Sho Okamoto

Monoclonal antibody against the fusion junction of a deletion-mutant epidermal growth factor receptor

A mouse monoclonal antibody (IgG2b), 3C10, was produced against the truncated epidermal growth factor receptor (EGFR), encoded by the (type III) in-frame deletion mutation of 801 nucleotides of EGFR affecting the external domain, known to be expressed in some human glioblastoma. As this mutation newly generates a glycine residue at the fusion point, a 14 amino acid peptide around the fusion junction including this glycine was chemically synthesised and used for immunisation of (B6 x DBA/2) F1 mice. Flow cytometric analysis showed 3C10 antibody staining of a mouse NIH/3T3 transfectant (ERM5) with the type III EGFR deletion-mutant gene, but not one with wild-type EGFR. The antibody immunoprecipitated the truncated EGFR protein with a molecular mass of approximately 140 kDa from ERM5 cells. Immunostaining of glioblastomas revealed binding in the case with the type III EGFR mutation, the five other specimens without the mutation being negative despite overexpression of EGFR in some cases.

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.

Production of a Single‐chain Variable Fragment Antibody Recognizing Type III Mutant Epidermal Growth Factor Receptor

The type III deletion mutant of the epidermal growth factor receptor (EGFR) is a potential target in diagnostic and therapeutic approaches for those glioblastomas characterized by its expression. We previously raised a mouse monoclonal antibody, 3C10 (IgG2b) specifically recognizing this mutant EGFR. In this study, a single‐chain variable fragment (scFv) antibody was produced. Partial determination of its N‐terminal amino acid sequence and preparation of adequate primers for variable heavy chain (VH) and variable light chain (VL) genes were performed to allow cloning by means of reverse transcriptase‐polymerase chain reaction. The genes cloned were assembled with a linker, (Gly4Ser)3, and ligated into a bacterial expression vector to express the scFv as cytoplasmic inclusion bodies. After appropriate refolding, the antibody activity of the VH‐VL scFv was examined in an enzyme‐linked immunosorbent assay. 3C10 scFv showed a selective reactivity with the mutant peptide, similarly to the parental 3C10 antibody. A mouse transfectant expressing the type III mutant EGFR and a glioblastoma with type III deletion‐mutant EGFR were positively stained by immunofluorescence. By Biacore analysis, the affinity (KA) of the parental 3C10 for the mutant peptide was 9.7x107M‐1, while that of 3C10 scFv was 2.45‐2.48x107M‐1, being approximately 4‐fold weaker. The results together suggested that the scFv antibody retained the appropriate structure to recognize a conformational epitope of the mutant receptor, similarly to the parental antibody.

Identification of novel biomarker candidates by proteomic analysis of cerebrospinal fluid from patients with moyamoya disease using SELDI-TOF-MS

BackgroundMoyamoya disease (MMD) is an uncommon cerebrovascular condition with unknown etiology characterized by slowly progressive stenosis or occlusion of the bilateral internal carotid arteries associated with an abnormal vascular network. MMD is a major cause of stroke, specifically in the younger population. Diagnosis is based on only radiological features as no other clinical data are available. The purpose of this study was to identify novel biomarker candidate proteins differentially expressed in the cerebrospinal fluid (CSF) of patients with MMD using proteomic analysis.MethodsFor detection of biomarkers, CSF samples were obtained from 20 patients with MMD and 12 control patients. Mass spectral data were generated by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) with an anion exchange chip in three different buffer conditions. After expression difference mapping was undertaken using the obtained protein profiles, a comparative analysis was performed.ResultsA statistically significant number of proteins (34) were recognized as single biomarker candidate proteins which were differentially detected in the CSF of patients with MMD, compared to the control patients (p < 0.05). All peak intensity profiles of the biomarker candidates underwent classification and regression tree (CART) analysis to produce prediction models. Two important biomarkers could successfully classify the patients with MMD and control patients.ConclusionsIn this study, several novel biomarker candidate proteins differentially expressed in the CSF of patients with MMD were identified by a recently developed proteomic approach. This is a pilot study of CSF proteomics for MMD using SELDI technology. These biomarker candidates have the potential to shed light on the underlying pathogenesis of MMD.

