Rolf Buslei

NeuN: a useful neuronal marker for diagnostic histopathology.:

The monoclonal antibody A60 specifically recognizes the DNA-binding, neuron-specific protein NeuN, which is present in most neuronal cell types of vertebrates. In this study we demonstrate the potential use of NeuN as a diagnostic neuronal marker using a wide range of formalin-fixed, paraffin-embedded human surgical and autopsy specimens from the central and peripheral nervous system. After microwave antigen retrieval, almost all neuronal populations revealed strong immunoreactivity for NeuN in nuclei, perikarya, and some proximal neuronal processes, whereas more distal axon cylinders and dendritic ramifications were not stained. The stain greatly enhanced the gray matter architecture. NeuN immunoreactivity was not detected in Purkinje cells, most neurons of the internal nuclear layer of the retina, and in sympathetic chain ganglia. We examined nine gangliogliomas and 14 dysembryoplastic neuroepithelial tumors, one ganglioneuroma, and one dysplastic cerebellar gangliocytoma. The neuronal component of all of these lesions showed marked immunoreactivity for NeuN. In addition, NeuN immunoreactivity was focally seen in one of seven medulloblastomas with prominent neuronal differentiation. There was no staining of non-neuronal structures. The results indicate that NeuN immunoreactivity is a sensitive and specific neuronal marker in formalin-fixed, paraffin-embedded tissues, and may be useful in diagnostic histopathology.

Common mutations of beta-catenin in adamantinomatous craniopharyngiomas but not in other tumours originating from the sellar region.

Dysregulation of the Wnt signalling pathway contributes to developmental abnormalities and carcinogenesis of solid tumours. Here, we examined β-catenin and adenomatous polyposis coli (APC) by mutational analysis in pituitary adenomas (n=60) and a large series of craniopharyngiomas (n=41). Furthermore, the expression pattern of β-catenin was immunohistochemically analysed in a cohort of tumours and cysts of the sellar region including pituitary adenomas (n=58), craniopharyngiomas (n=57), arachnoidal cysts (n=8), Rathke’s cleft cysts (n=10) and xanthogranulomas (n=6). Whereas APC mutations were not detectable in any tumour entity, β-catenin mutations were present in 77% of craniopharyngiomas, exclusively of the adamantinomatous subtype. All mutations affected exon 3, which encodes the degradation targeting box of β-catenin compatible with an accumulation of nuclear β-catenin protein. In addition, a novel 81-bp deletion of this exonic region was detected in one case. Immunohistochemical analysis confirmed a shift from membrane-bound to nuclear accumulation of β-catenin in 94% of the adamantinomatous tumours. Aberrant distribution patterns of β-catenin were never observed in the other tumour entities under study. We conclude that β-catenin mutations and/or nuclear accumulation serve as diagnostic hallmarks of the adamantinomatous variant, setting it apart from the papillary variant of craniopharyngioma.

In vitro and ex vivo evaluation of second-generation histone deacetylase inhibitors for the treatment of spinal muscular atrophy

Among a panel of histone deacetylase (HDAC) inhibitors investigated, suberoylanilide hydroxamic acid (SAHA) evolved as a potent and non‐toxic candidate drug for the treatment of spinal muscular atrophy (SMA), an α‐motoneurone disorder caused by insufficient survival motor neuron (SMN) protein levels. SAHA increased SMN levels at low micromolar concentrations in several neuroectodermal tissues, including rat hippocampal brain slices and motoneurone‐rich cell fractions, and its therapeutic capacity was confirmed using a novel human brain slice culture assay. SAHA activated survival motor neuron gene 2 (SMN2), the target gene for SMA therapy, and inhibited HDACs at submicromolar doses, providing evidence that SAHA is more efficient than the HDAC inhibitor valproic acid, which is under clinical investigation for SMA treatment. In contrast to SAHA, the compounds m‐Carboxycinnamic acid bis‐Hydroxamide, suberoyl bishydroxamic acid and M344 displayed unfavourable toxicity profiles, whereas MS‐275 failed to increase SMN levels. Clinical trials have revealed that SAHA, which is under investigation for cancer treatment, has a good oral bioavailability and is well tolerated, allowing in vivo concentrations shown to increase SMN levels to be achieved. Because SAHA crosses the blood–brain barrier, oral administration may allow deceleration of progressive α‐motoneurone degeneration by epigenetic SMN2 gene activation.

