Epigenetic repression of RASSF1A but not CASP8 in supratentorial PNET (sPNET) and atypical teratoid/rhabdoid tumors (AT/RT) of childhood.

Supratentorial primitive neuroectodermal tumors (sPNET) and atypical teratoid/rhabdoid tumors (AT/RT) of the CNS represent a biological and clinical enigma, despite advances in both molecular techniques and clinical management for these two rare embryonal brain tumors of childhood. Epigenetic changes hold great potential as possible disease mechanisms and may be manipulated therapeutically. We thus studied aberrant methylation of the genes RASSF1A and CASP8 and its consequence on expression in cell lines and primary tumors using a combination of semiquantitative methylation specific PCR (MSP), bisulfite sequencing and RT-PCR. In all, 17 samples of autopsy-derived normal appearing brain served as controls. Opposed to control tissues 19/24 sPNET and 4/6 AT/RT demonstrated aberrant methylation for the RASSF1A promoter region. Treatment of cell lines using 5-Aza-2'-deoxycytidine (5AZA) alone or in combination with trichostatin A (TSA) succeeded in re-establishing expression of RASSF1A in cell lines derived from a renal rhabdoid, an AT/RT and a medulloblastoma. A 5' CpG-rich region of CASP8 was methylated in normal tissues and in tumors. However, CASP8 showed inconsistent expression patterns in normal and tumor tissues. Our results indicate that aberrant methylation of the RASSF1A promoter region may be of importance in the origin and progression of sPNET and AT/RT while the analysed 5'-CpG rich region of the CASP8 gene does not seem to play an important role in these tumors. Further studies of epigenetic changes in these rare tumors are warranted as their biology remains obscure and treatment efforts have been rather unsuccessfull.

Evidence for specific phases in the development of human neuromelanin.

Neuromelanin is a dark-coloured pigment which forms in the dopamine neurons of the human midbrain. The age-related development and regulation of neuromelanin within these dopamine neurons has not been previously described. Optical density and area measurements of unstained neuromelanin in ventral substantia nigra neurons from 29 people spanning the ages of 24 weeks to 95 years old, demonstrated three developmental phases. Neuromelanin was not present at birth and initiation of pigmentation began at approximately 3 years of age, followed by a period of increasing pigment granule number and increasing pigment granule colouration until age 20. In middle and later life the colour of the pigment granules continued to darken but was not associated with any substantial growth in pigment volume. The identification of three phases and changes in the rate of neuromelanin production over time suggests the regulation of neuromelanin production and turnover, possibly through enzymatic processes.

[Clinical value of amino acid imaging in paediatric brain tumours. Comparison with MRI]. / Der klinische Wert der Darstellung kindlicher Hirntumore mit Aminosäuren: Vergleich mit MRT.

PURPOSE: To evaluate single photon emission computed tomography (SPECT) using the amino acid l-3-[123I]-alpha-methyl tyrosine (IMT) and contrast enhanced magnetic resonance imaging (MRI) as diagnostic tools in primary paediatric brain tumours in respect of non-invasive tumour grading. Patients, materials, methods: 45 children with primary brain tumours were retrospectively evaluated. IMT uptake was quantified as tumour/nontumour-ratio, a 4-value-scale was used to measure gadolinium enhancement on contrast enhanced MRI. Statistical analyses were performed to evaluate IMT uptake and gadolinium enhancement in low (WHO I/II) and high (WHO III/IV) grade tumours and to disclose a potential relationship of IMT uptake to disruption of blood brain barrier as measured in corresponding MRI scans. RESULTS: IMT uptake above background level was observed in 35 of 45 patients. IMT uptake was slightly higher in high grade tumours but the difference failed to attain statistical significance. Grading of individual tumours was neither possible by IMT SPECT nor by gadolinium enhanced MRI. CONCLUSION: IMT is accumulated in most brain tumours in children. Tumour grading was not possible using IMT or contrast enhancement as determined by MRI. Neither morphological nor functional imaging can replace histology in paediatric brain tumours.

Cell death mechanisms in multiple system atrophy.

