Masao Matsutani

Increased expression of podoplanin in malignant astrocytic tumors as a novel molecular marker of malignant progression

Podoplanin (aggrus) is a mucin-like transmembrane sialoglycoprotein that is expressed on lymphatic endothelial cells. Podoplanin is putatively involved in cancer cell migration, invasion, metastasis, and malignant progression and may be involved in platelet aggregation. Previously, we showed upregulated expression of podoplanin in central nervous system (CNS) germinomas, but not in non-germinomatous germ cell tumors, except for parts of immature teratomas in limited numbers. However, little information exists about its role in CNS astrocytic tumors. In this study, 188 astrocytic tumors (30 diffuse astrocytomas, 43 anaplastic astrocytomas, and 115 glioblastomas) were investigated using immunohistochemistry with an anti-podoplanin antibody, YM-1. In 11 of 43 anaplastic astrocytomas (25.6%) and in 54 of 115 glioblastomas (47.0%), podoplanin was expressed on the surface of anaplastic astrocytoma cells and glioblastoma cells, especially around necrotic areas and proliferating endothelial cells. However, the surrounding brain parenchyma was not stained by YM-1. On the other hand, podoplanin expression was not observed in diffuse astrocytoma (0/30: 0%). Furthermore, we investigated the expression of podoplanin using quantitative real-time PCR and Western blot analysis in 54 frozen astrocytic tumors (6 diffuse astrocytomas, 14 anaplastic astrocytomas, and 34 glioblastomas). Podoplanin mRNA and protein expression were markedly higher in glioblastomas than in anaplastic astrocytomas. These data suggest that podoplanin expression might be associated with malignancy of astrocytic tumors.

Combined chemotherapy and radiation therapy for CNS germ cell tumors: the Japanese experience

Among intracranial germ cell tumors, nongerminomatous tumors have proved refractory to conventional treatment with surgery and irradiation. The median survival is less than 2 years. Since 1983, chemotherapy has been delivered in Japan as an adjuvant therapy in patients with intracranial nongerminomatous germ cell tumors. Based on our clinical experience, we undertook a multi-institutional phase II study to establish post-surgical combined chemotherapy and radiation therapy for primary germ cell tumors in the brain. We adopted carboplatin-etoposide (CARB-VP) or cisplatin-etoposide (PE) combination chemotherapy for patients with germinomas and those with tumors that placed them in the intermediate prognosis group, and ifosphamide-cisplatin-etoposide (ICE) for patients with tumors that placed them in the poor prognosis group. One hundred and twelve patients were evaluated. Among patients with germinoma (n = 75), the rate or complete remission after combination therapy was 92.0%; it was 67.8% for patients in the intermediate prognosis group (n = 28). Tumor recurrence was noted in 9 patients with germinoma and 2 patients in the intermediate prognosis group. Of 9 patients with a poor prognosis, 4 experienced disease progression during treatment and died within 10 months. There were no serious complications attributable to the combination therapy. Our treatment protocols are effective for patients with germinomas and those with an intermediate prognosis.

Prognosis of intracranial germ cell tumours: effectiveness of chemotherapy with cisplatin and etoposide (CDDP and VP-16).

SummaryA co-operative study for patients with intracranial germ cell tumours was performed to analyze their prognosis and the effectiveness of Cisplatin/Etoposide (CDDP/VP-16) chemotherapy.A total of 46 patients; 30 primary and 16 recurrent cases were registered from 15 participating neurosurgical institutions in Japan. Based on histological criteria and tumour markers, they were classified into three groups; germinoma, germinoma with syncytiotro-phoblastic giant cell (STGC), and non-germinomatous malignant tumour.Sixteen patients were treated with CDDP/VP-16 chemotherapy alone and the other 30 patients were treated by a combination of surgery and/or radiation in addition to chemotherapy. Eleven out of 13 patients (85%) with germinoma showed a complete (n=10) or partial (n=1) response to CDDP/VP-16 chemotherapy even if their tumours were recurrent and there was evidence of CSF dissemination. For the germinoma with STGC and non-germinomatous malignant tumour, a high response rate; 100% for the former and 78% for the latter, could also be achieved in both the primary and the recurrent cases except in those cases of immature teratoma. Their survival times were still different between them. Two-year survival was 50% in germinoma with STGC and 48% in non-germinoma, while it was 88% in germinoma cases.

