CD74 regulates complexity of tumor cell HLA class II peptidome in brain metastasis and is a positive prognostic marker for patient survival.

Despite multidisciplinary local and systemic therapeutic approaches, the prognosis for most patients with brain metastases is still dismal. The role of adaptive and innate anti-tumor response including the Human Leukocyte Antigen (HLA) machinery of antigen presentation is still unclear. We present data on the HLA class II-chaperone molecule CD74 in brain metastases and its impact on the HLA peptidome complexity.We analyzed CD74 and HLA class II expression on tumor cells in a subset of 236 human brain metastases, primary tumors and peripheral metastases of different entities in association with clinical data including overall survival. Additionally, we assessed whole DNA methylome profiles including CD74 promoter methylation and differential methylation in 21 brain metastases. We analyzed the effects of a siRNA mediated CD74 knockdown on HLA-expression and HLA peptidome composition in a brain metastatic melanoma cell line.We observed that CD74 expression on tumor cells is a strong positive prognostic marker in brain metastasis patients and positively associated with tumor-infiltrating T-lymphocytes (TILs). Whole DNA methylome analysis suggested that CD74 tumor cell expression might be regulated epigenetically via CD74 promoter methylation. CD74high and TILhigh tumors displayed a differential DNA methylation pattern with highest enrichment scores for antigen processing and presentation. Furthermore, CD74 knockdown in vitro lead to a reduction of HLA class II peptidome complexity, while HLA class I peptidome remained unaffected.In summary, our results demonstrate that a functional HLA class II processing machinery in brain metastatic tumor cells, reflected by a high expression of CD74 and a complex tumor cell HLA peptidome, seems to be crucial for better patient prognosis.

AT-101 simultaneously triggers apoptosis and a cytoprotective type of autophagy irrespective of expression levels and the subcellular localization of Bcl-xL and Bcl-2 in MCF7 cells.

The effects of autophagy on cell death are highly contextual and either beneficial or deleterious. One prime example for this dual function of autophagy is evidenced by the cell responses to the BH3 mimetic AT-101 that is known to induce either apoptotic or autophagy-dependent cell death in different settings. Based on previous reports, we hypothesized that the expression levels of pro-survival Bcl-2 family members may be key determinants for the respective death mode induced by AT-101. Here we investigated the role of autophagy in the response of MCF7 breast cancer cells to AT-101. AT-101 treatment induced a prominent conversion of LC3-I to LC3-II and apoptotic cell death characterized by the appearance of Annexin-positive/PI-negative early apoptotic cells and PARP cleavage. Inhibition of the autophagy pathway, either through application of 3-MA or by lentiviral knockdown of ATG5, strongly potentiated cell death, indicating a pro-survival function of autophagy. Overexpression of wild type Bcl-xL significantly diminished the net amount of AT-101-induced cell death, but failed to alter the death-enhancing effects of the ATG5 knockdown. This was also observed with the organelle-specific variants Bcl-xL-ActA and Bcl-2-ActA (mitochondrial) as well as Bcl-xL-cb5 and Bcl-2-cb5 (ER) which all reduced AT-101-induced cell death, but did not affect the death-enhancing effects of 3-MA. Collectively, our data indicate that in apoptosis-proficient MCF7 cells, AT-101 triggers Bcl-2- and Bcl-xL-dependent apoptosis and a cytoprotective autophagy response that is independent of the expression and subcellular localization of Bcl-xL and Bcl-2.

The NADPH Oxidase Nox4 mediates tumour angiogenesis.

