Heterogeneity of histopathological presentation of pilocytic astrocytoma - diagnostic pitfalls. A review.

Pilocytic astrocytomas (PAs) are the most frequent primary astroglial tumours affecting children and adolescents. They occur sporadically or in association with a genetically determined syndrome - neurofibromatosis type 1. Classic PA usually manifests as a well-circumscribed, often cystic, slowly growing tumour, which corresponds to WHO grade I. The majority of pilocytic tumours arise along the neuraxis, predominantly in the cerebellum. They are associated with favourable long-term outcome or spontaneous regression, even after incomplete resection. However, the behaviour and prognosis might also be related to tumour histology and location. Pilomyxoid astrocytoma (PMA) represents a variant of classical PA with more invasive growth and increased risk of recurrences and dissemination. Typically, PAs exhibit distinct histology with biphasic architecture of loose, microcystic and compact, fibrillary areas. However, some tumours arise in an uncommon location and display heterogeneous histopathological appearance. The morphological pattern of PA can mimic some other glial neoplasms, including oligodendroglioma, pleomorphic xanthoastrocytoma, ependymoma or diffuse astrocytoma. Not infrequently, the advanced degenerative changes, including vascular fibrosis, and recent and old haemorrhages, may mimic vascular pathology. Sometimes, the neoplastic piloid tissue can resemble reactive gliosis, related to long-standing non neoplastic lesions. Not infrequently, PA exhibits histological features typical for anaplasia, including necrosis, mitoses and glomeruloid vascular proliferation that can suggest a diffuse high-grade glioma. However, even those PAs that lack distinct histological features of anaplasia can behave unpredictably, in a more aggressive manner, with leptomeningeal spreading. Genetic alterations resulting in aberrant signalling of the mitogen-activated protein kinase (MAPK) pathway have been considered to underlie the development of PAs. The most commonly identified KIAA1549-BRAF fusion is important for appropriate tumour molecular diagnosis. In this paper we summarize the clinicopathological presentation of PAs, with emphasis on their heterogeneous morphology, based on our own experience in the field of surgical neuropathology and the literature data. Diagnosis of pilocytic tumours requires careful analysis of clinical, histopathological and molecular features to avoid misinterpretation of these benign neoplastic lesions.

Aberrant DNA methylation of alternative promoter of DLC1 isoform 1 in meningiomas.

DLC1 encodes GTPase-activating protein with a well-documented tumor suppressor activity. This gene is downregulated in various tumors through aberrant promoter hypermethylation. Five different DLC1 isoforms can be transcribed from alternative promoters. Tumor-related DNA methylation of the DLC1 isoform 1 alternative promoter was identified as being hypermethylated in meningiomas in genome-wide DNA methylation profiling. We determined the methylation pattern of this region in 50 meningioma FFPE samples and sections of 6 normal meninges, with targeted bisulfite sequencing. All histopathological subtypes of meningiomas showed similar and significant increase of DNA methylation levels. High DNA methylation was associated with lack of DLC1 protein expression in meningiomas as determined by immunohistochemistry. mRNA expression levels of 5 isoforms of DLC1 transcript were measured in an additional series of meningiomas and normal meninges. The DLC1 isoform 1 was found as the most expressed in normal control tissue and was significantly downregulated in meningiomas. Transfection of KT21 meningioma cell line with shRNA targeting DLC1 isoform 1 resulted in increased activation of RHO-GTPases assessed with pull-down assay, enhanced cell migration observed in scratch assay as well as slight increase of cell metabolism determind by MTT test. Results indicate that isoform 1 represents the main pool of DLC1 protein in meninges and its downregulation in meningiomas is associated with hypermethylation of CpG dinucleotides within the corresponding promoter region. This isoform is functional GAP protein and tumor suppressor and targeting of its expression results in the increase of DLC1 related cell processes: RHO activation and cell migration.

Necrotic rhabdoid meningiomas with aggressive clinical behavior.

