CD133 expression is associated with small round blue cell tumour morphology in human central nervous system neoplasms.

AIMS: CD133 is considered to be a marker for brain tumour-initiating cells. However, most data on CD133 are derived from animal or in-vitro studies. The aim of this study was to characterize CD133 expression, and the distribution and morphological features of CD133(+) cells, in primary and secondary human central nervous system (CNS) neoplasms. METHODS AND RESULTS: Tumours were analysed by real-time reverse transcription polymerase chain reaction, western blot, flow cytometry and immunohistochemistry. Our results show that only small round blue cell tumours (SRBCTs) exhibit strong and consistent CD133 expression. Interestingly, glioblastomas, large-cell carcinomas and sarcomas were negative for CD133. Only glioblastomas with a focal small-cell component exhibited CD133 immunoreactivity in the SRBCT component. In addition, CD133 expression did not correlate with the expression of other markers associated with stem cell differentiation, including CD15 and nestin. CONCLUSIONS: CD133 expression in human CNS neoplasms is independent of the grade of malignancy but strongly correlates with SRBCT morphology. Together with recent findings showing that CD133 is quickly upregulated upon hypoxia and that CD133(-) cells can also exhibit stem cell properties, our data strongly question the suitability of CD133 as a brain tumour stem cell marker in vivo.

Atypical teratoid/rhabdoid tumors may show morphological and immunohistochemical features seen in choroid plexus tumors.

Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive embryonal tumors having a poor prognosis and are associated with mutations in the tumor suppressor gene hSNF5/SMARCB1/INI1. Differential diagnosis includes choroid plexus carcinoma which has occasionally been attributed as showing an inactivation of INI1/SMARCB1 nuclear staining in immunohistochemistry. However, these findings have been challenged by others. We therefore examined eight AT/RTs from six patients by immunohistochemistry for membranous expression of the inward rectifier potassium channel Kir7.1, which was in the central nervous system so far considered specific for choroid plexus tumors and normal choroid plexus epithelium. Two AT/RT cases exhibited membranous staining of Kir7.1, indicating a plexus epithelial differentiation of these tumors. The implications of these results on tumor diagnosis are discussed.

Immune surveillance of the normal human CNS takes place in dependence of the locoregional blood-brain barrier configuration and is mainly performed by CD3(+)/CD8(+) lymphocytes.

Despite the blood-brain barrier (BBB) the human CNS is continuously screened by blood-derived immunological cells. In certain brain areas the local BBB configuration grants passage of large molecules, whereas others are better shielded. We investigated whether these regional BBB compositions are paralleled by differences in the degree of cellular immunosurveillance by investigating tissue from 23 normal human brains for several CD markers, FoxP3, granzyme B, and perforin. Our results provide evidence that immunosurveillance is associated with locoregional BBB configuration and is mainly performed by CD3(+)/CD8(+)/granzyme B(-)/perforin(-) lymphocytes.

Patients with IDH1 wild type anaplastic astrocytomas exhibit worse prognosis than IDH1-mutated glioblastomas, and IDH1 mutation status accounts for the unfavorable prognostic effect of higher age: implications for classification of gliomas.

WHO grading of human brain tumors extends beyond a strictly histological grading system by providing a basis predictive for the clinical behavior of the respective neoplasm. For example, patients with glioblastoma WHO grade IV usually show a less favorable clinical course and receive more aggressive first-line treatment than patients with anaplastic astrocytoma WHO grade III. Here we provide evidence that the IDH1 status is more prognostic for overall survival than standard histological criteria that differentiate high-grade astrocytomas. We sequenced the isocitrate dehydrogenase 1 gene (IDH1) at codon 132 in 382 patients with anaplastic astrocytoma and glioblastoma from the NOA-04 trial and from a prospective translational cohort study of the German Glioma Network. Patients with anaplastic astrocytomas carried IDH1 mutations in 60%, and patients with glioblastomas in 7.2%. IDH1 was the most prominent single prognostic factor (RR 2.7; 95% CI 1.6-4.5) followed by age, diagnosis and MGMT. The sequence from more favorable to poorer outcome was (1) anaplastic astrocytoma with IDH1 mutation, (2) glioblastoma with IDH1 mutation, (3) anaplastic astrocytoma without IDH1 mutation and (4) glioblastoma without IDH1 mutation (p < 0.0001). In this combined set of anaplastic astrocytomas and glioblastomas both, IDH1 mutation and IDH1 expression status were of greater prognostic relevance than histological diagnosis according to the current WHO classification system. Our data indicate that much of the prognostic significance of patient age is due to the predominant occurrence of IDH1 mutations in younger patients. Immunohistochemistry using a mutation-specific antibody recognizing the R132H mutation yielded similar results. We propose to complement the current WHO classification and grading of high-grade astrocytic gliomas by the IDH1 mutation status and to use this combined histological and molecular classification in future clinical trials.