AIM: The aim of this work was to identify the role of the NADPH oxidase Nox4 for tumour angiogenesis in a slow-growing tumour model in mice. METHODS: Tumour angiogenesis was studied in tumours induced by the carcinogen 3-methylcholanthrene (MCA) in wild-type and Nox knockout mice. Mice were killed when the tumour reached a diameter of 1.5 cm and tumour tissue was used for histological and molecular analysis. RESULTS: 3-methylcholanthrene induced fibrosarcoma in wild-type, Nox1y/-, Nox2y/- and Nox4-/- mice. Histological analysis of vessel density using anti-CD31 staining showed a significant 38% reduction in tumour vascularization in fibrosarcomas of Nox4-/- mice. In contrast, tumour angiogenesis was doubled in Nox1 knockout mice, whereas knockout of Nox2 had no effect on tumour-vessel density. As underlying mechanisms, we identified a defect in hypoxia signalling in Nox4-/- mice. Hypoxia-inducible factor 1-alpha (Hif-1α) accumulation in the tumours was attenuated as was the expression of the Hif-1α-dependent pro-angiogenic genes vascular endothelial growth factor-A, glucose transporter 1 and adrenomedullin. CONCLUSION: By regulating the tumour-vessel density through stabilization of Hif-1α and induction of VEGF expression, Nox4 promotes tumour angiogenesis and may represent a novel target for anti-angiogenic tumour therapy.

Head and neck cancer relapse after chemoradiotherapy correlates with CD163+ macrophages in primary tumour and CD11b+ myeloid cells in recurrences.

BACKGROUND: We investigated the prognostic role of tumour-associated macrophages (TAMs) in patients with head and neck squamous cell carcinoma (HNSCC) treated with definitive chemoradiotherapy (CRT). METHODS: The expression of CD68+, CD163+ and CD11b+ cells was assessed using immunohistochemistry in n=106 pre-treatment tumour biopsy samples and was correlated with clinicopathological characteristics, including T-stage, N-stage, grading, tumour localisation, age and sex as well as local failure-free survival (LFFS), distant metastases-free survival (DMFS), progression-free (PFS), and overall survival (OS). Finally, TAMs expression and vessel density (CD31) were examined in n=12 available early local recurrence samples and compared with their matched primary tumours . The diagnostic images and radiotherapy plans of these 12 patients were also analysed. All local recurrences occurred in the high radiation dose region (⩾70 Gy). RESULTS: With a median follow-up of 40 months, OS at 2 years was 60.5%. High CD163 expression in primary tumours was associated with decreased OS (P=0.010), PFS (P=0.033), LFFS (P=0.036) and DMFS (P=0.038) in multivariate analysis. CD163 demonstrated a strong prognostic value only in human papillomavirus (p16(INK4))-negative patients. Early local recurrence specimens demonstrated a significantly increased infiltration of CD11b+ myeloid cells (P=0.0097) but decreased CD31-positive vessel density (P=0.0004) compared with their matched primary samples. CONCLUSIONS: Altogether, baseline CD163 expression predicts for an unfavourable clinical outcome in HNSCC after definitive CRT. Early local recurrences showed increased infiltration by CD11b+ cells. These data provide important insight on the role of TAMs in mediating response to CRT in patients with HNSCC.

BRAF V600E expression and distribution in desmoplastic infantile astrocytoma/ganglioglioma.

AIMS: Desmoplastic infantile astrocytoma/ganglioglioma (DIA/DIG) is a rare primary neuroepithelial brain tumour typically affecting paediatric patients younger than 24 months. Knowledge about genetic alterations in DIA/DIG is limited. However, a previous study on BRAF V600E mutation in paediatric glioma revealed a BRAF mutation in one of two tested DIAs/DIGs. The limited number of cases in that study did not allow any conclusion about mutation frequency of BRAF in this tumour entity. METHODS: We collected a series of 18 DIAs/DIGs for testing BRAF V600E mutational status by BRAF V600E immunohistochemistry (clone VE1). Cases with sufficient DNA were tested for BRAF V600E mutation by pyrosequencing. RESULTS: Three out of 18 DIAs/DIGs presented with VE1 binding. A considerable proportion of BRAF V600E mutated tumour cells was detected in the cortical tumour component, whereas the pronounced leptomeningeal tumoural stroma was predominantly negative for VE1 binding. Pyrosequencing confirmed BRAF V600E mutation in two of three VE1-positive cases. CONCLUSION: BRAF V600E mutation affects a subset of DIAs/DIGs and offers new therapeutic opportunities.