High-Dose Conformal Radiotherapy Influenced the Pattern of Failure But Did Not Improve Survival in Glioblastoma Multiforme

BACKGROUND AND PURPOSE Although glioblastoma multiforme is clearly radiation-resistant, there is evidence of a dose-dependent response relationship. The purpose of the study was to evaluate the impact of higher dose by rotational multileaf collimator (MLC) conformal radiation therapy. MATERIALS AND METHODS From 1984 to 1995, 38 consecutive cases with intracranial glioblastoma multiforme were treated using the rotational MLC conformal therapy. There were 25 men and 13 women with a median age of 47 years (12-73 years, mean 46.5 years). Median Karnofsky performance score was 80 (30-100, mean 78.2). Median tumor volume was 64 cc (8-800 cc, mean 110.3 cc). All underwent surgical intervention (only biopsy in 1, partial resection in 13, subtotal resection in 21, and gross total resection in 3). Radiation dose to was 60 to 80 Gy (median 68.5 Gy, mean 68.3 Gy) in 21 patients treated before 1990 and 90 Gy in the 17 patients thereafter. Biweekly i.v. chemotherapy was also administered for both arms. RESULTS The 1-year, 2-year, 5-year, and 10-year overall survival rates were 75%, 42%, 20%, and 15%, respectively. Univariate analysis showed the initial tumor volume, residual tumor volume, and Karnofsky performance score were statistically significant factors for survival. Only the residual tumor volume was statistically significant by multivariate analysis. The 5-year survival rate of patients with residual tumors of 5 cc or less in volume was as good as 37%. Survival of the 90-Gy Group appeared inferior to that of the Low-Dose Group, though no statistical difference was seen (the 3-year survival was 40% vs. 22%). Local failure was observed in 16 of the 19 recurrences in the Low-Dose Group, whereas it was observed in only 4 of the 13 recurrences in the 90-Gy Group. The difference in pattern of failure was statistically significant. Two patients of the High-Dose Group developed radiation necrosis and one died of it. CONCLUSIONS The high-dose conformal radiotherapy did not improve survival in the disease, but did change the pattern of failure.

Negative effects of wild-type p53 and s-Myc on cellular growth and tumorigenicity of glioma cells

SummaryHuman (U251, U87, U343) and rat glioma cell lines (C6, 9L) were examined by the reverse transcriptase-polymerase chain reaction and subsequent nucleotide sequencing analysis to see whether they express wild type (wt)-p53 or mutated form (mut)-p53 messages. Results showed that U87, U343, and C6 cells expressed wt-p53 messages whereas U251 and 9L cells expressed mut-p53 messages. All these cell lines were transfected with wt-p53 cDNA or the s-myc gene linked to the mouse mammary tumor virus (MMTV) promoter. Of several G418-resistant clones obtained from each transfection, a few expressed the s-Myc or wt-p53 proteins. Independent of mutations in the intrinsic p53 gene, the cellular growthin vitro and tumorigenicity in nude mice of these clones were drastically suppressed, the extent of suppression being correlated with the expression level of the transfected gene. Flow-cytometric analysis demonstrated that both p53 and s-Myc arrested the cell cycle at the G1/S boundary. These data suggest that these genes having negative effects on tumor cell proliferation could be used in gene therapy of gliomas, which are caused by alteration of the p53 gene or by some other genetic change.