CD8(+)/perforin/granzyme B(+) effector cells infiltrating cerebellum and inferior olives in gluten ataxia.

Up to 8% of patients with gluten sensitivity (GS) develop neurological symptoms such as ataxia, dementia, seizures or peripheral neuropathy. The underlying immunological mechanisms still remain to be elucidated. We here report the case of a 68-year-old male patient suffering from progressive ataxia and dementia associated with chronic diarrhea and both elevated IgG and IgA antigliadin-antibodies. At autopsy, frequent argyrophilic glial and neuronal inclusions within the basal nucleus of Meynert were considered as the structural correlative for the cognitive decline. Significant neuronal loss in the cerebellar cortex and the inferior olives was accompanied by infiltrating CD8(+)/perforin(+)/granzyme B(+) cells as well as reactive astrogliosis and microglial activation. These CD8(+) cytotoxic T and NK cells are likely to act as effector cells responsible for neuronal cell death in patients with gluten sensitivity and neurological disease and might therefore at least partly be responsible for cerebellar symptoms in gluten ataxia. In conclusion, our results, showing an absence of B- or plasma cells but multiple CD8(+) as well as granzyme B and perforin expressing cells in ataxia-associated brain areas, suggest that there are also prominent cytotoxic effects in neuropathogenesis of GS.

Modulation of TGF-beta activity by latent TGF-beta-binding protein 1 in human malignant glioma cells.

High biological activity of the transforming growth factor (TGF)-beta-Smad pathway characterizes the malignant phenotype of malignant gliomas and confers poor prognosis to glioma patients. Accordingly, TGF-beta has become a novel target for the experimental treatment of these tumors. TGF-beta is processed by furin-like proteases (FLP) and secreted from cells in a latent complex with its processed propeptide, the latency-associated peptide (LAP). Latent TGF-beta-binding protein 1 (LTBP-1) covalently binds to this small latent TGF-beta complex (SLC) and regulates its function, presumably via interaction with the extracellular matrix (ECM). We report here that the levels of LTBP-1 protein in vivo increase with the grade of malignancy in gliomas. LTBP-1 is associated with the ECM as well as secreted into the medium in cultured malignant glioma cells. The release of LTBP-1 into the medium is decreased by the inhibition of FLP activity. Gene-transfer mediated overexpression of LTBP-1 in glioma cell lines results in an increase inTGF-beta activity. Accordingly, Smad2 phosphorylation as an intracellular marker of TGF-beta activity is enhanced. Conversely, LTBP-1 gene silencing reduces TGF-beta activity and Smad2 phosphorylation without affecting TGF-beta protein levels. Collectively, we identify LTBP-1 as an important modulator of TGF-beta activation in glioma cells, which may contribute to the malignant phenotype of these tumors.

Comparative analysis of annexin-1 in neuroepithelial tumors shows altered expression with the grade of malignancy but is not associated with survival.

In various types of cancer, the expression of members of the annexin family of calcium- and phospholipid-binding anti-inflammatory proteins is dysregulated. Annexin-1 (ANXA1, lipocortin-1) is involved in proliferation, differentiation and apoptosis. It serves as a substrate for the epidermal growth factor receptor (EGFR), which is frequently amplified in primary gliomas. It is unclear how annexin-1 is expressed in various neuroepithelial tumors, and whether there is any association with tumor malignancy or survival. We studied annexin-1 expression in 394 glial neoplasms of all grades of malignancy and 81 normal brain samples by immunohistochemistry using tissue microarrays. The results were validated using western blot and reverse transcription-PCR (RT-PCR). In the normal human brain, the expression of annexin-1 is limited to ependymal cells and subependymal astrocytes, but is also upregulated in reactive astrocytes. Ependymomas and astrocytomas showed significantly higher mean annexin-1 expression levels in the cytoplasm compared with oligodendrogliomas (both: P<0.0001). In addition, nuclear staining of annexin-1 in oligodendroglial tumor cells was significantly reduced (P=0.0002), which may be used as a diagnostic tool for differentiating between astrocytomas and oligodendrogliomas. Although annexin-1 expression in ependymomas decreased with the grade of malignancy, diffuse astrocytomas showed a significant increase in cytoplasmic annexin-1-positive tumor cells. However, survival analysis showed that the expression of annexin-1 is not associated with patient survival. Similar to the EGFR amplification profile, primary glioblastomas had a higher annexin-1 expression level compared with secondary glioblastomas. Thus, annexin-1 upregulation in astrocytomas may contribute to tumor progression and its expression profile is similar to its substrate, EGFR, suggesting a possible regulation thereof.