Increased reelin promoter methylation is associated with granule cell dispersion in human temporal lobe epilepsy.

Mesial temporal sclerosis (MTS) is the most common lesion in chronic, intractable temporal lobe epilepsies (TLE) and characterized by segmental neuronal cell loss in major hippocampal segments. Another histopathological hallmark includes granule cell dispersion (GCD), an architectural disturbance of the dentate gyrus encountered in approximately 50% of patients with mesial temporal sclerosis. Reelin, which plays a key role during hippocampal development and maintenance of laminar organization, is synthesized and released by Cajal-Retzius cells of the dentate molecular layer, and previous studies have shown that Reelin transcript levels are downregulated in human temporal lobe epilepsies specimens. To investigate whether epigenetic silencing by Reelin promoter methylation may be an underlying pathogenetic mechanism of GCD, DNA was harvested from 3 microdissected hippocampal subregions (i.e. molecular and granule cell layers of the dentate gyrus and presubiculum) from 8 MTS specimens with GCD, 5 TLE samples without GCD, and 3 autopsy controls. Promoter methylation was analyzed after bisulfite treatment, cloning, and direct sequencing; immunohistochemistry was performed to identify Cajal-Retzius cells. Reelin promoter methylation was found to be greater in TLE specimens than in controls; promoter methylation correlated with GCD among TLE specimens (p < 0.0002). No other clinical or histopathological parameter (i.e. sex, age, seizure duration, medication or extent, of MTS) correlated with promoter methylation. These data support a compromised Reelin-signaling pathway and identify promoter methylation as an epigenetic mechanism in the pathogenesis of TLE.

Suberoylanilide hydroxamic acid (SAHA) has potent anti-glioma properties in vitro, ex vivo and in vivo

Current treatment modalities for malignant gliomas do not allow long‐term survival. Here, we identify suberoylanilide hydroxamic acid (SAHA), an inhibitor of histone deacetylases (HDAC), as an effective experimental anti‐glioma agent. Administration of SAHA to various glioma cell lines obtained from human, rat and mouse inhibited tumour cell growth in a range of 1–10 μm. This anti‐glioma property is associated with up‐regulation of the cell cycle control protein p21/WAF, as well as the induction of apoptosis. A novel tumour invasion model using slice cultures of rat brain corroborated the anti‐glioma properties of SAHA in the organotypic brain environment. In this model, glioma invasion compromised adjacent brain parenchyma, and this tumour‐associated cytotoxicity could be inhibited by SAHA. In addition, a 10‐fold dose escalation experiment did not challenge the viability of cultured brain slices. In vivo, a single intratumoural injection of SAHA 7 days after orthotopic implantation of glioma cells in syngeneic rats doubled their survival time. These observations identify chromatin‐modifying enzymes as possible and promising targets for the pharmacotherapy of malignant gliomas.

Improved delineation of brain tumors: an automated method for segmentation based on pathologic changes of 1H-MRSI metabolites in gliomas

