a higher MIB-1 index are both important factors related to postoperative tumor regrowth in patients who have undergone incomplete VS resection. These patients require frequent neuroimaging investigation during follow-up to assure early detection of tumor regrowth.

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.

Intraventricular pleomorphic xanthoastrocytoma with anaplastic features

Pleomorphic xanthoastrocytoma (PXA) is a rare astrocytic tumor that usually occurs in the superficial cerebral hemispheres of children and young adults and has a relatively favorable prognosis. We report an unusual case of supratentorial, intraventricular tumor in a 52‐year‐old man. The tumor was composed of pleomorphic cells, including giant cells, most of which were multinucleated, and small cells. In addition, frequent xanthic changes in the cytoplasm of the tumor cells, and widespread reticulin deposits and lymphocytic infiltrates in the stroma were characteristic features. Large areas of necrosis were also evident. However, mitotic figures were rare (1–2 mitoses per 10 high‐power fields). Many tumor cells were positive for GFAP, and a number were positive for neurofilament protein and synaptophysin, indicating their neuronal differentiation. In addition, occasional tumor cells were positive for CD34. p53 protein was entirely negative in the tumor cells. In diagnosing this tumor histopathologically, differentiation between PXA and giant cell glioblastoma (GCG), a rare variant of glioblastoma, was problematic. However, considering the overall histopathological picture, a final diagnosis of PXA with anaplastic features was made. The present case indicates that PXA can occur as an intraventricular tumor, and suggests that in some instances, it would be very difficult to differentiate PXA and GCG histopathologically.

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 glioblastoma

The present study aimed to identify genes associated with patient survival to improve our understanding of the underlying biology of gliomas. We investigated whether the expression of genes selected using random survival forests models could be used to define glioma subgroups more objectively than standard pathology. The RNA from 32 non‐treated grade 4 gliomas were analyzed using the GeneChip Human Genome U133 Plus 2.0 Expression array (which contains approximately 47 000 genes). Twenty‐five genes whose expressions were strongly and consistently related to patient survival were identified. The prognosis prediction score of these genes was most significant among several variables and survival analyses. The prognosis prediction score of three genes and age classifiers also revealed a strong prognostic value among grade 4 gliomas. These results were validated in an independent samples set (n = 488). Our method was effective for objectively classifying grade 4 gliomas and was a more accurate prognosis predictor than histological grading.

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.

Radiation-Induced Glioblastoma Following Radiotherapy for Pituitary Adenomas: Marked Response to Chemotherapy

Radiation-induced glioblastoma is particularly resistant to treatment, and therapeutic options are limited. We report a 48-year-old woman with a radiation-induced glioblastoma who had a complete response to nimustine hydrochloride. Our patient developed a left temporal meninigioma 22 years after and bi-temporal glioblastoma 30 years after a pituitary adenoma was treated with surgery and 50 Gy radiation therapy. She was treated with subtotal resection followed by four cycles of nimustine hydrochloride; a complete response was achieved. She relapsed 16 months after diagnosis of glioblastoma, and underwent further surgery and treatment with temozolomide. She survived for 26 months after the onset of glioblastoma. As the site was within the irradiated area, both meninigioma and glioblastoma were thought to be radiation-induced tumors. If further radiotherapy is not a therapeutic option for glioblastoma, chemotherapy may result in prolonged survival.