Biomarker Research for Moyamoya Disease in Cerebrospinal Fluid Using Surface-enhanced Laser Desorption/Ionization Time-of-flight Mass Spectrometry

Moyamoya disease (MMD) is a rare cerebrovascular disease characterized by steno-occlusive change in bilateral internal carotid arteries with unknown etiology. To discover biomarker candidates in cerebrospinal fluid from MMD patients, proteome analysis was performed by the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Three peptides, 4473Da, 4475Da, and 6253Da, were significantly elevated in MMD group. A positive correlation between 4473Da peptide and postoperative angiogenesis was determined. Twenty MMD patients were enrolled in this pilot study, including 11 pediatric cases less than 18 years of age (mean age, 8.67 years) and 9 adult MMD patients (mean age, 38.1 years). This study also includes 17 control cases with the mean age of 27.9 years old. In conclusion, 4473Da peptide is supposed to be a reliable biomarker of MMD. 4473Da peptide showed higher intensity peaks especially in younger MMD patients, and it was proved to be highly related to postoperative angiogenesis. Further study is needed to show how 4473Da peptide is involved with the etiology and the onset of MMD.

Treatment of Symptomatic Bilateral Cavernous Carotid Aneurysms: Long-term Results of 6 Cases

BACKGROUND Bilateral cavernous carotid aneurysms (CCAs) are very rare. A treatment strategy for symptomatic bilateral CCAs has not been established because of their complex pathogenesis. Here we report our treatment strategy and long-term results for 6 cases of symptomatic bilateral CCAs. METHODS From January 2007 to December 2013, we treated 6 patients (2 men and 4 women; mean age at first treatment, 56.0 years) with symptomatic bilateral CCAs. RESULTS All patients began to experience unilateral symptoms. Five of the 6 underwent high-flow bypass (HFB) with parent artery occlusion (PAO), and 1 received PAO only. Mean follow-up period after the first treatment was 61.3 months. All symptoms improved after the treatment. Five contralateral CCAs became enlarged during the follow-up period. Of these, 4 became symptomatic. One patient received superficial temporal-middle cerebral artery bypass with PAO, 2 received HFB with PAO, and 1 refused treatment. Final modified Rankin Scale scores were 0 in 4 patients, 1 in 1 patient, and 2 in 1 patient. There was no mortality in this series. CONCLUSIONS HFB with PAO is feasible as the first treatment for symptomatic bilateral CCAs. This treatment strategy steadies and simplifies future treatment of contralateral CCAs should they become symptomatic.

Default Mode Network Changes in Moyamoya Disease Before and After Bypass Surgery: Preliminary Report

OBJECTIVE Neurocognitive impairment is often reported in moyamoya disease. We aimed to detect default mode network (DMN) alterations using resting-state functional magnetic resonance imaging and their association with neurocognitive impairments. In addition, the influence of surgical treatment was individually evaluated. METHODS Seven patients with moyamoya disease underwent preoperative resting-state functional magnetic resonance imaging and neuropsychologic tests. We compared the resting-state networks (RSNs) of our patients with those obtained from relatively large cohort datasets (127 healthy controls) using group independent component analysis with dual regression analysis. We also explored correlations between RSN alterations and neuropsychologic scores. We evaluated individuals again 6 months after surgery to identify changes. RESULTS Patients had statistically significant differences in DMN connectivity compared with healthy controls. There were marked changes in functional connectivity of the ventral DMN of patients with low working memory and performance speed scores. These changes were characterized by increases and decreases in various locations. In contrast, patients with average or high neuropsychologic scores showed similar connectivity to the controls. In 5 patients who underwent vascular reconstruction surgery, DMN functional connectivity changed to resemble that of healthy controls. CONCLUSIONS In moyamoya disease, working memory and performance speed scores were inversely correlated to the degree of disruption of the DMN, suggesting a possible relationship between higher cognitive function and orderliness of fundamental brain networks. Vascular reconstruction surgery may contribute to normalization of brain networks. Analysis of RSNs may produce potential biomarkers for cognition in moyamoya disease.

Hands-on Simulation versus Traditional Video-learning in Teaching Microsurgery Technique

Bench model hands-on learning may be more effective than traditional didactic practice in some surgical fields. However, this has not been reported for microsurgery. Our study objective was to demonstrate the efficacy of bench model hands-on learning in acquiring microsuturing skills. The secondary objective was to evaluate the aptitude for microsurgery based on personality assessment. Eighty-six medical students comprising 62 men and 24 women were randomly assigned to either 20 min of hands-on learning with a bench model simulator or 20 min of video-learning using an instructional video. They then practiced microsuturing for 40 min. Each student then made three knots, and the time to complete the task was recorded. The final products were scored by two independent graders in a blind fashion. All participants then took a personality test, and their microsuture test scores and the time to complete the task were compared. The time to complete the task was significantly shorter in the simulator group than in the video-learning group. The final product scores tended to be higher with simulator-learning than with video-learning, but the difference was not significant. Students with high “extraversion” scores on the personality inventory took a shorter time to complete the suturing test. Simulator-learning was more effective for microsurgery training than video instruction, especially in understanding the procedure. There was a weak association between personality traits and microsurgery skill.