Expression of EAAT-1 distinguishes choroid plexus tumors from normal and reactive choroid plexus epithelium.

Microscopic distinction of normal choroid plexus (CP) from choroid plexus tumors (CPT) may be difficult, especially in small samples of well-differentiated CP papillomas. So far, there are no established markers that reliably distinguish normal and neoplastic CP epithelium. Recently, a correlation between expression/function of glial glutamate transporters EAAT-1 (GLAST) and EAAT-2 (Glt-1) and tumor proliferation has been reported. Furthermore, we previously found that CPTs frequently express EAAT-1, but not EAAT-2. We now compared expression of EAAT-1, EAAT-2 and GFAP in non-neoplastic CP (n = 68) and CPT (n = 79) by immunohistochemistry. Tissue of normal CP was obtained from 50 autopsy cases (20 normal and 30 pathologic brains) and 18 neurosurgical specimens that included 17 fetal, 21 pediatric and 30 adult cases. In non-neoplastic postnatal CP (n = 51), focal expression of EAAT-1 was found in only two pediatric cases (4%). In CPT, expression of EAAT-1 was found in 64 of 79 (81%) tumor samples and was significantly age-dependent (P < 0.0001). Hence, EAAT-1 expression distinguishes neoplastic from normal CP, both in children (P = 0.0032) and in adults (P < 0.0001). Immunostaining for EAAT-2 in selected samples from cases of different ages showed that normal CP (n = 15) or CPT (n = 16) lacked EAAT-2 expression. GFAP expression was found in 3 of 32 (10%) normal CP and in 28 of 73 (38%) tumor samples. In conclusion, in contrast to neoplastic CP samples, expression of EAAT-1 is exceptionally rare in non-neoplastic CP. Thus, EAAT-1 is superior to GFAP as a helpful diagnostic tool in CP samples.

Mature cerebellar teratoma in adulthood.

Extragonadal teratomas in adulthood are exceptionally rare and usually not located within the cerebellum. We here report on a 66-year-old male patient clinically presenting with chronic occipital headache and episodes of severe vertigo. Neuroradiological investigations revealed a hemorrhagic tumor mass in the cerebellar vermis which was surgically removed and histologically diagnosed as mature teratoma. Hence, the presented case is extraordinary with regard to age, late clinical onset of symptoms and cerebellar location. Late clinical manifestation of the tumor in this case is probably due to an acute late-onset hemorrhage within the tumor.

Malignant glioma cells counteract antitumor immune responses through expression of lectin-like transcript-1.

Glioblastoma, one of the most lethal tumors, is paradigmatic for tumor-associated immunosuppression. Lectin-like transcript-1 (LLT1) is a newly identified ligand for the inhibitory natural killer (NK) cell receptor CD161. Here, we report that glioma cells express LLT1 mRNA and protein in vitro and in vivo, whereas expression levels in normal brain are low. LLT1 expression in human gliomas increases with the WHO grade of malignancy. We further show that transforming growth factor-beta (TGF-beta) up-regulates the expression of LLT1 in glioma cells. Small interfering RNA (siRNA)-mediated down-regulation of LLT1 in LNT-229 and LN-428 cells promotes their lysis by NK cells. Thus, LLT1 acts as a mediator of immune escape and contributes to the immunosuppressive properties of glioma cells.

WT1 expression distinguishes astrocytic tumor cells from normal and reactive astrocytes.