Rhabdoid meningioma (RM) is a rare, aggressive variant of meningioma classified as a WHO Grade III malignancy. RM exhibits a striking histological resemblance to other rhabdoid tumors and strong tendency towards local recurrences, CSF dissemination, and/or remote metastasis. The majority of reported cases are of secondary rhabdoid transformation in recurrent meningiomas. We present two unusual cases of rhabdoid meningiomas diagnosed as a primary intracranial lesion in adults that were associated with extensive necrosis and an aggressive clinical course. On histological examination, the majority of the tumor mass was composed of necrotic tissue with focal clusters of neoplastic cells, often localized around blood vessels. Most tumor cells exhibited typical rhabdoid morphology with large, vesicular, often eccentrically located nuclei with distinct nucleoli and abundant cytoplasm containing eosinophilic hyaline inclusions. Classical meningothelial features with focal whorl formation were scarce and seen only in one case; in the second case the tumor was entirely rhabdoid. The differential diagnosis with atypical teratoid/rhabdoid tumors (AT/RTs) and other neoplasms, particularly metastatic carcinoma, was considered. Immunohistochemical and electron microscopic study were critical for the accurate diagnosis of the rhabdoid subtype of meningiomas. Rhabdoid cells stained diffusely positive for vimentin and S-100 protein and showed focal but strong expression of epithelial membrane antigen and cytokeratins. The rhabdoid areas of the tumors exhibited high mitotic activity with a MIB-1 labeling index of 80 - 90%. The diagnosis of rhabdoid meningioma was supported by evidence of SNF5 (INI1) protein expression. Ultrastructural examination demonstrated the presence of interdigitating cell processes joined by numerous desmosomes and paranuclear whorls of intermediate filaments typical of the rhabdoid phenotype. Our two cases of rhabdoid meningiomas were associated with lethal outcome within a few months of initial diagnosis. Extensive necrosis in rhabdoid meningioma might be considered an additional predictor of aggressive clinical behavior.

Light and electron microscopy characteristics of the muscle of patients with SURF1 gene mutations associated with Leigh disease.

AIMS: Leigh syndrome (LS) is characterised by almost identical brain changes despite considerable causal heterogeneity. SURF1 gene mutations are among the most frequent causes of LS. Although deficiency of cytochrome c oxidase (COX) is a typical feature of the muscle in SURF1-deficient LS, other abnormalities have been rarely described. The aim of the present work is to assess the skeletal muscle morphology coexisting with SURF1 mutations from our own research and in the literature. METHODS: Muscle samples from 21 patients who fulfilled the criteria of LS and SURF1 mutations (14 homozygotes and 7 heterozygotes of c.841delCT) were examined by light and electron microscopy. RESULTS: Diffuse decreased activity or total deficit of COX was revealed histochemically in all examined muscles. No ragged red fibres (RRFs) were seen. Lipid accumulation and fibre size variability were found in 14 and 9 specimens, respectively. Ultrastructural assessment showed several mitochondrial abnormalities, lipid deposits, myofibrillar disorganisation and other minor changes. In five cases no ultrastructural changes were found. Apart from slight correlation between lipid accumulation shown by histochemical and ultrastructural techniques, no other correlations were revealed between parameters investigated, especially between severity of morphological changes and the patient's age at the biopsy. CONCLUSION: Histological and histochemical features of muscle of genetically homogenous SURF1-deficient LS were reproducible in detection of COX deficit. Minor muscle changes were not commonly present. Also, ultrastructural abnormalities were not a consistent feature. It should be emphasised that SURF1-deficient muscle assessed in the light and electron microscopy panel may be interpreted as normal if COX staining is not employed.

Meningioma with the unique coexistence of secretory and lipomatous components: a case report with immunohistochemical and ultrastructural study.

Meningiomas exhibit a broad spectrum of differentiation potency corresponding to different histological subtypes. The separate secretory or lipomatous transformation of meningothelial cells is uncommonly encountered in meningiomas classified into distinct secretory or lipomatous variants. The coexistence of these two different histological subtypes is extremely rare. We report an exceptional case of secretory meningioma associated with extensive lipomatous component in a 58-year-old woman. CT scan and MRI of the brain showed a well-defined tumor mass in the right temporal lobe with areas of adipose tissue and extensive surrounding brain edema. Microscopically, the tumor was composed of two components: whorls of meningothelial cells with numerous PAS-positive hyaline inclusions (pseudopsammoma bodies) and numerous mature adipocyte-like cells. The presence of neutral fat was confirmed by oil-red-O staining. The hyaline inclusions and tumor cells surrounding them showed strong immunoreactivity for EMA and CEA. Ultrastructural findings confirmed both secretory and lipomatous differentiation of tumor cells. The majority of lipidized neoplastic cells shared the features of meningothelial cells and adipocytes. Our result supports the opinion that lipomatous component ought to be considered as an advanced lipidization of neoplastic meningothelial cells rather than true metaplastic transformation of meningothelial cells into mature fat tissue. The present case of meningioma demonstrates a unique coexistence of secretory and lipomatous meningothelial components, reflecting the multipotency of phenotypic transformation of primary meningothelial cells.

