Yoichi Nakazato

Anti-Human Olig2 Antibody as a Useful Immunohistochemical Marker of Normal Oligodendrocytes and Gliomas

Olig2 is a recently identified transcription factor involved in the phenotype definition of cells in the oligodendroglial lineage. The expression of Olig2 transcript has been demonstrated in human oligodendroglial tumors, although the protein expression has not been studied extensively. We developed a polyclonal antibody to human Olig2 and analyzed it immunohistochemically. The antibody depicted a single distinct band of predicted molecular weight by Western blotting, and did not cross-react with human Olig1. In normal human brain tissue, the nuclei of oligodendrocytes of interfascicular, perivascular, and perineuronal disposition were clearly labeled by the antibody. Similarly, the nuclei of oligodendroglial tumors were labeled. There was no apparent correlation between the staining intensity and histological grade. Astrocytic components within the tumors were generally less or not stained. Astrocytic tumors were also positive with the Olig2 antiserum to a lesser extent, and the difference between oligodendroglial and astrocytic tumors was demonstrated by a statistical analysis. Olig2 and glial fibrillary acidic protein were expressed in a mutually exclusive manner, and Olig2 expression was cell-cycle related. Neither central neurocytoma nor schwannoma cases were stained. Our antibody was demonstrated to be useful in recognizing normal oligodendrocytes on paraffin sections, and applicable in diagnosis of some brain tumors.

Systematic immunohistochemical profiling of 378 brain tumors with 37 antibodies using tissue microarray technology

We performed a systematic immunohistochemical study on 378 brain tumors using 37 antibodies and tissue microarray (TMA) technology. The aim of this study was to find new diagnostic biomarkers using antibodies established in our laboratory. Our TMA consisted of a grid of 1.5-mm cores that were extracted from individual donor blocks. Staining for each antibody was scored using a three-point system. We used hierarchical clustering analysis to interpret these data, which resulted in separation of all the brain tumors into seven groups. Although there were some exceptions, cases with the same histological diagnosis were generally grouped together. We then carried out statistical analyses to find the most useful antibodies for grouping of brain tumors. Ten antibodies [glial fibrillary acidic protein (GFAP), Olig2, vimentin, epithelial membrane antigen (EMA), cytokeratin (AE1/AE3), alpha-internexin, nestin, pinealocytes PP5, aquaporin-4 (AQP4) M13d and AQP4M13e] discriminated between astrocytomas and oligodendroglial tumors. Six antibodies [EMA, AE1/AE3, TUJ1, nestin, neurofilament protein-MH (NF-MH) and perivascular cells GP-1] showed significant differences between high-grade and low-grade gliomas. Our data have revealed new antibodies with potential diagnostic utility (Olig2, PP5, GP-1) and demonstrate that TMA technology is highly useful for evaluating newly established antibodies in brain-tumor research.

The identity of cells expressing MHC class II antigens in normal and pathological human brain

Major histocompatibility complex class II antigen (Ag) expression in human brain was investigated in autopsied human brain tissues, using anti‐human class II monoclonal antibodies. In normal brains, class II Ag was usually absent or was low in positivity. When it was found immunohistochemically, it appeared more frequently in the meninges (meningeal macro‐phages) and the neurohypophysis (pituicytes) than in the cerebral cortex (microglia and perivascular cells). The identity of the latter cell types was confirmed by immunoelectron microscopy. Class II‐positive microglial cells were usually present in the cerebral white matter, but in senile brains showing numerous senile plaques, their numbers were increased in the grey matter. In diseased brains, numerous reactive microglia and macrophages containing class II Ag were observed in the affected lesions of neural tissue destruction, neuronal degeneration, and inflammation. Astrocytes, which were identified with an antibody to glial fibrillary acidic protein, did not contain class II Ag, although a small number of reactive astrocytes showed an equivocal class II staining. Staining for class II Ag on cerebral endothelial cells was mostly negative; however, class II Ag was microscopically identified in a case of secondary CNS T‐cell lymphoma.

A distinct pattern of Olig2-positive cellular distribution in papillary glioneuronal tumors: a manifestation of the oligodendroglial phenotype?