Loss of FUBP1 expression in gliomas predicts FUBP1 mutation and is associated with oligodendroglial differentiation, IDH1 mutation and 1p/19q loss of heterozygosity.

AIMS: The Far Upstream Element [FUSE] Binding Protein 1 (FUBP1) regulates target genes, such as the cell cycle regulators MYC and p21. FUBP1 is up-regulated in many tumours and acts as an oncoprotein by stimulating proliferation and inhibiting apoptosis. Recently, FUBP1 mutations were identified in approximately 15% of oligodendrogliomas. To date, all reported FUBP1 mutations have been predicted to inactivate FUBP1, which suggests that in contrast to most other tumours FUBP1 may act as a tumour suppressor in oligodendrogliomas. METHODS: As no data are currently available concerning FUBP1 protein levels in gliomas, we examined the FUBP1 expression profiles of human glial tumours by immunohistochemistry and immunofluorescence. We analysed FUBP1 expression related to morphological differentiation, IDH1 and FUBP1 mutation status, 1p/19q loss of heterozygosity (LOH) as well as proliferation rate. RESULTS: Our findings demonstrate that FUBP1 expression levels are increased in all glioma subtypes as compared with normal central nervous system (CNS) control tissue and are associated with increased proliferation. In contrast, FUBP1 immunonegativity predicted FUBP1 mutation with a sensitivity of 100% and a specificity of 90% in our cohort and was associated with oligodendroglial differentiation, IDH1 mutation and 1p/19q loss of heterozygosity (LOH). Using this approach, we detected a to-date undescribed FUBP1 mutation in an oligodendroglioma. CONCLUSION: In summary, our data indicate an association between of FUBP1 expression and proliferation in gliomas. Furthermore, our findings present FUBP1 immunohistochemical analysis as a helpful additional tool for neuropathological glioma diagnostics predicting FUBP1 mutation.

Activation of executioner caspases is a predictor of progression-free survival in glioblastoma patients: a systems medicine approach.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. GBM cells are highly resistant to apoptosis induced by antitumor drugs and radiotherapy resulting in cancer progression. We assessed whether a systems medicine approach, analysing the ability of tumor cells to execute apoptosis could be utilized to predict the response of GBM patients to treatment. Concentrations of the key proapoptotic proteins procaspase-3, procaspase-9, Smac and Apaf-1 and the antiapopotic protein XIAP were determined in a panel of GBM cell lines and GBM patient tumor resections. These values were used as input for APOPTO-CELL, a systems biological based mathematical model built to predict cellular susceptibility to undergo caspase activation. The modeling was capable of accurately distinguishing between GBM cells that die or survive in response to treatment with temozolomide in 10 of the 11 lines analysed. Importantly the results obtained using GBM patient samples show that APOPTO-CELL was capable of stratifying patients according to their progression-free survival times and predicted the ability of tumor cells to support caspase activation in 16 of the 21 GBM patients analysed. Calculating the susceptibility to apoptosis execution may be a potent tool in predicting GBM patient therapy responsiveness and may allow for the use of APOPTO-CELL in a clinical setting.

Overexpression of the anti-apoptotic protein AVEN contributes to increased malignancy in hematopoietic neoplasms.

AVEN has been identified as an inhibitor of apoptosis, which binds to the adaptor protein, APAF-1, and thereby prevents apoptosome formation and mitochondrial apoptosis. Recent data have demonstrated high expression levels of AVEN messenger RNA in acute leukemias as well as a positive correlation between AVEN mRNA overexpression and poor prognosis in childhood acute lymphoblastic leukemia. On the basis of these data, we investigated the potential involvement of AVEN in tumorigenesis. First, we confirmed the overexpression of AVEN in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) patient samples. We then established a transgenic mouse model with T-cell-specific overexpression of AVEN, with which we demonstrated the oncogenic cooperation of AVEN with heterozygous loss of p53. Finally, we used a subcutaneous xenograft mouse model to show that AVEN knockdown in the T-ALL cell lines, MOLT-4 and CCRF-CEM, and in the acute myeloblastic leukemia cell line, Kasumi-1, leads to a halt in tumor growth owing to the increased apoptosis and decreased proliferation of tumor cells. Collectively, our data demonstrate that the anti-apoptotic molecule, AVEN, functions as an oncoprotein in hematopoietic neoplasms.