The presence and distribution of apoptotic cell death in multiple system atrophy (MSA) and morphologically related diseases were investigated by means of a modified terminal deoxynucleotidyl transferase-mediated nick end labeling method, comparing their distribution with that of glial cytoplasmic inclusions, immunohistochemically demonstrated bcl-2 protein, bax protein, CD95, TNFalpha, and p53-protein expression, as well as activated microglia. Apoptosis occurred almost exclusively in oligodendrocytes in multiple system atrophy and its general distribution was comparable to the already known oligodendroglial pathology in this disorder. Additionally, in about a quarter of glial cytoplasmic inclusions, there was upregulation of bcl-2-protein and coexpression with ubiquitin, suggesting a final attempt of involved cells to counteract apoptotic cell death. Bax protein was also demonstrated in oligodendroglial cells. A significant neuronal apoptosis was not observed in MSA; these cells might be destroyed secondarily to oligodendroglial apoptosis by necrosis or other forms of programmed cell death. These results emphasize the central role of oligodendroglial pathology in multiple system atrophy, making this disease unique among neurodegenerative diseases.

Comparative genomic hybridization in pineal germ cell tumors.

Fifteen primary pineal germ cell tumors (8 germinomas, 4 mixed teratomas-germinomas, 2 immature teratomas, and 1 yolk sac tumor) and 2 recurrences of the yolk sac tumor were studied by comparative genomic hybridization (CGH). An average of 1.8 chromosomal changes per germinoma (0.5 gains vs 1.3 losses), 5.5 per mixed teratoma-germinoma (3.0 gains vs 2.5 losses), 3.5 per immature teratoma (2.0 gains vs 1.5 losses), and 2.0 in the yolk sac tumor (2 gains vs 0 losses) were found; the first recurrence showed 7 (4 gains vs 3 losses), the second 13 imbalances (8 gains vs 5 losses). The most frequent imbalances were gains on 12p (40%), 8q (27%), and 1q (20%) as well as losses on 13q (47%), 18q (33%), 9q and 11q (20% each). Among germinomas, the most common chromosomal changes were -13q and -18q (38% each), in mixed teratomas-germinomas +8q (100%), +12p (75%), -13q (75%) and -9q (50%). Seven high-level gains were identified: 5 in mixed teratomas-germinomas (+8q: 3 cases, + 12p: 2 cases), 1 each in a germinoma (+2p) and an immature teratoma (+12p). Minimal common regions of over- and underrepresentation were found on +8q11.22-21.1, +12p11.1-12.1, -9q32-qter, -11q23.2-qter, -13q32-qter and -18q22-qter. Our findings suggest, that imbalances in cerebral germ cell tumors affect the same chromosomes as among their extracerebral counterparts, albeit in a considerably lower frequency among cerebral germinomas where +12p does not seem to play a major role.

A unique case of cerebral spleen.

We present the first case of cerebral splenosis, occurring in a 20-year-old man 15 years after posttraumatic splenectomy. He became symptomatic through seizures and was operated on for suspected meningioma of the right occipital pole. Histologic evaluation of the lesion revealed splenic tissue with matching immunohistochemical results. Because no penetrating head injuries were reported at the time of trauma, a hematogenous spread of splenic tissue has to be assumed.

Tumors of the choroid plexus.

Choroid plexus tumors are rare intraventricular papillary neoplasms derived from choroid plexus epithelium, which account for only between 0.4-0.6% of all intracranial and 2-3% of pediatric neoplasms. Plexus papillomas outnumber choroid plexus carcinomas by a ratio of 5:1 and around 80% of choroid plexus carcinomas arise in children. Plexus tumors are most common in the lateral and fourth ventricles; while 80% of lateral ventricle tumors present in children, fourth ventricle tumors are evenly distributed in all age groups. Clinically, choroid plexus tumors tend to cause hydrocephalus and increased intracranial pressure. Histologically, choroid plexus papillomas correspond to WHO grade I, choroid plexus carcinomas to WHO grade III. Immunohistochemically, cytokeratins and vimentin are expressed by virtually all choroid plexus papillomas and most choroid plexus carcinomas while transthyretin and S-100 protein are present in 80-90% of cases, less frequently, though, in choroid plexus carcinomas. Glial fibrillary acidic protein can be found focally in about 25-55% of choroid plexus papillomas and 20% of choroid plexus carcinomas. The mean Ki67/MIB1 labeling index for choroid plexus papillomas is 1.9%, for choroid plexus carcinomas 13. 8%. Choroid plexus papillomas typically show hyperdiploidy with gains particularly on chromosomes 7, 9, 12, 15, 17, and 18 while one choroid plexus carcinoma showed rearrangements of chromosomes 7p11-12, 9q11-12, 15q22, and 19q13.4. Choroid plexus papillomas can usually be cured by surgery alone with a 5-year survival rate of up to 100% with occasional recurrences while choroid plexus carcinomas grow more rapidly and have a less favorable outcome with a 5-year survival rate of 26-40%.