Mixed neuronal-glial tumors of the central nervous system display a wide spectrum of differentiation. Among them, the papillary glioneuronal tumor (PGNT) is characterized by pseudopapillary structures composed of astroglial cells covering hyalinized vessels, and by neurocytic, ganglioid and ganglion cells. In addition, a “nonspecific” cell type, not similar to either astrocytes or neurocytes, has been recognized since the initial reports. Recently, minigemistocytic cells and a population immunostained by anti-Olig2 antibody have also been recognized in PGNT. Olig2 is a transcription factor that is specific for the cellular phenotype of oligodendrocytes. The aim of this study was to further investigate the histological diversity of PGNT. We examined six cases of PGNT, each of which showed Olig2 immunopositivity. Minigemistocytes were encountered in three cases at close proximity to the Olig2-positive area. Olig2-positive cells were negative for glial fibrillary acidic protein (GFAP) and neuronal nuclear antigen by double immunostaining, and mainly occupied the interpapillary area laterally adjacent to the GFAP-positive cells. They had relatively small, round and vesicular nuclei, and were formerly regarded as neurocytic cells or nonspecific cellular elements. Fluorescence in situ hybridization targeting chromosome 1p failed to demonstrate any deletion. This study disclosed an additional cellular component of PGNT that is characterized by Olig2 positivity, suggestive of oligodendroglial phenotype, and the results also encourage us to investigate oligodendroglial participation in various glioneuronal tumors.

Immunohistochemical analysis of cleaved caspase-3 detects high level of apoptosis frequently in diffuse large B-cell lymphomas of the central nervous system

The purpose of the present paper was to examine the level of apoptosis and the relationships among apoptosis, apoptosis‐associated proteins, and proliferating potential in lymphoma tissues to clarify the characteristics of apoptosis in diffuse large B‐cell lymphomas (DLBCL) of the central nervous system (CNS). The formalin‐fixed, paraffin‐embedded tissues of CNS and non‐CNS DLBCL (20 cases each) were studied by terminal deoxynucleotidyl transferase‐mediated dUTP‐nick end labeling (TUNEL) and immunohistochemistry, using antibodies against single‐stranded DNA (ssDNA), cleaved caspase‐3, bcl‐2, bax, p53, Fas and Ki‐67. The cleaved caspase‐3 immunohistochemistry detected apoptosis of the lymphoma cells most sensitively compared to TUNEL and ssDNA immunohistochemistry. High expression (grade + + or + + +) of cleaved caspase‐3 was found more frequently in CNS DLBCL (11 cases, 55%)  than non‐CNS DLBCL (three cases, 15%; P = 0.009). Bax‐positivity of lymphoma cells was increased in six cases of CNS DLBCL, which also showed high positivity of cleaved caspase‐3. There was no significant correlation between the cleaved caspase‐3‐positivity and the Ki‐67 positivity. The present study indicates that the number of apoptotic cells and expression level of cleaved caspase‐3 were significantly higher in CNS DLBCL than non‐CNS DLBCL, and that the correlation of bax and cleaved caspase‐3 expression was often present in CNS DLBCL.

Expression of glucose transporter 5 by microglia in human gliomas

Our previous studies indicate that glucose transporter 5 (GLUT5) is a microglial marker in routine paraffin sections, and is rarely present in monocytes/macrophages of the peripheral organs. We examined the expression of GLUT5 in 91 cases of human gliomas to characterize the microglial phenotype in glioma tissues. Immunohistochemistry was performed on formalin‐fixed, paraffin‐embedded sections using such antibodies as a GLUT5 antibody, two markers for activated microglia: major histocompatibility complex (MHC) class II Ag and macrophage scavenger receptor class A (MSR‐A), and MIB‐1 antibody. The immunoreactivity of GLUT5 was present in three microglial phenotypes: ramified (resting), activated, and ameboid (macrophagic) microglia in most of the cases. A double‐labelling study of astrocytic tumours using GLUT5 and MIB‐1 antibodies demonstrated a proportion of proliferating microglia. However, no morphological difference between MIB‐1‐positive, microglial cells and MIB‐1‐negative, microglial cells was found. The number of GLUT5‐positive microglia was significantly (P < 0.001) higher in astrocytic tumours than in oligodendroglial tumours. Many GLUT5‐positive microglia (up to 52% in total cells) were often observed in pilocytic astrocytomas, where microglial cells were predominantly ramified, and the number of MHC class II‐ or MSR‐A‐positive microglia was less than GLUT5‐positive microglia. Thus, the present study indicated that intrinsic microglia can be a source of microglia/macrophages cell populations in astrocytic tumours, and that pilocytic astrocytomas often have a high proportion of microglial cells with mild activation.

Histopathological and immunohistochemical study of the enteric innervations among various types of aganglionoses including isolated and syndromic Hirschsprung disease