Immunohistochemical analysis of the mutant epidermal growth factor, ΔEGFR, in glioblastoma

The naturally occurring mutated form of the epidermal growth factor receptor, ΔEGFR (also named EGFRvIII and de2-7EGFR), greatly enhances glioblastoma (GBM) cell growth in vivo through several activities, such as down-regulating p27 and up-regulating BclX(L) while increasing signaling through the RAS-MAPK and PI3-K cascades. More than half of GBMs, especially of the de novo type, overexpress EGFR, and 50%–70% of these express ΔEGFR. However, little is known about the distribution of ΔEGFR-expressing tumor cells within surgical specimens. In order to address this clinically important issue, we performed immunohistochemical analyses of 53 GBMs obtained during surgery using the anti-ΔEGFR monoclonal antibody, DH8.3. We also simultaneously analyzed wild-type EGFR expression in these tissues using the anti-EGFR monoclonal antibody, EGFR. 113. ΔEGFR and wild-type EGFR expression were observed in 20/53 (38%) and 29/53 (55%), respectively. Nineteen (95%) of the ΔEGFR-positive tumors also expressed wild-type EGFR; one case was ΔEGFR-positive but wild-type EGFR-negative. In 13/20 (65%) of the ΔEGFR-positive tumors, tumor cells were scattered diffusely within the tumors, 6/20 showed geographical distribution of ΔEGFR-positive tumor cells, and one case showed homogeneous staining. In the wild-type EGFR-positive cases, almost all tumor cells expressed EGFR. The differential distribution of cells expressing the two receptors observed here may suggest either that ΔEGFR arises at a low frequency from wild-type EGFR-expressing cells, perhaps during the process of gene amplification, or that there is a paracrine-type of interaction between them.

Podoplanin expression in primary central nervous system germ cell tumors: a useful histological marker for the diagnosis of germinoma

Podoplanin, a mucin-like transmembrane sialoglycoprotein, promotes platelet aggregation and may be involved in cancer cell migration, invasion, metastasis, and malignant progression. Podoplanin/aggrus is highly expressed in testicular seminoma, suggesting that it may be a sensitive marker for testicular seminomas. Here we investigated the expression of podoplanin in central nervous system (CNS) germ cell tumors (GCTs) by immunohistochemical staining of tumor samples from 62 patients. In 40 of 41 (98%) germinomas (including germinomatous components in mixed GCTs), podoplanin was diffusely expressed on the surface of germinoma cells; lymphocytes, interstitial cells, and syncytiotrophoblastic giant cells were negative for podoplanin. Except for immature teratomas (12/17; 71%), podoplanin expression was absent in non-germinomatous GCTs, including seven teratomas, seven embryonal carcinomas, seven yolk sac tumors, and seven choriocarcinomas. In immature teratomas, focal podoplanin staining was observed in fewer than 10% of immature squamous and columnar epithelial cells. Thus, podoplanin expression may be a sensitive immunohistochemical marker for germinoma in CNS GCTs. As such, it may be useful for diagnosis, for monitoring the efficacy of treatment, and as a potential target for antibody-based therapy.

Expression of vascular endothelial growth factor in human brain tumors

Abstract Compared to normal brain an increased expression of vascular endothelial growth factor (VEGF) has been reported in many types of brain tumors. However, the numbers of samples analyzed and information about the cellular distribution of VEGF have been limited. Here we used novel monochlonal antibodies against VEGF to analyze, using immunohistochemistry, Western blotting and enzyme-linked immunosorbent assay, its expression in 108 human brain tumors that included astrocytic tumors, meningiomas, pituitary adenomas, primary intracranial germ cell tumors and neuronal tumors. The results showed that 37 of 48 astrocytic tumors (77%) and 15 of 19 meningiomas (79%) were immunoreactive for VEGF, consistent with previous reports. However, in contrast to a previous report that analyzed only VEGF mRNA; all of our 15 pituitary adenomas showed specific immunoreactivity for VEGF. We also extended the studies to previously unanalyzed neoplasms: 13 of 15 primary intracranial germ cell tumors (82%), and 7 of 10 neuronal tumors (70%) were immunoreactive for VEGF. Direct protein analysis by Western blotting confirmed the expression of VEGF in those tumors, and showed differential expression of the isoforms of VEGF protein; a pituitary adenoma expressed both VEGF165 and VEGF189 proteins, a central neurocytoma expressed only VEGF165, while an immature teratoma expressed only VEGF189. The data herein show that VEGF is expressed in a wide spectrum of brain tumors and suggest differences among tumor entities in the mechanisms of VEGF up-regulation as well as their employment of distinct VEGF isoforms for neovascularization.