Shunt-related abdominal metastasis of cerebral teratocarcinoma: report of an unusual case and review of the literature.

OBJECTIVE AND IMPORTANCE: Internal drainage of cerebrospinal fluid to the abdominal cavity via a ventriculoperitoneal shunt (VPS) is a common procedure for therapy of obstructive hydrocephalus; because this condition is often caused by brain tumors blocking the natural cerebrospinal fluid pathways, the VPS as an artificial anastomosis can provide the means for the spreading of tumor cells by the cerebrospinal fluid. We report the case of a VPS-related abdominal metastasis of a teratocarcinoma and review the pertaining literature. CLINICAL PRESENTATION AND INTERVENTION: A 24-year-old man with a history of three brain tumors that were operated on when the patient was 14, 21, and 23 years of age developed an acute ileus 7 months after VPS insertion for cerebral teratocarcinoma. Intraoperatively, a massive abdominal tumor was observed, which turned out to be a peritoneal metastasis of the aforesaid brain tumor. The patient died as a result of his illness 1 month later. RESULTS: To date, 58 VPS-related metastases of brain tumors have been described. The male-to-female ratio is 1.6:1, the mean age at shunt insertion is 12.2 years, and the interval between shunt operation and diagnosis of metastases is 16.8 months. During the observation time, 69.2% of the patients died as a result of their illness or abdominal metastases. The most common sources of the metastases were germinomas (27.7%), medulloblastomas (19.1%), and endodermal sinus tumors (10.3%). CONCLUSION: The presented case is only the second VPS-related abdominal spreading of a cerebral teratocarcinoma. Metastases via VPS are rare but should be considered as a possible complication and mode of systemic spread in patients with primary intracranial malignancy.

Factor XIIIa expression in granulomatous lesions due to sarcoidosis or mycobacterial infection.

The a-subunit of the clotting factor XIII (FXIIIa) has previously been shown to be synthesized by cells of monocyte lineage such as macrophages and histiocytes. Thus, besides clot retraction, a possible role of FXIIIa has also been postulated in inflammation. In order to test this hypothesis, FXIIIa-expression in granulomatous lesions due to sarcoidosis and mycobacterial infection was investigated. In the 12 cases (six cases each) examined, FXIIIa-positive macrophages were consistently detected by immunohistochemistry. They were predominantly observed in the periphery of granulomas, whereas the centers were generally devoid of these cells. We did not find any difference in the distribution of FXIIIa-positive cells in both conditions; thus FXIIIa did not improve the differential diagnosis between sarcoidosis and tuberculosis. However, FXIIIa-producing macrophages seemed to contribute to the centripetal fibrosis in granuloma. These results further suggest that the basic pathogenetic mechanisms in granuloma formation are very similar, regardless of their origin from sarcoidosis or tuberculosis.

Factor XIIIa-immunoreactivity in tumors of the central nervous system.

The clotting factor XIIIa (FXIIIa) has been shown to be present both in tumor cells and in tumor-associated macrophages of different neoplasms such as Hodgkin's disease, giant cell tumor of bone, malignant fibrous histiocytoma, meningeal tumors, and hemangiopericytoma. The biological significance of these findings, however, are still unclear. This study investigates the immunohistochemical distribution of FXIIIa in 186 tumors of the central nervous system (CNS) in order to evaluate its possible diagnostic or prognostic significance in neuro-oncology. High-grade gliomas such as glioblastoma, gliosarcoma, astrocytoma (grade III WHO), and ependymoma (III) as well as meningiomas and meningeal hemangiopericytomas consistently contained factor XIIIa-positive cells, whereas low-grade glial tumors did not do so. One desmoplastic medulloblastoma and one anaplastic schwannoma also showed FXIIIa-positive cells. With the exception of hemangiopericytomas, however, the major source of FXIIIa expression in all these tumors consisted of a subpopulation of tumor-associated macrophages, the exact role of which still remains unclear. Because of its non-discriminatory staining in a wide variety of CNS tumors, the differential diagnostic contribution of FXIIIa in neuro-oncology seems to be limited.