We investigated enteric innervations in 15 isolated and five syndromic cases of Hirschsprung disease (HSCR) with immunohistochemistry for the S100 protein (S100), class III α‐tubulin (TUJ1), peripherin, neuronal nitric oxide synthase (nNOS) and CD34. The number of neurites per smooth muscle unit of the circular muscle layer (CML) was counted in the longitudinal sections. TUJ1 was the best marker to detect whole neuritic networks of the enteric nervous system. There were differences in the innervation patterns between isolated rectosigmoid aganglionosis (RS) and long segment aganglionosis (LS) including total colonic aganglionosis and extensive aganglionosis. In the aganglionic bowel (AGB) of LS, no nerve fibers innervated smooth muscle units in the CML in the area from the small bowel to the terminal descending colon. In the rectosigmoid region of every type of isolated HSCR, we observed transmural nerve fibers forming meshworks in the CML with TUJ1 and S100 antibodies. In RS, the neurites running parallel with smooth muscle cells gradually decreased in number in the distal portion. However, in the rectosigmoid AGB in LS, those neurites were absent and most neurites perpendicularly crossed the CML. Hypertrophic nerve trunks (HNT) in the submucous and myenteric plexuses were observed more frequently in the rectosigmoid region than in the rostral portion. Based on these data, it is suggested that the neuritic meshworks in the CML of the rectosigmoid AGB might derive from not only the sacral plexus, via HNT, but also intrinsic neurons in the oligoganglionic bowel. All of the syndromic HSCR were RS. In the AGB of RS with Down syndrome, the distribution of neurite meshworks in the CML is markedly reduced. In the AGB of RS with mental retardation suspected of having Mowat–Wilson syndrome, the density of intramuscular innervation was comparatively higher. In the rostral portion to the AGB of syndromic HSCR, myenteric ganglia were clearly small in size, and more numerous per smooth muscle unit with scarce internodal strands. These dysplastic features fall under neither hyperganglionosis nor hypoganglionosis classifications. We considered that syndromic HSCR might occur on the basis of a dysplastic enteric nervous system caused by genetic alteration.

Pineal parenchymal tumor of intermediate differentiation with cytologic pleomorphism

We report a case of pineal parenchymal tumor in a 33‐year‐old man incidentally detected by radiological examination. The MRI showed an unhomogeneously enhanced, small tumor (approximately 1 cm in size) in the pineal region. A tumor specimen was obtained at endoscopic biopsy. Routine histology showed a highly cellular tumor characterized by a predominance of small cells showing high nuclear : cytoplasmic ratio and moderate nuclear atypia, pleomorphism including giant cells and an absence of pineocytomatous rosettes. Mitotic figures were rare (approximately 1 per 10 high‐power fields). Tumor necrosis was not evident. Immunohistochemically, the neoplastic cells showed positivity for neural markers (neurofilament protein, synaptophysin) and pinealocyte‐associated antibodies (PP1, PP5, PP6), but not for glial fibrillary acidic protein or S‐100. The MIB‐1 labeling index was relatively high (6.3%). Ultrastructurally, there was some evidence of pinealocytic differentiation, such as vesicle‐crowned rodlets (synaptic ribbons) and paired twisted filaments in neoplastic cells. Thus, the tumor was confirmed as a pineal parenchymal tumor of intermediate differentiation by histology, immunohistochemistry and electron microscopy. This case indicates that marked cytologic pleomorphism can occur in pineal parenchymal tumors of intermediate differentiation.

Amplification of the epidermal growth factor receptor gene in glioblastoma: An analysis of the relationship between genotype and phenotype by CISH method

We examined epidermal growth factor receptor (EGFR) overexpression and EGFR gene amplification using immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) in 109 glioblastomas, including 98 primary glioblastomas and 11 secondary glioblastomas. EGFR overexpression and EGFR gene amplification were found in 33% and 24% of glioblastoma, respectively, and all of those cases were primary glioblastoma. Large ischemic necrosis was significantly more frequent in primary glioblastomas than in secondary glioblastomas (54% vs. 18%), but pseudopalisading necrosis was not (65% vs. 54%). EGFR gene amplification was detected significantly more frequently in cases with both types of necrosis. Although glioblastomas with EGFR gene amplification invariably exhibited EGFR overexpression at the level of the whole tumor, tumor cells with EGFR gene amplification did not always show EGFR overexpression at the level of individual tumor cells. Cases of “strong” EGFR overexpression on IHC could be regarded as having EGFR gene amplification, and cases without EGFR overexpression could not. Cases of “weak” EGFR overexpression should be tested with CISH to confirm the presence of EGFR gene amplification. We found that 54% of glioblastomas with EGFR gene amplification were composed of areas with and without EGFR gene amplification; however, there were no obvious differences in morphology between tumor cells with and without EGFR gene amplification. Although small cell architecture might be associated with EGFR gene amplification at the level of the whole tumor, it did not always suggest amplification of the EGFR gene at the level of individual tumor cells. In one case, it seemed to suggest that a clone with EGFR gene amplification may arise in pre‐existing tumor tissue and extend into the surrounding area. In cases of overall EGFR amplification, CISH would be a useful tool to decide the tumor border in areas infiltrated by tumor cells.

Automatic quantification of the MIB-1 immunoreactivity in brain tumors

Abstract Although MIB-1 labeling index (LI) was frequently used as a proliferation indicator, it can differ among observers. In this study, we evaluated the observer-variability and we propose to use GunmaLI, an image analytical software, to measure LI.