Yuichi Hirose

Chromosomal Abnormalities Subdivide Ependymal Tumors into Clinically Relevant Groups

Ependymoma occurs most frequently within the central nervous system of children and young adults. We determined relative chromosomal copy-number aberrations in 44 ependymomas using comparative genomic hybridization. The study included 24 intracranial and 20 spinal cord tumors from pediatric and adult patients. Frequent chromosomal aberrations in intracranial tumors were gain of 1q and losses on 6q, 9, and 13. Gain of 1q and loss on 9 were preferentially associated with histological grade 3 tumors. On the other hand, gain on chromosome 7 was recognized almost exclusively in spinal cord tumors, and was associated with various other chromosomal aberrations including frequent loss of 22q. We conclude that cytogenetic analysis of ependymomas may help to classify these tumors and provide leads concerning their initiation and progression. The relationship of these aberrations to patient outcome needs to be addressed.

The p38 mitogen-activated protein kinase pathway links the DNA mismatch repair system to the G2 checkpoint and to resistance to chemotherapeutic DNA-methylating agents.

ABSTRACT Although human cells exposed to DNA-methylating agents undergo mismatch repair (MMR)-dependent G2 arrest, the basis for the linkage between MMR and the G2 checkpoint is unclear. We noted that mitogen-activated protein kinase p38α was activated in MMR-proficient human glioma cells exposed to the chemotherapeutic methylating agent temozolomide (TMZ) but not in paired cells made MMR deficient by expression of a short inhibitory RNA (siRNA) targeted to the MMR protein Mlh1. Furthermore, activation of p38α in MMR-proficient cells was associated with nuclear inactivation of the cell cycle regulator Cdc25C phosphatase and its downstream target Cdc2 and with activation of the G2 checkpoint, actions which were suppressed by the p38α/β inhibitors SB203580 and SB202590 or by expression of a p38α siRNA. Finally, pharmacologic or genetic inhibition of p38α increased the sensitivity of MMR-proficient cells to the cytotoxic actions of TMZ by increasing the percentage of cells that underwent mitotic catastrophe as a consequence of G2 checkpoint bypass. These results suggest that p38α links DNA MMR to the G2 checkpoint and to resistance to chemotherapeutic DNA-methylating agents. The p38 pathway may therefore represent a new target for the development of agents to sensitize tumor cells to chemotherapeutic methylating agents.

Tissue Microdissection and Degenerate Oligonucleotide Primed-Polymerase Chain Reaction (DOP-PCR) Is an Effective Method to Analyze Genetic Aberrations in Invasive Tumors

We amplified various amounts of DNA derived from frozen SF210 and U251NCI human glioblastoma cells, carried out comparative genomic hybridization (CGH) using degenerate oligonucleotide primed-PCR (DOP-PCR) products as test probes, and compared results to analyses performed with probes prepared by standard nick translation. Next we extracted DNA from hematoxylin-eosin (HE)- and methyl green (MG)-stained, microdissected sections of formalin-fixed and paraffin-embedded U251NCI cells, amplified and labeled it by DOP-PCR, and subjected it to CGH. Finally, we used the same methods in multiple samples from a single human mixed glioma tissue. DOP-PCR products from 50 pg to 250 ng of DNA were equally effective in generating the same CGH profiles as the standard method. DOP-PCR products from microdissected pieces of MG-stained cells were effective probes for CGH, but HE-stained samples were not desirable. As the proportion of HE-stained sample increased, CGH profiles deteriorated. DOP-PCR products from microdissected pieces of MG-stained paraffin sections of glioma tissue produced CGH profiles compatible with their histological features. CGH performed with DOP-PCR products from microdissected paraffin blocks allows for the accurate investigation of the cytogenetic characteristics from invasive tumors and of cytogenetic heterogeneity within neoplastic tissue.

Oncolytic Virus-Mediated Manipulation of DNA Damage Responses: Synergy With Chemotherapy in Killing Glioblastoma Stem Cells

