Yasuo Iwadate

Molecular Classification and Survival Prediction in Human Gliomas Based on Proteome Analysis

The biological features of gliomas, which are characterized by highly heterogeneous biological aggressiveness even in the same histological category, would be precisely described by global gene expression data at the protein level. We investigated whether proteome analysis based on two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry can identify differences in protein expression between high- and low-grade glioma tissues. Proteome profiling patterns were compared in 85 tissue samples: 52 glioblastoma multiforme, 13 anaplastic astrocytomas, 10 atrocytomas, and 10 normal brain tissues. We could completely distinguish the normal brain tissues from glioma tissues by cluster analysis based on the proteome profiling patterns. Proteome-based clustering significantly correlated with the patient survival, and we could identify a biologically distinct subset of astrocytomas with aggressive nature. Discriminant analysis extracted a set of 37 proteins differentially expressed based on histological grading. Among them, many of the proteins that were increased in high-grade gliomas were categorized as signal transduction proteins, including small G-proteins. Immunohistochemical analysis confirmed the expression of identified proteins in glioma tissues. The present study shows that proteome analysis is useful to develop a novel system for the prediction of biological aggressiveness of gliomas. The proteins identified here could be novel biomarkers for survival prediction and rational targets for antiglioma therapy.

Cathepsin D Is a Potential Serum Marker for Poor Prognosis in Glioma Patients

Cathepsin D is an aspartyl protease involved in protein catabolism and tissue remodeling which can be secreted from cancer cells. To identify a potential serum marker for gliomas, we investigated the gene expression levels of cathepsin D in 87 tissue samples and measured the protein concentrations in sera of glioma patients. The tissue samples consisted of 43 glioblastomas, 13 anaplastic astrocytomas, 22 astrocytomas, and 9 normal brain tissues. The results of real-time quantitative reverse transcription-PCR analysis showed that cathepsin D transcript levels became significantly higher as the glioma grade advanced (P = 0.0466, glioblastoma and anaplastic astrocytoma; P = 0.0008, glioblastoma and astrocytoma; P = 0.0271, glioblastoma and normal brain tissue; unpaired t test). Immunohistochemical analysis with anti-cathepsin D antibody revealed dense and spotty staining in the tumor cells with high transcript levels. The low expression of cathepsin D significantly correlated with long survival of the glioma patients. Furthermore, the glioblastoma patients with high gene expression of cathepsin D lived significantly shorter than those with low expression (P = 0.0104, Cox-Mantel log-rank test) and frequently had leptomeningeal dissemination (P = 0.0016, chi2 test). The multivariate analysis confirmed that the cathepsin D expression level was an independent predictor for short survival (P = 0.0102, Cox proportional hazard regression model). Measurement of the serum cathepsin D concentrations by ELISA showed a significant increase in the patients with high-grade gliomas as compared with the low-grade tumors (P = 0.0081, chi2 test). These results collectively suggest that cathepsin D could be a potential serum marker for the prediction of aggressive nature of human gliomas.

High linear energy transfer carbon radiation effectively kills cultured glioma cells with either mutant or wild-type p53

PURPOSE A mutation in the p53 gene is believed to play an important role in the radioresistance of many cancer cell lines. We studied cytotoxic effects of high linear energy transfer (LET) carbon beams on glioma cell lines with either mutant or wild-type p53. METHODS AND MATERIALS Cell lines U-87 and U-138 expressing wild-type p53 and U-251 and U-373 expressing mutant p53 were used. These cells were irradiated with 290 MeV/u carbon beams generated by the Heavy Ion Medical Accelerator in the National Institute of Radiologic Science or X-rays. A standard colony-forming assay and flow cytometric detection of apoptosis were performed. Cell cycle progression and the expression of p53, p21, and bax proteins were examined. RESULTS High LET carbon radiation was more cytotoxic than low LET X-ray treatment against glioma cells. The effects of the carbon beams were not dependent on the p53 gene status but were reduced by G(1) arrest, which was independent of p21 expression. The expression of bax remained unchanged in all four cell lines. CONCLUSION These results indicate that high LET charged particle radiation can induce cell death in glioma cells more effectively than X-rays and that cell death other than p53-dependent apoptosis may participate in the cytotoxicity of heavy charged particles. Thus, it might prove to be an effective alternative radiotherapy for patients with gliomas harboring mutated p53 gene.

Three-staged stereotactic radiotherapy without whole brain irradiation for large metastatic brain tumors.