Particularly in small brain biopsies, it might be difficult to distinguish reactive astrogliosis from low-grade or infiltration zones of high-grade astrocytomas. So far no immunohistochemical marker allows a reliable distinction. Recently, the over-expression of Wilms' tumor gene product WT1 was reported in astrocytic tumor cells. However, no sufficient data on WT1 expression in normal or reactive astrocytes are available. Therefore, we investigated WT1 expression in paraffin-embedded brain sections from 28 controls, 48 cases with astrogliosis of various etiology and 219 astrocytomas [World Health Organization (WHO) grades I-IV] by immunohistochemistry. In normal brains and in astrogliosis, expression of WT1 was restricted to endothelial cells. In astrocytomas, WT1-positive tumor cells were found in pilocytic astrocytomas (66.7% of cases), diffuse astrocytomas (52.7%) WHO grade II (52.7%), anaplastic astrocytomas (83.4%) and glioblastomas (98.1%). Overall, the majority of all astrocytic neoplasms (84.5%) expressed WT1. Establishing a cut-off value of 0% immunoreactive tumor cells served to recognize neoplastic astrocytes with 100% specificity and 68% sensitivity and was associated with positive and negative predictive values of 1 and 0.68, respectively. Therefore, WT1 expression in astrocytes indicates a neoplastic origin and might represent an important diagnostic tool to differentiate reactive from neoplastic astrocytes by immunohistochemistry.

Severe hypoxia and multiple infarctions resembling Creutzfeldt-Jakob disease.

Although neuropathological examination is still required for the definite diagnosis of Creutzfeldt-Jakob disease (CJD), specialised clinical assessment predicts probable CJD. Here we present a 73-year-old female patient presenting with rapid cognitive decline, visual, acoustic and cerebellar disturbances, ataxia and EEG changes compatible with early CJD stages. MRI revealed hyperintensities within the thalami, hypothalami, corpora mammillaria, the tectum and the cortex. Initial neuropathological examination showed severe cortical and subcortical spongiosis. However, both immunohistochemistry and Western blotting showed no pathological prion protein. Finally, small infarctions affecting the tectum, tegmentum, corpora mammillaria and global hypoxic-ischaemic changes could be identified as the probable reason for the changes interpreted as CJD-related pathology. Hypoxic-ischaemic CNS alterations mainly affecting the supply area of the basilar artery should be ruled out in case of probable CJD. In addition, severe spongiosis can be misleading in the histological examination, suggesting the diagnosis of a prion-induced spongiform encephalopathy.

Regeneration and tolerance factor: a novel mediator of glioblastoma-associated immunosuppression.

Regeneration and tolerance factor (RTF) was originally identified in placenta where it is thought to be essential for fetal allograft survival. Here we report that RTF mRNA and protein are also expressed in human glioma cells in vitro and in vivo. Suppression of RTF expression by RNA interference promotes the lysis of glioma cells by natural killer (NK) and T cells in vitro. Moreover, RTF-depleted glioma cells are less tumorigenic than control cells in nude mice in vivo. Depletion of NK cells in these animals abolished this effect. RTF is thus a novel aberrantly expressed molecule which confers immune privilege to human malignant gliomas.

Elevated HLA-E levels in human glioblastomas but not in grade I to III astrocytomas correlate with infiltrating CD8+ cells.

HLA-E is a ligand for the immune-inhibitory NKG2A receptor expressed on NK and T cells. To investigate HLA-E expression and immune cell infiltration in human astrocytic tumors in vivo, we analyzed normal CNS controls and astrocytomas of all WHO grades by immunohistochemistry. Both, CD8(+) immune cell infiltration and HLA-E expression were significantly higher in astrocytic tumors than in normal brain. Further, HLA-E expression levels and immune cell infiltration were significantly correlated in WHO grade IV glioblastomas. Thus, HLA-E overexpression in glioblastomas may be triggered by T and NK cell infiltration.

Inflammatory myofibroblastic tumor of the ulnar nerve. Case report and review of the literature.

Inflammatory myofibroblastic tumors with involvement of cranial and peripheral nerves are exceedingly rare. The authors present the case of a 67-year-old man with an inflammatory myofibroblastic tumor of the left ulnar nerve, which was identified intraoperatively and mimicked a malignant neoplastic lesion. Histopathological examination revealed loosely structured fibrous tissue and collagen deposits intermingled with patchy infiltrates of lymphocytes, plasma cells, and histiocytes penetrating the endo- and epineurium of the affected nerve fascicles. There was strong expression of vimentin and actin in spindle cells throughout the lesion. The histiocytes were CD68- and major histocompatibility complex class II-positive, but lacked CD1a expression. A review of the literature revealed nine histopathologically confirmed cases of inflammatory myofibroblastic tumors involving peripheral or cranial nerves in which slight differences in histopathological features and surgical management were found, which are discussed here.

Expression of interleukin-16 by tumor-associated macrophages/activated microglia in high-grade astrocytic brain tumors.