Heavily lipidized, calcified giant cell glioblastoma in an 8-year-old patient, associated with neurofibromatosis type 1 (NF1): report of a case with long-term survival.

Giant cell glioblastoma (GCG-BM) with predominance of bizarre, multinucleated giant cells is a rare subtype of glioblastoma, however, its clinical behavior and histological features are still not fully understood. We report an unusual case of a heavily lipidized form of giant cell glioma corresponding mostly to GCGBM in a young patient with neurofibromatosis 1 (NF1). Histologically, the tumor revealed numerous characteristic histopathological features of giant cell glioblastoma including cellular pleomorphism with numerous giant tumor cells, pseudopalisades around necrotic foci and mitotic activity, accompanied by additional unique morphological elements such as massive lipidization of the neoplastic cells, abundant microcalcifications and angiomatous pattern of vascularization. Such aberrant morphology might be associated with the unusually long survival period of 12 years without clinical evidence of tumor recurrence. The coexistence of intracerebral heavily lipidized, calcified giant cell glioblastoma with NF1 has not been previously reported in literature.

Immunohistochemical studies in diagnosis of the uncommon cases of tumours of the central nervous system.

There is a growing evidence that tumoursof the central nervous system (CNS) exhibit some immunophenotypic aberrations pointing to the multipotential cell differentiation. However, the immunohistochemistry remains still very helpful in differential diagnosis and nosologic classification of the CNS neoplasms. The purpose of this paper is to present the immunomorphological pattern of some rare neoplasms of neuroepithelial origin that over last years were recognised and classified as new clinico-pathological entities. Histological and immuniohistochemical features of three cases including pleomorphic xanthoastrocytoma, chordoid glioma and central neurocytoma are reported with special references to immunohistochemical differentiation of these neoplasmswith other tumours of similar morphology but different histogenesis.

Phenotypic characteristics of GFAP-immunopositive oligodendroglial tumours Part I: immunohistochemical study.

Oligodendrogliomas are believed to derive from oligodendrocyte lineage but the expression of different immunohistochemical markers indicates some variability in their differentiation potency. It has been documented that some heterogeneity of the tumour cells exists and that oligodendrogliomas can display a spectrum of histological, immunohistochemical and fine structural features. The expression of glial fibrillary acidic protein (GFAP) in various types of neoplastic cells in oligodendroglial tumours has been well established, however the nature of these cells in relation to tumour malignancy remains controversial. The current histopathological and immunohistochemical study (with a panel of antibodies for GFAP, vimentin, S-100 protein, MBP, NSE) has been performed on biopsy specimens from 12 cases of GFAP-immunopositive oligodendroglial tumours to evaluate their phenotypic characteristics. The majority of tumours showed a variable pattern of GFAP expression in morphologically different tumour cells responding to typical neoplastic oligodendrocytes (gliofibrillary oligodendrocytes-GFOC), miniature form of gemistocytes (minigemistocytes) and neoplastic or reactive astrocytes. The majority of cases exhibited negative staining for vimentin whereas there was no evident correlation between GFAP expression and other immunohistochemical markers. The present immunohistochemical findings support the opinion that the majority of GFAP-positive neoplastic cells in oligodendroglial tumours represent the transitional cell types between oligodendroglial and astrocyte lineage. The difficulties in differential diagnosis of oligodendroglial tumours exhibiting the various patterns of GFAP expression are emphasised.

Phenotypic characteristics of GFAP-positive oligodendroglial tumours. Part II: ultrastructural study.

