Norimoto Nakahara

Human Gene Therapy for Malignant Gliomas (Glioblastoma Multiforme and Anaplastic Astrocytoma) by In Vivo Transduction with Human Interferon β Gene Using Cationic Liposomes

Transfer of interferon beta gene via cationic liposomes has been found to induce regression of experimental glioma. We performed a pilot clinical trial of safety and effectiveness of this interferon beta gene therapy in five patients with malignant glioma (glioblastoma multiforme or anaplastic astrocytoma). Transgene expression and antitumor activity were detected in four patients. Two patients showed a partial response (>50% tumor reduction) and two others had stable disease 10 weeks after beginning therapy. One patient could not be evaluated because of previous treatment with gamma-knife therapy. This study suggests the feasibility and safety of interferon beta gene therapy, which may become an important treatment option for patients with malignant glioma.

EGFR mutations in patients with brain metastases from lung cancer: Association with the efficacy of gefitinib

Gefitinib--a specific inhibitor of epidermal growth factor receptor (EGFR)-associated tyrosine kinase--has demonstrated efficacy in a subgroup of patients with non-small-cell lung carcinoma (NSCLC) who fail conventional chemotherapy. It is also reported to have an antitumor effect in brain metastases from NSCLC. Additionally, EGFR mutations have shown a strong association with gefitinib sensitivity for NSCLC. Here, we assessed the efficacy of gefitinib in brain metastases from NSCLC and evaluated the association of this efficacy with EGFR mutations. We retrospectively reviewed eight cases in which patients were suffering from brain metastases before the initiation of gefitinib treatment. Brain tumor response could be evaluated by MRI in these patients; EGFR gene analyses were also available. We evaluated whether objective tumor response was observed after gefitinib treatment and assessed the efficacy of gefitinib as effective, noneffective, or not assessable in consideration of the influence of previous radiotherapy. Of the eight patients, the efficacy of gefitinib was assessed as effective in three and as noneffective in three. All three patients demonstrating effective efficacy had EGFR mutations in the tyrosine kinase domain (deletion mutation in two patients and point mutation in one patients), whereas none of the three patients demonstrating noneffective efficacy had EGFR mutations. Gefitinib appears to be effective in treating brain metastases in a subgroup of patients. Our data suggested a possible association between the efficacy of gefitinib in the treatment of brain metastases and EGFR mutations.

Activation of JAK/STAT signalling in neurons following spinal cord injury in mice

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathway is one of the most important in transducing signals from the cell surface to the nucleus in response to cytokines. In the present study, we investigated chronological alteration and cellular location of JAK1, STAT3, phosphorylated (p)‐Tyr1022/1023‐JAK1, p‐Tyr705‐STAT3, and interleukin‐6 (IL‐6) following spinal cord injury (SCI) in mice. Western blot analysis showed JAK1 to be significantly phosphorylated at Tyr1022/1023 from 6 h after SCI, peaking at 12 h and gradually decreasing thereafter, accompanied by phosphorylation of STAT3 at Tyr705 with a similar time course. ELISA analysis showed the concentration of IL‐6 in injured spinal cord to also significantly increase from 3 h after SCI, peaking at 12 h, then gradually decreasing. Immunohistochemistry revealed p‐Tyr1022/1023‐JAK1, p‐Tyr705‐STAT3, and IL‐6 to be mainly expressed in neurons of the anterior horns at 12 h after SCI. Pretreatment with a JAK inhibitor, AG‐490, suppressed phosphorylation of JAK1 and STAT3 at 12 h after SCI, reducing recovery of motor functions. These findings suggest that SCI at the acute stage produces IL‐6 mainly in neurons of the injured spinal cord, which activates the JAK/STAT pathway, and that this pathway may be involved with neuronal response to SCI.

Intraoperative Tractography and Motor Evoked Potential (MEP) Monitoring in Surgery for Gliomas Around the Corticospinal Tract

BACKGROUND Our goal is to indicate the importance of combining intraoperative tractography with motor-evoked potential (MEP) monitoring for glioma surgery in motor eloquent areas. METHODS Tumor removal was performed in 28 patients with gliomas in and around the corticospinal tract (CST), in an operation theater equipped with an integrated high-field intraoperative magnetic resonance imaging and a neuronavigation system. Diffusion-tensor imaging-based tractography of the CST was implemented preoperatively and intraoperatively. When the surgically manipulated area came close to the corticospinal pathway, MEP responses were elicited by subcortical stimulation. Responsive areas were compared with the locations of fibers traced by preoperative and intraoperative tractography. Imaging and functional outcomes were reviewed. RESULTS Intraoperative tractography demonstrated significant inward or outward shift during surgery. MEP responses were observed around the tract at various intensities, and the distance between MEP responsive sites and intraoperative tractography was significantly correlated with the stimulation intensity (P < 0.01). The distance from preoperative tractography was not correlated. A more than subtotal resection was achieved in 24 patients (85.7%). Transient motor deterioration was seen in 12 patients (42.8%), and a permanent deficit was seen in 1 patient (3.5%). CONCLUSIONS We found that intraoperative tractography demonstrated the location of the CST more accurately than preoperative tractography. The results of the linear regression between distance and stimulation intensity were informative for guiding approaches to tumor remnants without impinging on the CST. The combination of intraoperative tractography and MEP monitoring can enhance the quality of surgery for gliomas in motor eloquent areas.