BACKGROUND Although both the alkylating agent temozolomide (TMZ) and oncolytic viruses hold promise for treating glioblastoma, which remains uniformly lethal, the effectiveness of combining the two treatments and the mechanism of their interaction on cancer stem cells are unknown. METHODS We investigated the efficacy of combining TMZ and the oncolytic herpes simplex virus (oHSV) G47Δ in killing glioblastoma stem cells (GSCs), using Chou-Talalay combination index analysis, immunocytochemistry and fluorescence microscopy, and neutral comet assay. The role of treatment-induced DNA double-strand breaks, activation of DNA damage responses, and virus replication in the cytotoxic interaction between G47Δ and TMZ was examined with a panel of pharmacological inhibitors and short-hairpin RNA (shRNA)-mediated knockdown of DNA repair pathways. Comparisons of cell survival and virus replication were performed using a two-sided t test (unpaired). The survival of athymic mice (n = 6-8 mice per group) bearing GSC-derived glioblastoma tumors treated with the combination of G47Δ and TMZ was analyzed by the Kaplan-Meier method and evaluated with a two-sided log-rank test. RESULTS The combination of G47Δ and TMZ acted synergistically in killing GSCs but not neurons, with associated robust induction of DNA damage. Pharmacological and shRNA-mediated knockdown studies suggested that activated ataxia telangiectasia mutated (ATM) is a crucial mediator of synergy. Activated ATM relocalized to HSV DNA replication compartments where it likely enhanced oHSV replication and could not participate in repairing TMZ-induced DNA damage. Sensitivity to TMZ and synergy with G47Δ decreased with O(6)-methylguanine-DNA-methyltransferase (MGMT) expression and MSH6 knockdown. Combined G47Δ and TMZ treatment extended survival of mice bearing GSC-derived intracranial tumors, achieving long-term remission in four of eight mice (median survival = 228 days; G47Δ alone vs G47Δ + TMZ, hazard ratio of survival = 7.1, 95% confidence interval = 1.9 to 26.1, P = .003) at TMZ doses attainable in patients. CONCLUSIONS The combination of G47Δ and TMZ acts synergistically in killing GSCs through oHSV-mediated manipulation of DNA damage responses. This strategy is highly efficacious in representative preclinical models and warrants clinical translation.

Grade II astrocytomas are subgrouped by chromosome aberrations

Grade II astrocytoma is defined as a low-grade tumor, yet patients have a wide range of survival and tumors can quickly progress to high-grade astrocytoma/glioblastoma. Previous studies using comparative genomic hybridization (CGH) failed to demonstrate frequent copy number aberrations (CNA) in these tumors. This may be related to technical difficulties because infiltrating astrocytic tumors are often intermixed with normal brain tissue. We developed methods to exclude most normal tissue and use small amounts of DNA for CGH by microdissecting small regions of tumor from paraffin sections and amplifying extracted DNA using degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). Using this method, we examined 30 grade II astrocytoma cases. We found CNA in 25 cases (83%), with a mean of two CNA per case. The most frequent CNA were gains on 7q (12 cases), 5p (5 cases), 9 (5 cases), and 19p (3 cases), and losses on 19q (7 cases), 1p (6 cases), and Xp (3 cases). Gain on 7q and losses on 1p/19q were mutually exclusive. This is the first report on the genetic characterization of low-grade astrocytomas using CGH from microdissected and formalin-fixed tissue. The comparatively large number of cases in this study allows us to suggest that these tumors are genetically subgrouped.

The study of flow diversion effects on aneurysm using multiple enterprise stents and two flow diverters.

Background: Computer-based simulation is necessary to clarify the hemodynamics in brain aneurysm. Specifically for endovascular treatments, the effects of indwelling intravascular devices on blood stream need to be considered. The most recent technology used for cerebral aneurysm treatment is related to the use of flow diverters to reduce the amount of flow entering the aneurysm. To verify the differences of flow reduction, we analyzed multiple Enterprise stents and two kinds of flow diverters. Materials and Methods: In this research, we virtually modeled three kinds of commercial intracranial stents (Enterprise, Silk, and Pipeline) and mounted to fit into the vessel wall, and deployed across the neck of an IC-ophthalmic artery aneurysm. Also, we compared the differences among multiple Enterprise stents and two flow diverters in a standalone mode. Results: From the numerical results, the values of wall shear stress and pressure are reduced in proportion to the size of mesh, especially in the inflow area. However, the reduced velocity within the aneurysm sac by the multiple stents is not as significant as the flow diverters. Conclusions: This is the first study analyzing the flow alterations among multiple Enterprise stents and flow diverters. The placement of small meshed stents dramatically reduced the aneurysmal fluid movement. However, compared to the flow diverters, we did not observe the reduction of flow velocity within the aneurysm by the multiple stents.

The Role of Norepinephrine and Estradiol in the Pathogenesis of Cardiac Wall Motion Abnormality Associated With Subarachnoid Hemorrhage

Background and Purpose— The majority of patients with ventricular wall motion abnormality (WMA) associated with subarachnoid hemorrhage (SAH) are postmenopausal women. In addition to elevated catecholamine, the role of estrogen in the pathogenesis of WMA has recently been implicated. The objective of this study is to clarify the interrelation among catecholamine, estrogen, and WMA in patients with SAH. Methods— A retrospective analysis was performed on the medical records of 77 patients with SAH (23 men, 54 women) whose plasma levels of epinephrine, norepinephrine, and estradiol had been measured and echocardiograms had been obtained within 48 hours of SAH onset. Results— Twenty-four patients (31%) were found to sustain WMA on admission. Multivariate regression analysis revealed that decreased estradiol (P=0.018; OR, 0.902) and elevated norepinephrine levels (P=0.027; OR, 1.002) were associated with WMA. After quadrichotomization of 77 patients based on sex/WMA, plasma norepinephrine levels were markedly elevated in men with WMA, whereas estradiol levels were markedly decreased in women with WMA. Plasma norepinephrine and estradiol levels were not correlated. Fifty-four female patients with SAH were further quadrichotomized based on norepinephrine/estradiol levels with a threshold value of 1375 pg/mL for norepinephrine and 11 pg/mL for estradiol. The incidence of WMA in the high-norepinephrine/low-estradiol group was significantly higher than the low-norepinephrine/high-estradiol group. Conclusions— To our knowledge, this is the first study to evaluate the interrelation among catecholamine, estrogen, and SAH-induced WMA. Lack of estradiol in postmenopausal women may predispose them to develop WMA after poor-grade SAH. However, the precise role of multiple sex hormones in SAH-induced WMA should be evaluated in future prospective studies.