Immunoglobulin deposition in sural nerves of AIDS patients with distal-symmetric HIV-associated polyneuropathy.

The most frequent neurological diagnosis in peripheral nerve function of HIV-positive individuals is distal-symmetric polyneuropathy (DSPN). In this study we investigated the histopathology as well as the immunohistochemical expression of immunoglobulins IgA, IgG and IgM in post-mortem sural nerve tissue gained from 11 patients who had suffered from DSPN in the clinical course of AIDS (CDC 3C). We found that all 11 sural nerves showed signs of demyelination while in 6 out of 11 cases axonal degeneration could also be detected. Immunohistochemical expression of at least one immunoglobulin was found in all but two cases with deposits uniformly being located immediately beneath the basement membrane of capillary blood vessels and within the perineurium while endoneurial staining was discernable in three cases. The most commonly expressed immunoglobulin was IgA which was identified in 7 cases, followed by IgG and IgM which were positive in 6 and 5 cases, respectively. All three immunoglobulins were found to be expressed simultaneously in only two cases. Thus, our study shows that immunoglobulin deposits among other factors may be implicated in altering the function of sural nerves or enhance their vulnerability. In peripheral nerves they may be responsible for some of the common alterations in the development of AIDS-associated distal symmetric polyneuropathy.

[Adhesions and interference images of unfixed erythrocytes]. / Adhäsions- und Interferenzfiguren unfixierter Erythrozyten.

For the analysis of deformability in microcirculatory investigations an exact understanding of red blood cell (RBC) geometry is required. To extend knowledge we introduce a new morphological feature of resting unfixed erythrocytes by means of an inverted reflection contrast microscope (RCM). By assessing the interference patterns caused by RCM erythrocytes can be classified according to the depth of their central concavity which depends on the flexibility of the RBC. Moreover, the RBC adhesion can be directly observed. We found out that: 1. Five types of normocytes can be distinguished in RCM. 2. In phase contrast the size distribution of RBC without central concavity (type 5, 11.53% of all normocytes) shows peaks at 48 microns2 and 52 microns2. 3. Image analysis reveals two size categories of relative adhesion areas. One category consists of type 1 and 2 (relative adhesion area 25.63%), the other of the types 3 to 5 (relative adhesion area 39.91%). Besides, RCM allows the reliable identification of pathologic erythrocytes in unstained specimens.

Evaluation on blood platelets by the image analysis system VIDAS 2.5.

This paper introduces a program written on the image analysis system VIDAS 2.5. It enables the automatic quantification of high numbers of adhesion areas of vital human platelets, thus allowing statistical analysis. These adhesion areas were observed by reflection contrast microscopy (RCM), which generates images of an intense contrast and serves as a prerequisite for an evaluation by image analysis. However, RCM-photographs of the observed platelets have highly varying mean greyvalues and greyranges. These common problems for self-operating identification are excluded by two procedures within the program: 1. calibration of the scanning process for an optimal use of the available greyvalues provided by the negative, camera, and the image analysis system; and 2. relation of the threshold for discrimination of adhesion areas to the statistic parameters of the histogram within each individual digitized image. Images processed according to these prerequisites were transferred to the VIDAS implemented routines for identification and measurement of areas. Thus, image analysis combined with RCM offers a tool for basic and clinical platelet research, which is shown by an example of stimulation and inhibited stimulation of platelet activation.

Frequent but borderline methylation of p16 (INK4a) and TIMP3 in medulloblastoma and sPNET revealed by quantitative analyses.