CT and MRI features of intracranial germ cell tumors.

SummaryThe computed tomographic (CT) and magnetic resonance imaging (MRI) features of 73 histologically proven primary intracranial germ cell tumors were analysed. CT images were available for all 73 patients, and 22 of them were also examined by MRI. The tumors were classified as germinoma, mature teratoma, immature or malignant teratoma, yolk sac tumor, choriocarcinoma, embryonal carcinoma and mixed type. Germinoma was revealed as a high or slightly high-density area on plain CT scan, and was enhanced homogeneously. MRI revealed iso or slightly low signal intensity on T1-weighted images, and iso-or high intensity on T2-weighted images. Mature teratoma, which had a clear margin on neuroradiological images, was characterized by mixed density on CT scans, often showing large cysts and area of calcification. Immature or malignant teratoma had a similar pattern to that of mature teratoma, but the cystic components and area of calcification tended to be less and smaller respectively. The tumor margin was obscure in malignant teratoma, and perifocal edema was observed in some cases. The shape of yolk sac tumors was irregular. Plain CT scan revealed an iso or low-density mass with good heterogeneous enhancement. Perifocal edema was observed in some cases. In mixed germ cell tumors, MRI imaging was useful for detecting teratomatous components, particularly fatty components. Although definite histological diagnosis cannot be achieved by CT and/or MRI alone, detailed analysis of neuroradiological images are useful for predicting the histological diagnosis.

Primary central nervous system lymphoma in Japan--a retrospective, co-operative study by CNS-Lymphoma Study Group in Japan.

This manuscript reports the results of the first cooperative study on primary central nervous system lymphoma (PCNSL) in Japan. Of 196 patients registered, 170 were judged as having PCNSL. No patients were immunocompromised. Of the 170 patients with PCNSL, 93 were males and 77 were females. The mean was 56.7 years. One hundred and nineteen tumors were confirmed histopathologically, and 51 were diagnosed by neuroimaging alone. All the tumors were non-Hodgkins lymphoma. According to the Working Formulation for Clinical Usage (WF), 96 out of 119 tumors were classifiable: 53 were diffuse large cell type (55.2%), 17 immunoblastic type (17.7%), 9 diffuse small cleaved type (9.4%), 6 diffuse mixed type (6.3%), 5 polymorphous type (5.2%), 5 small lymphocytic type (5.2%) and 1 small non-cleaved type (1.0%). Of 21 tumors studied immunohistochemically, 18 were B-cell type and 3 were T-cell type. Irradiated patients (144) survived significantly longer than non-irradiated patients, (median survival time, MST: 19.2 and 2.7 months, respectively; p<0.001). There was a remarkable difference in survival among patients of the intermediate lymphomas; MST (18 months) of patients with large cell lymphoma was significantly shorter than MST (over 96 months) of patients with other intermediate grade lymphomas (small cleaved and mixed) (p < 0.001) and had no significant difference from MST (9 months) of patients with high grade lymphomas. If patients were irradiated with more than 40 Gy, higher doses and different modes of irradiation brought no further survival advantage. Chemotherapy was performed in 87 of 144 irradiated patients (60.4%). No regimens were effective in prolonging survival. Of 144 irradiated patients, a complete or partial response to initial treatment was demonstrated in 91 (63.2%) and 43 patients (29.9%), respectively. Improvement in performance status was confirmed in 82 patients (57.0%). Despite a good response to initial treatments. 88 out of 144 evaluable patients have died of PCNSL (MST: 19 months). Multivariate analysis based on the Cox hazard model revealed that histology of tumor, age at onset, performance status, and radiotherapy were prognostic factors. Neither chemotherapy nor mode of surgery was a beneficial factor.