PAX2 is an antiapoptotic molecule with deregulated expression in medulloblastoma.

PAX2 is a paired box transcription factor possessing a fundamental role in the embryogenesis of hindbrain and urinary tract. PAX genes are proto-oncogenes, PAX2 expression may contribute to the pathogenesis of renal cell carcinoma. Because of the expression of PAX2 in the developing hindbrain and its essential role in cerebellar development, it has been hypothesized that PAX2 may also be involved in medulloblastoma tumorigenesis. We investigated the expression pattern of PAX2 and various genes of the neuronal lineage in medulloblastoma and glioma cell lines. We found high expression of PAX2 mRNA and PAX2 protein in medulloblastoma cells and some glioma cell lines independent of their neuronal lineage gene expression signature. Gene suppression of PAX2 decreased the expression of the PAX2 transcriptional target GDNF in Daoy cells and had a profound cytotoxic effect in vitro on Daoy medulloblastoma and T98G glioma cells. Expression of PAX2 was then assessed in two separate medulloblastoma tissue microarrays with a total of 61 patient samples by immunohistochemistry. PAX2 expression was detected in the majority of medulloblastoma samples and correlated with less differentiated histology. Therefore, PAX2 is a biomarker for a more aggressive medulloblastoma phenotype and may represent a novel therapeutic target.

Bone morphogenetic protein-7 is a MYC target with prosurvival functions in childhood medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in children. It is known that overexpression and/or amplification of the MYC oncogene is associated with poor clinical outcome, but the molecular mechanisms and the MYC downstream effectors in MB remain still elusive. Besides contributing to elucidate how progression of MB takes place, most importantly, the identification of novel MYC-target genes will suggest novel candidates for targeted therapy in MB. A group of 209 MYC-responsive genes was obtained from a complementary DNA microarray analysis of a MB-derived cell line, following MYC overexpression and silencing. Among the MYC-responsive genes, we identified the members of the bone morphogenetic protein (BMP) signaling pathway, which have a crucial role during the development of the cerebellum. In particular, the gene BMP7 was identified as a direct target of MYC. A positive correlation between MYC and BMP7 expression was documented by analyzing two distinct sets of primary MB samples. Functional studies in vitro using a small-molecule inhibitor of the BMP/SMAD signaling pathway reproduced the effect of the small interfering RNA-mediated silencing of BMP7. Both approaches led to a block of proliferation in a panel of MB cells and to inhibition of SMAD phosphorylation. Altogether, our findings indicate that high MYC levels drive BMP7 overexpression, promoting cell survival in MB cells. This observation suggests the potential relevance of targeting the BMP/SMAD pathway as a novel therapeutic approach for the treatment of childhood MB.

Erythropoietin receptor expression in normal and neoplastic choroid plexus.