Certain risk groups among tumors of the central nervous system (CNS) in children take an almost inevitably fatal course. The elucidation of molecular mechanisms offers hope for improved therapy. Aberrant methylation is common in malignant brain tumors of childhood and may have implications for stratification and therapy. Methylation of p16 (INK4A), p14 (ARF), TIMP3, CDH1, p15 (INK4B )and DAPK1 in medulloblastoma (MB) and ependymoma has been discussed controversially in the literature. DUTT1 and SOCS1 have not previously been analyzed. We examined methylation in MB, sPNET and ependymoma using methylation-specific PCR (MSP), quantitative Combined Bisulfite Restriction Analysis (COBRA) and direct and clone sequencing of bisulfite PCR products. We detected methylation of p16 (INK4A) (17/43), p14 (ARF) (11/42) and TIMP3 (9/44) in MB and others by MSP. CDH1 was not only methylated in MB (31/41), but also in normal controls. Evaluation of MSP results by quantitative COBRA and sequencing yielded methylation between the detection limits of COBRA (1%) and MSP (0.1%). Only p16 (INK4A )and TIMP3 were methylated consistently in medulloblastomas (p16 (INK4A ) 14%, TIMP3 11%) and p16 (INK4A) also in anaplastic ependymomas (1/4 tumors). Methylation ranged from 1-5%. Evaluation of methylation using MSP has thus to be supplemented by quantitative methods. Our analyses raise the issue of the functional significance of low level methylation, which may disturb the delicate growth factor equilibrium within the cell. Therapeutic and diagnostic implications urge into depth analyses of methylation as a mechanism, which might fill some of the gaps of our understanding of brain tumor origin.

Chromosomal imbalances and NF2 mutational analysis in a series of 10 spinal nerve sheath myxomas.

AIMS: To report the demographic, clinical and molecular profile of a series of intraspinal nerve sheath myxomas. Nerve sheath myxomas are diagnostically challenging, mainly cutaneous spindle cell neoplasms exhibiting Schwann cell differentiation. They are frequently mistaken for neurothekeomas and their genetic features are essentially unknown. METHODS AND RESULTS: Ten spinal nerve sheath myxomas with a preferential location in the lumbar spine (70%) were investigated. Presenting symptoms consisted of sciatic pain (100%), muscle weakness and paraesthesia (60% each). Intraoperatively, all tumours were attached to a spinal nerve. Chromosomal imbalances by comparative genomic hybridization were found in 8/10 cases, consisting of -22q (80%) and -19 (30%). Polymerase chain reaction analysis of the NF2 gene (exons 1-16) revealed two tumours with mutations in exon 8 and 14, respectively. CONCLUSIONS: Although these 10 nerve sheath myxomas exhibited Schwann cell differentiation and frequently showed loss of chromosome 22q typically encountered in peripheral nerve tumours, only two cases demonstrated mutations of the NF2 gene. This may indicate involvement of other tumour suppressor genes on 22q in nerve sheath myxomas and shows that they are more closely related at the molecular level to sporadic schwannomas, underscoring the presumption that they are true nerve sheath tumours.

Evidence for specific phases in the development of human neuromelanin.

Neuromelanin is a dark-coloured pigment which forms in the dopamine neurons of the human midbrain. Here we describe the age-related development and regulation of neuromelanin within these dopamine neurons. 10 microm sections from formalin-fixed midbrain from 29 people spanning the ages of 24 weeks to 95 years old were either stained with a basic Nissl substance stain (0.5% cresyl violet), or processed unstained. After locating the substantia nigra using the stained sections, digital photos were taken of individual ventral substantia nigra neurons in the unstained sections, and the cellular area occupied by pigment, and optical density were measured using computer software. These measurements demonstrated three developmental phases. Neuromelanin was not present at birth and initiation of pigmentation began at approximately 3 years of age, followed by a period of increasing pigment granule number and increasing pigment granule colouration until age 20. In middle and later life the colour of the pigment granules continued to darken but was not associated with any substantial growth in pigment volume. The identification of three phases and changes in the rate of neuromelanin production over time suggests the regulation of neuromelanin production and turnover, possibly through enzymatic processes.

Immunohistochemical profile and chromosomal imbalances in papillary tumours of the pineal region.