INTRODUCTION: Macrophages/microglial cells are considered as immune cells in the central nervous system. Interleukin (IL)-16 is a proinflammatory cytokine produced by activated monocytic cells. MATERIALS AND METHODS: Expression of IL-16 was analyzed by immunohistochemistry in human astrocytic brain tumors and the rat C6 glioblastoma tumor model. IL-16 was detected in both human astrocytic brain tumors and rat C6 glioma. RESULTS: Compared with human control brains, a significant increase in the percentages of parenchymal IL-16+ macrophages/microglia was observed already in grade II astrocytomas, indicating that IL-16+ immunostaining could be a descriptor of a macrophage/microglia subset in astrocytic brain tumors. A further increase was observed at the transition from grade II to III astrocytomas. This increase in IL-16 immunoreactivity correlated with WHO grades of human astrocytic brain tumors. CONCLUSIONS: Therefore, IL-16 might be a so far unknown factor in the regulation of the local inflammatory milieu of human and experimental astrocytomas.

TGF-beta and metalloproteinases differentially suppress NKG2D ligand surface expression on malignant glioma cells.

NKG2D ligands (NKG2DL) are expressed by infected and transformed cells. They transmit danger signals to NKG2D-expressing immune cells, leading to lysis of NKG2DL-expressing cells. We here report that the NKG2DL MHC class I-chain-related molecules A and B (MICA/B) and UL16-binding proteins (ULBP) 1-3 are expressed in human brain tumours in vivo, while expression levels are low or undetectable in normal brain. MICA and ULBP2 expression decrease with increasing WHO grade of malignancy, while MICB and ULBP1 are expressed independently of tumour grade. We further delineate two independent mechanisms that can explain these expression patterns: (i) transforming growth factor-beta (TGF-beta) is upregulated during malignant progression and selectively downregulates MICA, ULBP2 and ULBP4 expression, while MICB, ULBP1 and ULBP3 are unaffected. (ii) Cleavage of MICA and ULBP2 is reduced by inhibition of metalloproteinases (MP), whereas no changes in the expression levels of other NKG2DL were detected. Consequently, NKG2DL-dependent NK cell-mediated lysis is enhanced by depletion of TGF-beta or inhibition of MP. Thus, escape from NKG2D-mediated immune surveillance of malignant gliomas in vivo may be promoted by the inhibition of MICA and ULBP2 expression via an autocrine TGF-beta loop and by MP-dependent shedding from the cell surface. Loss of MICA and ULBP2, in contrast to other NKG2DL, may be particularly important in glioma immune escape, and differential regulation of human NKG2DL expression is part of the immunosuppressive properties of human malignant glioma cells.

Cavernoma of the trochlear nerve.

Here we present the case of a 53-year old man with progressive double vision due to isolated left trochlear nerve palsy. Cranial magnetic resonance imaging (MRI) showed a small tumor within the left quadrigeminal cistern that did not increase in size after several months. Explorative neurosurgical intervention revealed a left trochlear nerve cavernoma. The lesion was microsurgically excised followed by end-to-end anastomosis of the trochlear nerve. After a one-year follow up, double vision totally disappeared and cranial MRI showed no recurrence. Cerebral cavernous malformations usually become symptomatic in seizures or focal neurological deficits after intracerebral hemorrhage. Rarely, cavernomas arise from cranial nerves. To the authors' knowledge, this is the first report on a symptomatic cavernous malformation arising from the trochlear nerve and on its successful surgical management.

Multiple thromboembolic events in fetofetal transfusion syndrome in triplets contributing to the understanding of pathogenesis of hydranencephaly in combination with polymicrogyria.

Over the last 180 years, several theories concerning the origin of hydranencephaly have been proposed with an emphasis on infectious, aplastic, and vascular etiologies. In this report, we present a case of triplets with fetofetal transfusion syndrome of which 2 fetuses (1 and 2) developed almost similar hydranencephaly, whereas the third exhibited the features of a fetus papyraceus (3). In the monochorial triamniotic placenta, multiple arteriovenous anastomoses were detected, representing a probable route for the transmission of thrombi originating from fetus 3 causing visceral lesions in fetus 2. Hydranencephaly was histologically characterized by necrosis, macrophage invasion, and endothelial proliferation. In addition, polymicrogyria was seen in fetuses 1 and 2. The combination of multiple visceral thromboembolic events and the death of fetus 3 approximately in the 11th week of gestation suggested a vascular thrombotic pathogenesis of hydranencephaly. Polymicrogyria can be considered as postmigratory laminar necrosis. Our findings contribute to the pathogenetic understanding of combined hydranencephaly and polymicrogyria.