BACKGROUND: The erythropoietin receptor (EpoR) is expressed widely throughout the human CNS, including the choroid plexus. Recent studies have shown that EpoR is also expressed in various human tumors, including carcinomas, meningiomas and gliomas. Thereby, the Epo-EpoR pathway plays a role in inhibition of apoptosis and tumor growth, infiltration, angiogenesis and metastasis as well as treatment resistance and is a potential target in oncological treatment. Lower levels of EpoR have been associated with shorter survival in high grade gliomas and higher risk of tumor recurrence in meningiomas. METHODS: Since the EpoR status in human choroid plexus tumors (CPT) is not known, we investigated 57 CPT from 43 cases including 14 recurrent tumors and compared them with 23 samples of normal choroid plexus (CP). CPT samples consisted of choroid plexus papillomas/CPP (n = 41), atypical CPP (n = 15) and choroid plexus carcinoma/CPC (n = 1). EpoR expression was determined by immunohistochemistry using semi-quantitative scoring for staining intensity and was validated in exemplary cases using western blot and RT-PCR. RESULTS: EpoR expression was observed in all samples of normal and neoplastic CP with significantly lower expression levels in CPT (p < 0.001). CONCLUSION: No significant correlation was found between EpoR expression and age, gender, WHO grade, number of mitosis or tumor recurrence. EpoR expression in CPT is in line with its expression in normal CP and with previous reports on EpoR expression in other glial neoplasms. Association of EpoR levels in CPT with survival, as known in astrocytic gliomas, remains to be determined.

Spatio-temporal deleted in colorectal cancer (DCC) and netrin-1 expression in human foetal brain development.

AIMS: Deleted in colorectal cancer (DCC) and its ligand netrin-1 are known as axonal guidance factors, being involved in angiogenesis, migration and survival of precursor cells in the embryonic mammalian central nervous system (CNS). So far, little is known about the distribution of those molecules in human CNS development. METHODS: We investigated 22 human foetal brain specimens (12th and 28th week of gestation) for DCC and netrin-1 expression by means of immunohistochemistry, immunofluorescence and confocal laser microscopy. Statistical analysis was performed by applying a semi-quantitative score, including staining intensity and frequency and correlation with foetal age. RESULTS: DCC and netrin-1 were differentially expressed throughout the developing human foetal telencephalic and cerebellar cortical layers. Netrin-1 exhibited the highest levels in telencephalic germinal layers, whereas the strongest DCC immunoreactivity was seen in the developing cortical plate. Netrin-1 and DCC were predominantly present on cerebellar external granule layer cells. Distinct co-expression was seen in maturing foetal brainstem nuclei, cerebellar external granular layer and the choroid plexus. In contrast, endothelial cells showed strong netrin-1 expression with subsidiary DCC immunoreactivity. Pontine and telencephalic axonal fibre tracts also demonstrated strong netrin-1 expression. CONCLUSIONS: We show that DCC and netrin-1 are ubiquitously expressed in the human foetal brain; however, both exhibit a distinct spatio-temporal expression pattern. Together with the data from animal experiments, our findings might indicate also an important role for DCC and netrin-1 in human foetal CNS development.

Secreted protein, acidic and rich in cysteine (SPARC) expression in astrocytic tumour cells negatively correlates with proliferation, while vascular SPARC expression is associated with patient survival.

AIMS: Secreted protein, acidic and rich in cysteine (SPARC) is a regulator of cell-matrix interaction and has been associated with tumour stage and patient survival in various malignancies. As no large-scale study has yet been undertaken, we investigated human brain and astrocytomas for SPARC expression and associations with tumour grade, proliferation, vascular density and patient survival. METHODS: A spectrum of 188 WHO grade I-IV astrocytic tumours and 24 autopsy cases were studied by immunohistochemistry for SPARC, MIB-1 proliferation index and CD31-positive vessels. SPARC protein expression was confirmed by quantitative real-time polymerase chain reaction and Western blot in 13 cases. RESULTS: In normal brain, SPARC is expressed in cortical marginal glia, cerebellar Bergmann glia and focally in white matter but is absent in neurones or vessels. High SPARC expression levels in the cytoplasm of astrocytic tumour cells decreased with the grade of malignancy but showed an increase with grade of malignancy in tumour vessels. SPARC negatively correlated with tumour proliferation but not with vascular density. While cytoplasmic SPARC staining was not associated with survival, vascular SPARC showed a significant association in the group of grade II-IV tumours (P = 0.02) and also in grade II astrocytomas alone (P = 0.01) with vascular SPARC associated with worse prognosis. CONCLUSIONS: SPARC is highly expressed in astrocytomas and decreases with tumour progression. We confirm an association of increased SPARC expression and decreased proliferation. While there is no association between the level of SPARC in the tumour cells and patient survival, increased tumour vascular SPARC expression is associated with decreased patient survival.