The histopathology of papillary tumours of the pineal region (PTPR) closely resembles that of ependymomas and choroid plexus tumours. Therefore, immunohistochemical staining profiles were investigated in a series of 15 PTPR. In addition to cytokeratin, synaptophysin and glial fibrillary acidic protein expression, PTPR were examined for the presence of dot- or ring-like epithelial membrane antigen (EMA) immunoreactivity typically encountered in ependymoma, staining for inwardly rectifying potassium channel Kir7.1 and stanniocalcin-1 (specifically expressed in choroid plexus tumours) as well as microtubule-associated protein-2 (MAP-2). Furthermore, comparative genomic hybridization was performed in five PTPR. Cytokeratin was expressed in all PTPR examined, whereas glial fibrillary acidic protein and synaptophysin staining were absent. Dot- or ring-like EMA immunoreactivity was only observed in 1 out of 15 PTPR. Membranous Kir7.1 and cytoplasmic stanniocalcin-1 staining were present in the minority of PTPR (3/15 and 4/15, respectively). In contrast, MAP-2 immunoreactivity was encountered in 13 out of 15 PTPR, but was significantly less frequently observed in a series of choroid plexus tumours (7/37). PTPR mainly presented with chromosomal losses affecting chromosomes 10 (4/5 cases) and 22q (3/5 cases) as well as gains on chromosomes 4 (4/5 cases), 8 (3/5 cases), 9 (3/5 cases) and 12 (3/5 cases). To conclude, the majority of PTPR can be distinguished from ependymomas and choroid plexus tumours by absent staining for epithelial membrane antigen, Kir7.1 and staniocalcin-1 as well as the presence of distinct MAP-2 immunoreactivity. Antibodies directed against these antigens are thus expected to be valuable markers in the diagnosis of papillary tumours located in the vicinity of the third ventricle.

O-(2-[18F]fluorethyl)-L-tyrosine PET in the clinical evaluation of primary brain tumours.

PURPOSE: The aim of this study was to evaluate the differential uptake of O-(2-[18F]fluorethyl)-L-tyrosine (FET) in suspected primary brain tumours. METHODS: Positron emission tomography (PET) was performed in 44 patients referred for the evaluation of a suspected brain tumour. Acquisition consisted of four 10-min frames starting upon i.v. injection of FET. Tumour uptake was calculated as the ratio of maximal tumour intensity to mean activity within a reference region (FETmax). RESULTS: FET uptake above the cortical level was observed in 35/44 lesions. All histologically confirmed gliomas and many other lesions showed FET uptake to a variable extent. No uptake was observed in nine lesions (one inflammatory lesion, one dysembryoplastic neuroepithelial tumour, one mature teratoma, six lesions without histological confirmation). An analysis of uptake dynamics was done in the patients with increased FET uptake (22 gliomas, three lymphomas, three non-neoplastic lesions, three lesions with unknown histology and four other primaries). Upon classification of tumours into low (i.e. WHO I and II) and high grade (i.e. WHO III and IV), a significant difference in FETmax between the two categories was observed only in the first image frame (0-10 min p.i.), with FETmax=2.0 in low-grade and 3.2 in high-grade tumours (p<0.05); no significant differences were found in frame 4 (30-40 min p.i.), with FETmax=2.4 vs 2.7. Similar results were obtained when the analysis was applied only to astrocytic tumours (2.0 vs 3.1 in the first frame; 2.4 vs 2.6 in the fourth frame). CONCLUSION: These initial results indicate that FET PET is a useful method to identify malignant brain lesions. It appears that high- and low-grade brain tumours exhibit a different uptake kinetics of FET. A kinetic analysis of FET PET may provide additional information in the differentiation of suspected brain lesions.

Epidemiological features of brain tumors in the first 3 years of life.

We investigated the age-related location, gender distribution, and histology of 75 brain tumors in children under 3 years of age seen in our department between 1984 and 1997. These were characterized by a higher overall incidence in boys (42/33 cases; ratio 1.3) and a prevalence for a supratentorial location (48/27 cases; ratio 1.7); the most common histological entities were astrocytoma (25.3%) and ependymoma (17.3%), followed by medulloblastoma (13.3%) and PNET (10.8%); 44% were high-grade tumors corresponding to WHO grades III and IV. In the 1st (22 cases), 2nd (25 cases) and 3rd (28 cases) years of life, the boy-girl ratios were 1.0, 1.5 and 1.3, respectively, while there was a decrease with age in the frequency of supratentorial (ratios 3.4, 1.1, and 1.2) and high-grade tumors (77.3%, 36.0%, and 32.1%). In the 1st year of life the most common neoplasms were PNETs (22.7%) and in the 2nd year both astrocytomas and ependymomas (24.0 % each); astrocytomas (35.7%) prevailed in the 3rd year of life.