PURPOSE To evaluate the efficacy and toxicity of staged stereotactic radiotherapy with a 2-week interfraction interval for unresectable brain metastases more than 10 cm(3) in volume. PATIENTS AND METHODS Subjects included 43 patients (24 men and 19 women), ranging in age from 41 to 84 years, who had large brain metastases (> 10 cc in volume). Primary tumors were in the colon in 14 patients, lung in 12, breast in 11, and other in 6. The peripheral dose was 10 Gy in three fractions. The interval between fractions was 2 weeks. The mean tumor volume before treatment was 17.6 +/- 6.3 cm(3) (mean +/- SD). Mean follow-up interval was 7.8 months. The local tumor control rate, as well as overall, neurological, and qualitative survivals, were calculated using the Kaplan-Meier method. RESULTS At the time of the second and third fractions, mean tumor volumes were 14.3 +/- 6.5 (18.8% reduction) and 10.6 +/- 6.1 cm(3) (39.8% reduction), respectively, showing significant reductions. The median overall survival period was 8.8 months. Neurological and qualitative survivals at 12 months were 81.8% and 76.2%, respectively. Local tumor control rates were 89.8% and 75.9% at 6 and 12 months, respectively. Tumor recurrence-free and symptomatic edema-free rates at 12 months were 80.7% and 84.4%, respectively. CONCLUSIONS The 2-week interval allowed significant reduction of the treatment volume. Our results suggest staged stereotactic radiotherapy using our protocol to be a possible alternative for treating large brain metastases.

Glucose and methionine uptake and proliferative activity in meningiomas

Despite similar benign histological appearances, proliferative activity of meningiomas varies tumor to tumor, and even region to region in a tumor. To predict proliferative potential before surgery, we compared regional uptake of 2-[18F]fluoro-2-deoxyglucose ([18F]FDG) and L-[methyl-11C]methionine ([11C]MET) with histological indices of tumor proliferative activity in 17 specimens from six patients with meningioma obtained by PET guided stereotactic biopsies. Uptake of [11C]MET, an index of protein synthesis rate, significantly correlated not only with the count of nucleolar organizer regions (NORs), a histological index of protein synthesis, but also with Ki-67 index, a histological index of proliferative activity. On the other hand, [18F]FDG uptake showed no significant correlation with Ki-67 index or clinical malignancy. These results suggest that [11C]MET-PET is a useful tool for predicting tumor proliferative potential in meningiomas.

Postoperative recurrence and the role of adjuvant chemotherapy in patients with pulmonary large-cell neuroendocrine carcinoma

OBJECTIVES The prognosis for patients with large-cell neuroendocrine carcinoma is generally very poor. In this study, we describe the clinical features of recurrent tumors of large-cell neuroendocrine carcinoma and discuss the role of adjuvant chemotherapy and management of recurrence in patients with large-cell neuroendocrine carcinoma. METHODS We retrospectively analyzed clinical data from 79 patients and evaluated the prognosis of patients with platinum-based adjuvant chemotherapy, recurrence patterns, patient response to chemotherapy or radiation therapy, and prognosis in patients who experienced relapse. RESULTS Of 72 patients, 36 had confirmed recurrent tumors upon follow-up examinations. Of those with recurrent tumors, 33 patients (91.7%) had their first recurrent tumors within 3 years. Patients who underwent platinum-based adjuvant chemotherapy had a significantly lower rate of tumor recurrence and a higher rate of disease-free survival than those who had non-platinum-based adjuvant chemotherapy or no adjuvant chemotherapy. Multivariate analyses revealed that platinum-based adjuvant chemotherapy, pathologic stage, and the presence of second cancer are independent prognostic factors. Three patients with limited resection of the primary tumor had poor prognosis with recurrence. Postoperatively, 11 of the 36 patients without recurrence (30.6%) had metachronous second primary cancers, of which 4 patients had more than 1 site. CONCLUSIONS Patients with large-cell neuroendocrine carcinoma had frequent recurrence following resection of the primary tumor, and those without recurrence often developed metachronous second primary cancers. Platinum-based adjuvant chemotherapy after surgery may be useful for preventing recurrence in patients with large-cell neuroendocrine carcinoma.

Neuroradiological characteristics of pineocytoma and pineoblastoma

Abstract We reviewed neuroradiological images in two histologically proven cases of pineocytoma and three of pineoblastoma to delineate the characteristic features of these rare tumours. CT revealed isodense or slightly hyperdense masses with central or peripheral calcification; enhancement with contrast medium tended to be homogeneous in pineocytomas and heterogeneous in pineoblastomas. In the pineocytomas, T1-weighted images revealed rounded, sometimes or slightly lobulated low-signal masses with strong, homogeneous contrast enhancement. Their margin was clear, without invasion of adjacent structures. In the pineoblastomas, however, T1-weighted images revealed multilobulated tumours with heterogeneous contrast enhancement. All three pineoblastomas had poorly defined margins with adjacent structures such as the posterior thalamus or corpus callosum, suggesting a more invasive nature. T2-weighted images revealed nonspecific high signal lesions in all five case.