In this study, we developed a method to improve the delineation of intrinsic brain tumors based on the changes in metabolism due to tumor infiltration. Proton magnetic resonance spectroscopic imaging ((1)H-MRSI) with a nominal voxel size of 0.45 cm(3) was used to investigate the spatial distribution of choline-containing compounds (Cho), creatine (Cr) and N-acetyl-aspartate (NAA) in brain tumors and normal brain. Ten patients with untreated gliomas were examined on a 1.5 T clinical scanner using a MRSI sequence with PRESS volume preselection. Metabolic maps of Cho, Cr, NAA and Cho/NAA ratios were calculated. Tumors were automatically segmented in the Cho/NAA images based on the assumption of Gaussian distribution of Cho/NAA values in normal brain using a limit for normal brain tissue of the mean + three times the standard deviation. Based on this threshold, an area was calculated which was delineated as pathologic tissue. This area was then compared to areas of hyperintense signal caused by the tumor in T2-weighted MRI, which were determined by a region growing algorithm in combination with visual inspection by two experienced clinicians. The area that was abnormal on (1)H-MRSI exceeded the area delineated via T2 signal changes in the tumor (mean difference 24%) in all cases. For verification of higher sensitivity of our spectroscopic imaging strategy we developed a method for coregistration of MRI and MRSI data sets. Integration of the biochemical information into a frameless stereotactic system allowed biopsy sampling from the brain areas that showed normal T2-weighted signal but abnormal (1)H-MRSI changes. The histological findings showed tumor infiltration ranging from about 4-17% in areas differentiated from normal tissue by (1)H-MRSI only. We conclude that high spatial resolution (1)H-MRSI (nominal voxel size = 0.45 cm(3)) in combination with our segmentation algorithm can improve delineation of tumor borders compared to routine MRI tumor diagnosis.

Diffusion tensor imaging and optimized fiber tracking in glioma patients: Histopathologic evaluation of tumor-invaded white matter structures

Fiber tracking is increasingly used to plan and guide neurosurgical procedures of intracranial tumors in the vicinity of functionally important areas of the brain. However, valid data concerning the reliability of tracking with respect to the actual pathoanatomical situation are lacking. We retrospectively correlated fiber tracking based on magnetic resonance (MR) DT imaging with the histopathological data of 25 patients with WHO grade II and III gliomas. Fiber tracking using the Fiber Assignment by Continuous Tracking (FACT) method was performed to investigate the integrity of white matter tracts in the surrounding border zone of the lesions. The tracking procedure was stopped when fractional anisotropy (FA) thresholds = 0.1, 0.15, 0.2, 0.25, and 0.3, or a tract turning angle >60 degrees were encountered. In 9 patients we were able to reconstruct brain fiber tracts at biopsy loci (2-32% tumor infiltration) using an FA threshold of 0.15 and 0.2, but not for a threshold of 0.25 or 0.3. The neurological outcome demonstrated potential tumor cell infiltration of functionally intact brain fiber tracts in the range of 2-8%. These findings may be useful in planning therapeutic approaches to gliomas in the vicinity of eloquent brain regions.

Nuclear beta-catenin accumulation as reliable marker for the differentiation between cystic craniopharyngiomas and rathke cleft cysts: a clinico-pathologic approach.

Clinical and histopathologic differentiation of cystic lesions from the sellar region, that is, craniopharyngiomas (CPs) and Rathke cleft cysts (RCCs), is challenging and has paramount importance with respect to variable clinical manifestation and adapted surgical treatment strategies in both entities. Here, we retrospectively evaluated clinico-pathologic findings in 81 patients presenting with a cystic tumor located in the sellar region. All patients underwent transsphenoidal or transcranial resections. Microscopic inspection of surgical specimens identified CP in 51 patients, and RCC in 30 patients. Amongst the panel of immunohistochemical marker proteins used for histopathologic analysis, nuclear accumulation of β-catenin was detectable only in CP. On the basis of the histopathologic and immunohistochemical analysis, clinical presentation (sex, age, ophthalmologic, and endocrinologic deficits), imaging (tumor location, size, and calcification), as well as a description of cyst contents obtained during operation were retrospectively evaluated. In purely cystic CPs, an isointense signal was more frequent in T1-weighted magnetic resonance images and calcification of the tumor capsule in computed tomography scans. In addition, the size of RCC was smaller and this tumor entity was more often located within the sella. Aberrant (nuclear) immunohistochemical staining for β-catenin appeared, however, as most reliable factor for the differentiation between purely cystic CPs and RCCs, whereas tumor location, tumor size, and calcification of the tumor capsule were less consistent parameters. The data are compatible with distinct pathogenic pathways associated with these related histopathologic entities.