Peptide-pulsed dendritic cell vaccination targeting interleukin-13 receptor α2 chain in recurrent malignant glioma patients with HLA-A*24/A*02 allele.

BACKGROUND AIMS Dendritic cell (DC)-based vaccination targeting tumor-associated antigens is an attractive approach to overcoming the limitations of current treatments for malignant gliomas (MG). Interleukin-13 receptor α2 chain (IL-13Rα2) is a promising target because of its abundant and specific expression in MG. We conducted a phase I trial of DC vaccination in patients with recurrent MG using two IL-13Rα2-derived peptides restricted to HLA-A*0201 and -A*2402. The objective was to evaluate the safety and clinical and immunologic responses. METHODS Eight recurrent MG patients were enrolled. DC were generated from peripheral blood and pulsed with HLA-matched peptide; 1 × 10(7) DC were administered every 2 weeks for a maximum of six immunizations. The T-cell response in peripheral blood was evaluated by tetramer and ELISPOT assays in HLA-A*2402 patients. RESULTS All enrolled patients except one completed at least four DC vaccinations. No severe adverse events were observed. A positive T-cell response was detected in two out of three evaluable HLA-A*2402 patients. One patient achieved stable disease for 16 months and another patient showed a dramatic regression for one lesion for 4 months. CONCLUSIONS The regimen was feasible and safe, and the HLA-A*24-restricted peptide exhibited a capacity to induce immune responses. These results warrant further studies to evaluate whether add-on regimens to post-operative chemoradiotherapy delays recurrence in newly diagnosed MG patients.

Effective induction of antiglioma cytotoxic T cells by coadministration of interferon-|[beta]| gene vector and dendritic cells

As type I interferons (IFNs) enhance the stimulatory activity of dendritic cells (DCs), we hypothesized that transfection of glioma cells with the IFN-β gene in the presence of DCs would provide particularly effective antitumor activity by both facilitating apoptosis of glioma cells and presenting the resulting glioma antigens to T cell by DCs, thereby inducing specific T-cell responses against glioma cells. A mouse glioma cell line 203G was first transfected with cDNA encoding IFN-β using cationic liposomes, then cocultured with syngeneic bone marrow-derived DCs and naïve splenic T cells. The 203G cells were almost completely killed following 96-hour coculture with DCs and T cells, and strong tumor-specific cytotoxic T-lymphocyte (CTL) activity accompanied by high level interleukin (IL)-12 and IFN-γ production was observed in culture. In addition, omission of either IFN-β gene delivery, DCs or T cells from the coculture completely abrogated the induction of the CTL activity, suggesting that the combination of these components was required to elicit an optimal effect. On the basis of these in vitro data, syngeneic animals bearing subcutaneous 203G tumors received intratumoral injections of IFN-β gene and DCs. Suppression of the tumor growth by this combinational therapy was superior to treatment with DC or IFN-β gene solely. This combination may constitute a new therapeutic strategy to induce potent antiglioma immune responses.

Antitumor Effect of an Adeno-associated Virus Vector Containing the Human Interferon-β Gene on Experimental Intracranial Human Glioma

We constructed an adeno‐associated virus (AAV) vector containing the human interferon‐β(HuIFN‐β) gene (AAV‐IFN‐β) and investigated its antitumor effect against human glioma cells (U251‐SP) inoculated into the brain of nude mice. Prior to this, we examined human glioma cells transduced with AAV‐IFN‐β using video‐enhanced contrast differential interference contrast (VEC‐DIC) microscopy. Infection of AAV‐IFN‐β induced apoptosis and secondary necrosis in human glioma cells. In in vivo experiments, we confirmed production of HuIFN‐β and induction of heat‐shock protein (HSP) in glioma cells transduced with AAV‐IFN‐β. Growth of the experimental gliomas was completely inhibited by six injections of AAV‐IFN‐β, starting 7 days after transplantation of glioma cells. In addition, the survival of mice treated with AAV‐IFN‐β was remarkably prolonged. These results indicate that AAV‐IFN‐β induces apoptosis of glioma cells and has a strong antitumor effect in this experimental glioma model.