Identification of the small interstitial deletion at chromosome band 1p34–p35 and its association with poor outcome in oligodendroglial tumors

To narrow down the putative tumor‐suppressor gene locus and to assess the predictability of clinical courses by genomic alterations, we analyzed 46 oligodendroglial tumors for loss of heterozygosity (LOH) in the distal region of the short arm of chromosome 1. LOH at 1p was found in 43 tumors (93.5%), including all 28 oligodendrogliomas, all eight oligo‐astrocytomas, six of eight anaplastic oligodendrogliomas, and in one of two anaplastic oligo‐astrocytomas. Thirty‐seven tumors showed LOH patterns consistent with a large terminal deletion, whereas six tumors showed LOH suggesting interstitial deletions. Our data also showed two small regions of overlap at 1p34–p35 (∼5.7 Mb) and at 1p36.1–p36.2 (∼12 Mb). Among the six tumors with interstitial deletion, the proximal region was deleted in five tumors, whereas the distal region was deleted in only half of them. Overall, 91% of tumors showed deletion including this proximal region. To examine the clinical significance of the LOH pattern, the samples were classified into three groups: tumors without 1p LOH (Group 1, n = 3), tumors with an interstitial deletion (Group 2, n = 6), and tumors with a large terminal deletion (Group 3, n = 37). Both overall and progression‐free survival of patients in Group 2 was extremely poor compared with those included in Group 3 (P = 0.0006 and P = 0.003, respectively). As to the clinical response to chemotherapy, nimustine prevented tumor recurrence in Group 3 (P = 0.034) but not in Group 2. Our results demonstrate that a putative tumor‐suppressor gene(s) in oligodendroglial tumors is localized at 1p34–p35 and that small interstitial deletions, in contrast to large terminal deletions, are strongly predictive of both chemoresistance and aggressive characteristics of these tumors.

Epithelial‑mesenchymal transition in glioblastoma progression (Review)

Epithelial-mesenchymal transition (EMT) is a reversible biological process that occurs in epithelial cells. EMT ultimately leads to the acquisition of a mesenchymal phenotype, characterized by increased cell motility and resistance to genotoxic agents. These processes mostly overlap with the acquirement of stem cell properties in differentiated tumor cells. With regard to gliomas, the clinical picture is heterogeneous, even within the same grades and histological categories of the disease. Furthermore, the areas of invasion and responses to radiochemotherapy are markedly different among cases, and occasionally even in the same patient. Such phenotypic diversity in glioma tissues may be caused by various microenvironmental factors, as well as intrinsic genetic alterations. The current review focuses on the EMT-inducing factors that are present in gliomas; these typically vary from those observed in epithelial cancers, as no basement membrane is present. Furthermore, the most important cell-cell contact factor, E-cadherin, is rarely expressed in gliomas. The microenvironment that induces EMT in gliomas is characterized by hypoxia and the enrichment of myeloid cells following stimulation by transforming growth factor-β. Anti-vascular endothelial growth factor therapy, including the use of bevacizumab, may be a suitable candidate to modulate the glioma microenvironment.

Efficacy of the bystander effect in the herpes simplex virus thymidine kinase-mediated gene therapy is influenced by the expression of connexin43 in the target cells.

Tumoricidal “bystander effect” observed in the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) gene therapy was studied between different rat glioma cell lines (9L and C6 cells) under both in vitro and in vivo conditions. For that purpose, mixed populations of wild-type cells (9Lwt and C6wt) and respective HSVtk gene–transduced cells (9Ltk and C6tk) were examined for their sensitivity to GCV. A potent in vitro bystander effect was observed in 9Lwt/9Ltk and 9Lwt/C6tk combinations but not in C6wt/9Ltk and C6wt/C6tk combinations. In vivo bystander effect studied in a subcutaneous tumor model in athymic nude mice was also potent in 9Lwt/9Ltk and 9Lwt/C6tk combinations. Because the expression of connexin43, a major protein in the connexin family gene products, in 9L cells is much higher than that in C6 cells, the results suggest that the amount of connexin in target (wild-type) cells but not in effector ( HSVtk gene–bearing) cells is important for the generation of the bystander effect. This hypothesis was further confirmed by the observation that in vitro bystander effect in C6wt/C6tk combination was potentiated by transduction of the connexin43 gene to the target cells. Cancer Gene Therapy (2001) 8, 414–420