Proton Magnetic Resonance Spectroscopic Imaging Integrated into Image-guided Surgery: Correlation to Standard Magnetic Resonance Imaging and Tumor Cell Density

OBJECTIVE: In this study, we attempted to improve the delineation of the infiltration zone in gliomas using proton magnetic resonance spectroscopic imaging (1H MRSI). In conventional magnetic resonance imaging (MRI), the boundaries of gliomas sometimes are underestimated. 1H MRSI is a noninvasive tool that can be used to investigate the spatial distribution of metabolic changes in brain lesions. The purpose was to correlate tumor cell density from histopathological specimens with metabolic levels and the coregistered metabolic maps. METHODS: We developed a method to integrate spectroscopic data depicted as metabolic maps of biochemically pathological tissue into frameless stereotaxy. In seven patients harboring gliomas, we performed 1H MRSI with high spatial resolution and evaluated the spectral data. An algorithm was developed for user-independent calculation of pathological voxels and for visualization as metabolic maps. These maps were integrated into a three-dimensional MRI data set used for frameless stereotaxy. Stereotactic biopsies were taken from three different areas in and around the tumor involving the maximum pathological change, the border zone, and an area from outside the spectroscopically suspicious area. These specimens were correlated to the exact voxel positions in the stereotactic image space and evaluated histopathologically. RESULTS: In all cases, the implementation of the metabolic maps into frameless stereotaxy was successful, and stereotactic biopsies were acquired by use of the spectral data. A relation could be demonstrated between the metabolic changes and tumor cell density ranging from 60 to 100% in the maximum pathological area to 5 to 15% in the border zone. Interestingly, the tumor areas defined by the metabolic maps and histopathologically confirmed by biopsy exceeded the T2-weighted signal change in all cases, ranging from 6 to 32% in the examined volume. CONCLUSION: Our preliminary data suggest that 1H MRSI may be useful in combination with frameless stereotaxy to define more exactly the tumor infiltration zone in glioma surgery compared with conventional anatomic MRI alone.

Microtubule-associated protein-2 immunoreactivity: a useful tool in the differential diagnosis of low-grade neuroepithelial tumors

Complex and variable morphological phenotypes pose a major challenge to the histopathological classification of neuroepithelial tumors. This applies in particular for low-grade gliomas and glio-neuronal tumors. Recently, we and others have identified microtubule-associated protein-2 (MAP2) as an immunohistochemical marker expressed in the majority of glial tumors. Characteristic cell morphologies can be recognized by MAP2 immunoreactivity in different glioma entities, i.e., process sparse oligodendroglial versus densely ramified astrocytic elements. Here, we describe MAP2-immunoreactivity patterns in a large series of various neuroepithelial tumors and related neoplasms (n=960). Immunohistochemical analysis led to the following conclusions: (1) specific pattern of MAP2-positive tumor cells can be identified in 95% of glial neoplasms; (2) ependymal tumors do not express MAP2 in their rosette-forming cell component; (3) tumors of the pineal gland as well as malignant embryonic tumors are also characterized by abundant MAP2 immunoreactivity; (4) virtually no MAP2 expression can be observed in the neoplastic glial component of glio-neuronal tumors, i.e. gangliogliomas; (5) malignant glial tumor variants (WHO grade III or IV) exhibit different and less specific MAP2 staining patterns compared to their benign counterparts (WHO grade I or II); (6) with the exception of melanomas and small cell lung cancers, MAP2 expression is very rare in metastatic and non-neuroepithelial tumors; (7) glial MAP2 expression was not detected in 56 non-neoplastic lesions. These data point towards MAP2 as valuable diagnostic tool for pattern recognition and differential diagnosis of low-grade neuroepithelial tumors.