Inhibition of Aurora-B function increases formation of multinucleated cells in p53 gene deficient cells and enhances anti-tumor effect of temozolomide in human glioma cells

Cell division is an elemental process, and mainly consists of chromosome segregation and subsequent cytokinesis. Some errors in this process have the possibility of leading to carcinogenesis. Aurora-B is known as a chromosomal passenger protein that regulates cell division. In our previous studies of giant cell glioblastoma, we reported that multinucleated giant cells resulted from aberrations in cytokinesis with intact nuclear division occurring in the early mitotic phase, probably due to Aurora-B dysfunction. In this study, as we determined p53 gene mutation occurring in multinucleated giant cell glioblastoma, we investigated the role of Aurora-B in formation of multinucleated cells in human neoplasm cells with various p53 statuses as well as cytotoxity of glioma cells to temozolomide (TMZ), a common oral alkylating agent used in the treatment of gliomas. The inhibition of Aurora-B function by small-interfering (si)RNA led to an increase in the number of multinucleated cells and the ratios of G2/M phase in p53-mutant and p53-null cells, but not in p53-wild cells or the cells transduced adenovirally with wild-p53. The combination of TMZ and Aurora-B-siRNA remarkably inhibited the cell viability of TMZ-resistant glioma cells. Accordingly, our results suggested that Aurora-B dysfunction increases in the appearance of multinucleated cells in p53 gene deficient cells, and TMZ treatment in combination with the inhibition of Aurora-B function may become a potential therapy against p53 gene deficient and chemotherapeutic-resistant human gliomas.

Vaccination with tumor cell lysate-pulsed dendritic cells augments the effect of IFN-β gene therapy for malignant glioma in an experimental mouse intracranial glioma

Interferon‐β (IFN‐β) has been used as an antitumor drug against human glioma, melanoma and medulloblastoma since the 1980s. Recently, we developed a new gene therapy using the IFN‐β gene against malignant gliomas and then began clinical trials in 2000. Since stimulation of immune system was one mechanism of antitumor effect induced by IFN‐β gene therapy, we hypothesized that combination of IFN‐β gene therapy with immunotherapy might increase its effectiveness. In the present study, we tested whether combination therapy with IFN‐β gene therapy and immunotherapy using tumor cell lysate‐pulsed dendritic cells (DCs) would increase the efficacy of IFN‐β gene therapy. In an experimental mouse intracranial glioma (GL261), which cannot be cured by either IFN‐β gene therapy or DC immunotherapy alone, IFN‐β gene therapy following DC immunotherapy resulted in a significant prolongation in survival of the mice. Moreover, when this combination was performed twice, 50% of treated mice survived longer than 100 days. Considering these results, this combination therapy may be one promising candidate for glioma therapy in the near future.

Radioimmunoscintigraphy of Intracranial Glioma Xenograft with a Technetium-99m-Labeled Mouse Monoclonal Antibody Specifically Recognizing Type III Mutant Epidermal Growth Factor Receptor

The type III mutant epidermal growth factor receptor (EGFR) is expressed on the cell surface of a subset of glioma, but not of normal tissues. In this study, we investigated the in vivo kinetics of 3C10 mouse monoclonal antibody (mAb), specifically recognizing the type III mutant EGFR (EGFRvIII), using athymic nude mice bearing the intracranial glioma xenograft overexpressing the EGFRvIII.Human glioma cell line, U87MG, expressing the wild type EGFR and the transfectant, named U87MGċΔEGFR, expressing the EGFRvIII, were transplanted subcutaneously or intracranially to nude mice. 3C10 mAb labeled with a technetium-99m (99mTc) was intravenously injected into these nude mice and then the mice were sacrificed at 24 h later, and the 99mTc-uptake by xenografts and major normal organs was measured to determine the biodistribution of mAb. Furthermore, at 3, 6 and 24 h after injection of 99mTc-labeled 3C10 mAb, whole-body scintigraphy was obtained with a gamma camera to localize the tumor site.3C10 mAb significantly accumulated to U87MG ċ ΔEGFR xenografts transplanted subcutaneously or intracranially in nude mice, showing high tumor-to-blood ratio of 10.30 and 4.01, respectively. In contrast, uptake of control antibody in the intracranial tumor was as low as 0.43. In scintigrams, intracranially transplanted U87MG ċ ΔEGFR xenografts were detectable at 3 h after injection of 99mTc-labeled 3C10 mAb.These results suggest that intravenously injected 3C10 mAb specifically accumulated to the subcutaneous or intracranial glioma xenograft expressing the EGFRvIII and 3C10 mAb is a potential diagnostic and therapeutic agent for patients with gliomas expressing the EGFRvIII.