Five cases of anaplastic oligodendrogliomas containing numerous GFAP-positive cells have been analysed by electron microscopy to establish the fine structural characteristics of neoplastic cells. Ultrastructurally, all tumours have revealed monotonous appearance typical of oligodendrogliomas, however some structural variability, particularly with reference to astrocytic differentiation, has been observed. The majority of neoplastic cells have shown the fine structural features of oligodendrocytes, accompanied by various numbers of intermediate cytoplasmic filaments. These filaments have been usually distributed in the perinuclear, less often in the peripheral, parts of the cytoplasm. The cells exhibiting features common to both oligodendroglial and astroglial cells might be regarded as an intermediate morphological form between these two cell types. True neoplastic astrocytes could be encountered only sporadically. The present electron microscopic studysupports the opinion that GFAP-positive oligodendroglial tumours contain heterogeneous neoplastic cell populations with the transitional cell types between oligodendroglial and astroglial lineage.

Ultrastructural characteristics of necrotic and apoptotic mode of neuronal cell death in a model of anoxia in vitro.

Increasing evidence suggests that two distinct modes of cell death, known as apoptosis and necrosis, are involved in many different pathological states. The morphological pattern of postanoxic changes has been widely studied in various experimental models, however the exact mechanism of neuronal cell death induced by ischaemic/anoxic insult is still not fully understood. The aim of this study was to determine the detailed ultrastructural criteria of postanoxic neuronal changes in in vitro model of anoxia. The electron-microscopic examination of organotypic cultures of rat hippocampus, exposed to 10- and 20-minute anoxic insult, revealed the morphological features typical for both necrotic and apoptotic neuronal cell death. Numerous neurones revealed a typical picture of passive necrotic lysis, such as advanced swelling of intracellular organelles associated with cell membrane disruption, whereas others clearly reflected an active apoptotic form of cell injury, consisting of condensation of nuclear chromatin with early preservation of cell membranes. However, there was also a subset of damaged cells sharing several features typical for both necrosis and apoptosis. These results add additional evidence to the previous studies suggesting not only that neurones injured by anoxic insult can die in a pure necrotic or apoptotic way but also that a continuum might exist between apoptosis and necrosis in certain pathological conditions.

Epithelioid schwannomas of the acoustic nerve.

Epithelioid schwannomas occur predominantly in relation to peripheral nerves and are associated with histological and clinical malignancy. However, a variant of the epithelioid schwannoma involving cranial nerves is extremely rare. In this study we report three cases of epithelioid schwannomas originating from the acoustic nerves and located in the cerebello-pontine angles. In the first case, the tumor was histopathologically entirely solid and demonstrated biphasic pattern with both spindle-shaped cells and a population of round or polygonal epithelioid cells. The second one consisted of the smaller part exhibiting typical Antoni B and A tissue and large areas containing clusters and bundles of epithelioid cells. Purely epithelioid schwannoma composed predominantly of cords or nests of round and polygonal epithelioid cells were observed in the third case. All schwannomas revealed marked polymorphism and nuclear hyperchromasia. Immunohistochemical studies showed a diffuse, strong positivity for S-100 protein in the cytoplasm of the spindle and epithelioid tumor cells. These two populations of cells were positively stained for vimentin, but were negative for EMA, cytokeratin and HMB45. Patchy GFAP-immunoreactivity was also noticed at the peripheral parts of the tumors. The authors discuss differential diagnosis of this unusual variant of schwannoma in relation to malignant transformation of the epithelioid component.

Aluminum enhances glutamate-mediated neurotoxicity in organotypic cultures of rat hippocampus.

Both, glutamate (GLU) and aluminum (Al) have been implicated in neuronal damage and/or death in certain human neurodegenerative disorders. Recent evidence suggests that aluminum (Al) may potentiate the increase in glutamate-induced intracellular calcium overload. The present ultrastructural study was undertaken to determine the effect of Al on the development of GLU-mediated neurotoxicity in tissue culture conditions. The experiments were performed on organotypic cultures of rat hippocampus treated with low, subtoxic concentration of GLU (50 microM) and AlCl3 (400 microM) added to the growth medium separately or in combination. The exposure of cultures to GLU in the presence of Al3+ ions for up to 24 hours resulted in the development of typical excitotoxic neuronal changes, whereas separate GLU treatment at subtoxic doses or single Al application did not produce any apparent tissue damage. The neuronal lesions resulting from the combined application of GLU plus Al consisted predominately of more or less pronounced mitochondrial abnormalities, which are characteristic for early excitotoxic events. Severe swelling of the mitochondria led to the disruption of their internal structure and finally resulted in an apparent microvacuolization of the perikaryal cytoplasm of some pyramidal neurons. The present morphological data evidenced that Al is capable to potentiate the GLU-induced degenerative changes in hippocampal neurons in vitro. This supports the view of a possible role of Al in the process of neurodegeneration and suggests that Al may participate in the development of glutamate-mediated excitotoxic neuronal injury under certain pathological conditions.