A case of subarachnoid hemorrhage from ruptured oncotic fusiform aneurysms from choriocarcinoma metastasis treated with aneurysmectomy and vessel reconstruction

BACKGROUND Oncotic aneurysm is a rare condition with a high mortality rate. Because no consensus has been reached regarding therapeutic strategy for ruptured oncotic aneurysm, treatment remains challenging. CASE DESCRIPTION A 35-year-old woman developed sudden onset of severe headache. Computed tomography showed subarachnoid hemorrhage and cerebral angiography revealed 2 fusiform aneurysms in the distal portion of the left middle cerebral artery. Aneurysmectomy with vessel reconstruction using a superficial temporal artery graft was performed to maintain blood flow to the distal middle cerebral artery. Pathologic examination of the aneurysm and wall of the resected recipient middle cerebral artery showed infiltrating trophoblasts. Immunostaining for human chorionic gonadotropin was positive in the aneurysm specimen. On the basis of an elevated concentration of serum human chorionic gonadotropin, choriocarcinoma with ruptured intracranial oncotic aneurysms was diagnosed. After further systemic examination for carcinoma, chemotherapy was initiated. CONCLUSIONS Aneurysmectomy, resection of the parent artery with irregular walls and reconstruction to the distal recipient artery with normal intima should be considered to secure patency of the anastomosis and prevent the recurrence of oncotic aneurysm. Subsequent chemotherapy is essential to prevent carcinomatous meningitis and disease progression.

Intraoperative evaluation of local cerebral hemodynamic change by indocyanine green videoangiography: prediction of incidence and duration of postoperative transient neurological events in patients with moyamoya disease

OBJECTIVETransient neurological events (TNEs) occur frequently in the acute phase after direct bypass surgery for moyamoya disease (MMD), but there is currently no way to predict them. FlowInsight is a specialized software for analyzing indocyanine green (ICG) videoangiography taken with a surgical microscope. The purpose of this study was to investigate whether intraoperative evaluation of local hemodynamic changes around anastomotic sites using FlowInsight could predict the incidence and duration of TNEs.METHODSFrom patients who were diagnosed with MMD in our hospital between August 2014 and March 2017 and who underwent superficial temporal artery-middle cerebral artery bypass surgery, we investigated 25 hemispheres (in 22 patients) in which intraoperative ICG analysis was performed using FlowInsight. To evaluate the local cerebral hemodynamics before and after anastomosis, regions of interest were set at 3 locations on the brain surface around the anastomotic site, and the mean cerebral blood flow (CBF), mean gradation (Grad), mean transit time (MTT), and mean time to peak (TTP) were calculated from the 3 regions of interest. Furthermore, the change rate in CBF (ΔCBF [%]) was calculated using the formula (postanastomosis mean CBF - preanastomosis mean CBF)/preanastomosis mean CBF. ΔGrad (%), ΔMTT (%), and ΔTTP (%) were similarly calculated.RESULTSPostoperative stroke without TNE occurred in 2 of the 25 hemispheres. These 2 hemispheres (in 2 patients) were excluded from the study, and data from the remaining 23 hemispheres (in 20 patients) were analyzed. For each parameter (ΔCBF, ΔGrad, ΔMTT, and ΔTTP) calculated by FlowInsight, the difference between the groups with and without TNEs was significant. The median values for ΔCBF and ΔGrad were significantly higher in the TNE group than in the no-TNE group (ΔCBF 30.13 vs 3.54, p = 0.0106; ΔGrad 62.05 vs 10.78, p = 0.00435), whereas the median values for ΔMTT and ΔTTP were significantly lower in the TNE group (ΔMTT -16.90 vs -7.393, p = 0.023; ΔTTP -29.07 vs -7.02, p = 0.00342). Comparison of the area under the curve (AUC) for each parameter showed that ΔTTP had the highest AUC and was the parameter with the highest diagnostic accuracy (AUC 0.857). The Youden index revealed that the optimal cutoff value of ΔTTP was -11.61 (sensitivity 77.8%, specificity 71.4%) as a predictor of TNEs. In addition, Spearmans rank correlation coefficients were calculated, and ΔCBF, ΔGrad, ΔMTT, and ΔTTP each showed a strong correlation with the duration of TNEs. The larger the change in each parameter, the longer the TNEs persisted.CONCLUSIONSIntraoperative ICG videoangiography findings were correlated with the occurrence and duration of TNEs after direct bypass surgery for MMD. Screening for cases at high risk of TNEs can be achieved by ICG analysis using FlowInsight.