The application of intraoperative near-infrared indocyanine green videoangiography and analysis of fluorescence intensity in cerebrovascular surgery.

Objective: To evaluate the usefulness and limitations of the intraoperative near-infrared (NIR) indocyanine green videoangiography (ICG-VA) and analysis of fluorescence intensity in cerebrovascular surgery. Methods: Forty-eight patients received ICG-VA during various surgical procedures from May 2010 to August 2010. Included among them were 45 cases of cerebral aneurysms and 3 cases of cerebral arteriovenous malformations (AVMs). The infrared fluorescence module integrated into the surgical microscope was used to visualize fluorescent areas in the surgical field. An integrated analytical visualization tool constantly analyzed the fluorescence video sequence and generated it in the form of an intensity diagram for objective interpretation. Results: Overall, the procedure of ICG VA was done 158 times in 48 patients. There was no adverse effect of ICG dye. In cerebral aneurysm cases, the images obtained were of high resolution. In 4 cases, incomplete clipping was detected by ICG-VA and allowed suitable adjustment to completely obliterate the aneurysm. In 3 aneurysm cases, the intensity diagram of ICG VA provided valuable information. ICG-VA identified the feeding arteries, the draining veins, and nidus in all 3 AVM cases, which was confirmed by an immediate analysis of fluorescence intensity. Conclusions: ICG-VA provides high resolution images allowing real-time assessment of the blood flow in surgical field. The intensity analysis function, in addition, is a useful adjunct to improve the accuracy of the clipping and decrease the complication rates in cerebral aneurysm cases. In cerebral AVM cases, with the help of color map and intensity diagram function, the superficial feeders, drainers, and nidus can be identified easily.

Intraoperative near-infrared indocyanine green–videoangiography (ICG–VA) and graphic analysis of fluorescence intensity in cerebral aneurysm surgery

We present our preliminary experience with intraoperative near-infrared indocyanine green-videoangiography (ICG-VA) and analysis of blood flow dynamics using fluorescence intensity assessment in cerebral aneurysm clipping surgery. Thirty-nine patients with 43 intracranial aneurysms underwent microsurgical clipping. Intraoperative ICG-VA was performed before and after clip application. An infrared fluorescence module integrated into a surgical microscope was used to visualize fluorescence in the surgical field and we recorded the emitted fluorescent light. An integrated analytical visualization tool simultaneously analyzed the video sequence and converted it into an intensity diagram, which allowed an objective evaluation of the results rather than the subjective assessment of fluorescence using ICG-VA. Overall, ICG-VA was performed 137 times. Incomplete clipping was detected in four patients, which allowed suitable adjustment to completely obliterate the aneurysm. In 12 patients, perforators arising close to, or from, the aneurysmal neck were identified in the surgical field. In three patients, the ICG-VA intensity diagram provided valuable information leading to modification of the primary surgical maneuver. ICG-VA provides high resolution images allowing real-time assessment of the blood flow in the parent artery and arterial branches, including the perforators. The intensity diagram is useful for providing a more objective record of the hemodynamics than the traditional ICG-VA, which relies more on subjective assessment and may allow interobserver variability. We conclude that ICG-VA, combined with the intensity diagram, can reduce the morbidity and complications associated with aneurysm clipping and improve patient outcomes.

Immunohistochemical examination of c-Met protein expression in astrocytic tumors

Abstract Hepatocyte growth factor/scatter factor (HGF/ SF), which has various physiological functions, and its receptor c-Met, the human c-met proto-oncogene product, are thought to be determinant in the pathological processes of various malignancies. To investigate the possible role of HGF/SF in the progression of development of astrocytic tumors, we examined the expression of c-Met in these tumors. Immunohistochemistry using the streptavidin-biotin peroxidase complex method and immunofluorescence double staining with anti-c-Met polyclonal and anti-glial fibrillary acidic protein monoclonal antibodies were performed. Positive c-Met expression was detected in 31 of the 42 astrocytic tumors and some of the control cases analyzed. c-Met-positive cells showed morphological characteristics of astrocytes. Especially in the cases of high-grade tumors, c-Met positivity was abundant in cells in both vascular-rich and peripheral regions of the tumors but not in the cells with distinctly malignant features. Immunofluorescence double staining revealed that the c-Met-positive cells were in part of astrocytic origin. We suggest that c-Met-positive cells are affected by some factors in the lesions where the pathological processes are in a state of development. Our studies indicated that c-Met expression might take part in glioma invasion but not in the development of malignancy.