Xanthomatous changes in atypical and anaplastic meningiomas. Light and electron microscopic investigations.

Xanthomatous changes may occur in meningiomas of different histological type, however their incidence in combination with histological features of atypical or anaplastic meningioma has not been previously documented. In this report we present clinicopathologic, immunohistochemical and ultrastructural studies in the surgical cases of two atypical and three anaplastic meningiomas exhibiting prominent xanthomatous changes. In all tumors the xanthomatous cells were seen in association with typical meningioma structures such as meningothelial whorls or psammoma bodies as well as within the tumor parts displaying pleomorphism, patternless growth, increased cellularity, presence of necroses and mitoses or brain invasion. Ultrastructural study revealed a wide-range of lipid-containing cells, reflecting a continuum of gradual transition between polymorphic meningioma cells and xanthomatous cells. Commonly, the lipidized cells exhibited different degrees of plasmalemmal interdigitations and desmosomal junctions. Our study allowed us to confirm the meningothelial origin of xanthomatous cells in atypical and anaplastic meningiomas. Moreover, the ultrastructural observations of lysosomes in the majority of xanthomatous cells and the immunoreactivity for the CD68 antigen indicated their macrophage characteristics. It seems that a mixed meningeal/macrophage nature of xanthomatous cells can be related to the functional and structural multipotentiality of the primary leptomeningeal cells.

Disseminated spinal and cerebral ependymoma with unusual histological pattern: clinicopathological study of a case with retrograde tumor spread.

The subject of this study is a case of anaplastic ependymoma originally arising from the central canal of the lower spinal cord followed by the 13 years history of events of upper spinal dissemination and retrograde intracranial spread. The specimens from four subsequent surgeries generally displayed the same microscopic features of neoplastic tissue and were consistent with the diagnosis of anaplastic ependymoma. The histological diagnosis was based upon the high cellularity, considerable nuclear atypia and pleomorphism, brisk mitotic activity, focally exhibited vascular endothelial proliferation and extensive necrosis. Apart from the typical pattern of ependymoma, the tumors contained areas composed almost entirely of large, uniform clear cells or pseudogemistocytes indicating the morphological heterogeneity of neoplastic cells population. The surgical specimens from four surgical resections shared light microscopic similarities suggesting spinal and intracranial dissemination from the primary spinal tumor. Since the retrograde spread via the cerebrospinal fluid (CSF) pathway is extremely rare, the authors of this study discuss the mechanism of such way of tumor metastases.

In vivo brain microdialysis as a tool in studies of neuroprotective effects of cyclosporin A in acute excitotoxicity.

The mitochondrial permeability transition (MPT) resulting from calcium-induced opening of cyclosporin A (CsA)-sensitive megachannels, leading to deenergisation of mitochondria and release of pro-apoptotic cytochrome c, has been implicated in the pathomechanism of excitotoxic neurodegeneration. The aim of this work was to test neuroprotective potential of CsA in the model of N-methyl-D-aspartate-(NMDA)-induced excitotoxicity in vivo, and to verify utility of microdialysis of the rabbit hippocampus in vivo for these mechanistic studies. In vitro experiments demonstrated that the early rapid phase of Ca(2+)-induced swelling of isolated brain mitochondria, and of accompanying cytochrome c release, was strongly inhibited by 0.5 microM CsA. In the in vivo experiments 1 mM NMDA was applied for 20 min to the hippocampus in a control, or 5 microM CsA-containing dialysis medium via transhippocampal microdialysis probes, and changes in extracellular Ca2+ concentration and in NO release were monitored. Application of NMDA induced a prolonged decrease in the extracellular concentration of Ca2+, reflecting influx of Ca2+ to stimulated neurones. CsA only slightly enhanced this effect. NMDA induced also release of NO to the dialysis medium. Morphological examination 30 min after NMDA application visualised swelling of dendritic mitochondria and cisternae of endoplasmatic reticulum of pyramidal neurones in the CA1 sector of the hippocampus in the vicinity of microdialysis probes. CsA prevented mitochondrial swelling. After 24 h degeneration of the CA1 pyramidal neurones close to a microdialysis probes was observed, which was partially prevented in CsA-treated rabbits. These results indicate that the mechanism of CsA nuroprotection may be at least in part ascribed to prevention of MPT. Microdialysis of the rabbit hippocampus combined with NMDA excitotoxicity appeared to be useful in mechanistic studies of CsA neuroprotection.

Giant cervico-thoracic schwannoma with long clinical history. Case report.

An unusual case of a giant intraspinal schwannoma in a 45-year-old woman with 14-year history of preoperative symptoms was presented. MRI of the spine revealed an intradural, extramedullary tumor extending from the intervertebral space C4/C5 to T4 vertebral body level (2 x 1.2 x 12 cm) and filling almost the entire spinal canal. Microscopical examination showed a typical neurinoma pattern with two distinct zones of Antoni A and Antoni B tissue. Some areas exhibited nuclear atypia and hyperchromasia reflecting the degenerative changes in this slowly growing nerve sheath tumor. A rich pericellular reticulin network was seen in the areas composed of Antoni A tissue. Immunohistochemically, the tumor cells were strongly positive for S-100 protein. The diagnostic difficulties in the presented case of longstanding schwannoma resulted in the late surgical treatment. The importance of the early diagnosis of spinal nerve sheath tumors for the patient's quick recovery is stressed.

Ultrastructural features of the astrocytes involved in the phagocytic response to excitotoxins and anoxia in vitro.

The present ultrastructural studies demonstrate the morphological evidence of phagocytic response of astroglia to excitotoxins and anoxia in vitro. The studies were performed on organotypic cultures of the rat hippocampus exposed to various excitatory amino acids (QUIN, KA, GLU) or pure nitrogen atmosphere. In all these pathological conditions, the extensive neuronal degeneration was accompanied by marked astroglial reaction. The astrocytes revealed ultrastructural abnormalities consisting of swelling of their cytoplasm followed by more or less advanced fibrillar changes. Numerous reactive astrocytes demonstrated morphological evidence of phagocytic activity during the process of neurodegeneration. They exhibited the presence of ingested degenerating neurons or neuronal debris and accumulation of dense bodies. The results support a possible phagocytic role of astroglia after neuronal injury in various pathological states. The phagocytic response of astrocytes may interfere with other glial functions in CNS and may play a role in tissue recovery.

Necrosis and apoptosis of tumor cells in embolized meningiomas: histopathology and P53, BCL-2, CD-68 immunohistochemistry.

The preoperative embolization of intracranial meningiomas, used in selected patients to reduce tumor vascularity and blood loss during surgery, may produce ischemic changes and/or tumor necrosis. The aim of the present study was to determine the relationship between necrosis within the embolized tumors and expression of two apoptosis-associated proteins (p53 and bcl-2) and macrophage-monocyte CD-68 antigen. Four biopsy specimens of embolized meningiomas, including three benign and one atypical tumor, were revived histopathologically and examined immunohistochemically using the monoclonal antibodies to p53, bcl-2 proteins and CD-68 antigen. The observations showed that the p53-immunopositive cells were most frequent in perinecrotic and ischemic areas than in non-ischemic, intact parts of tumors. The bcl-2 protein was expressed predominantly in well-preserved regions lacking ischemic tumor cells, whereas in close proximity to the necrosis only a few bcl-2 positive cells could be detected. Anti-CD-68 immunostained cells were distributed around or within the necrotic foci. Our results indicate that the expression of apoptosis-related proteins correlates with ischemic cell injury induced by preoperative tumor embolization.

Disorganization of cortical structure and the brain tumors.

In the course of histopathological investigation of the temporal lobe sections, selected from 63 patients treated surgically for intractable epilepsy and finally presented with primary temporal tumors, we found 12 cases expressed both neoplastic process' and developmental disorders. The temporal mass lesions consisting of neuro-glial or pure glial tumors were associated with some developmental abnormalities such as cortical dysplasia, neuronal heterotopias and additional cortical neoplastic nodules. The possible "dual pathology" concerning these lesions are discussed in this paper.