Immunohistochemical analysis of CDX2 expression in normal choroid plexus epithelium and choroid plexus tumors.

BACKGROUND: The Wnt and BMP signaling pathways are involved in the morphogenesis of both gastrointestinal and choroid plexus epithelium. In the intestine, Wnt signaling represses the expression of the tumor suppressor gene CDX2 via SOX9, a transcription factor, which is also expressed in the choroid plexus. Recently, an inverse correlation between CDX2 expression and tumor grade, tumor stage and lymph node metastasis in colorectal adenocarcinomas has been reported. Besides intestinal tissues, expression of CDX2 has also been reported in various other epithelial tissues and carcinomas. To date, no data exist on expression of CDX2 in normal and neoplastic choroid plexus epithelium. AIM: To investigate CDX2 expression in normal and neoplastic choroid plexus. MATERIALS AND METHODS: Paraffin-embedded samples from 60 normal choroid plexus, including 23 fetal tissue samples and from 65 choroid plexus tumors (47 choroid plexus papillomas WHO grade I, 16 atypical choroid plexus papillomas and 2 choroid plexus carcinomas WHO grade III) were examined by immunohistochemistry. Samples from normal choroid plexus were collected from 45 autopsy cases and from 15 neurosurgical specimens. RESULTS: Normal and neoplastic choroid plexus lacked CDX2 expression. CONCLUSION: In our series, immunohistochemistry shows no evidence for a role of CDX2 in development or differentiation of normal choroid plexus from the 9th gestational week until adulthood. Since choroid plexus tumors reliably lack CDX2 immunoreactivity, this marker may be helpful in distinguishing cerebral metastases from CDX2-positive adenocarcinomas and choroid plexus neoplasms.

Basement membrane remodelling and segmental fibrosis in sporadic inclusion body myositis.

Sporadic inclusion body myositis (sIBM) is a debilitating idiopathic inflammatory myopathy. Little is known about the pathogenetic mechanisms that lead to myofiber degeneration. In the present study, we evaluated the integrity of the myofiber basement membrane in non-necrotic myofibers invaded by inflammatory infiltrates. We used 100 ten mum thick serial sections obtained from biopsies of 5 patients suffering from sIBM. Biopsies from 5 patients suffering from polymyositis served as controls. We performed sequential HE staining and immunolabeling using anti-CD68, -CD8, -merosin, -laminin alpha4 chain, and -collagen IV antibodies. In sIBM, we detected a total of 89 non-necrotic myofibers that were invaded by inflammatory cells. The invasive process and its sequelae were segmental in nature and included destruction of the myofiber basement membrane, and eventually, partial replacement by fibrosis of the invaded myofiber. In polymyositis, we found only two myofibers that were affected in this way. In sIBM, basement membrane remodelling and irreversible replacement by fibrosis of myofibers appear to represent the end result of a process in which the balance between injury and repair are disrupted.

Erythropoietin receptor is expressed in meningiomas and lower levels are associated with tumour recurrence.

AIMS: The Epo-EpoR pathway plays a role in tumour growth, metastasis and treatment resistance and is a potential target in oncological treatment. As the EpoR status in human meningiomas is unknown, our aim was to characterize EpoR expression in these tumours. METHODS: We examined 131 meningioma samples of all WHO grades from 116 patients by immunohistochemistry for EpoR. Among these, 25 meningiomas showed brain invasion and 29 patients had a further tumour recurrence. A group of 20 patients without tumour recurrence served as controls. In 12 cases we were able to compare both the primary and the following recurrent tumours. The presence of EpoR in meningiomas was confirmed by RT-PCR and Western blot. RESULTS: EpoR was expressed in all meningiomas. Statistical analysis revealed that the mean expression levels of EpoR were significantly lower in primary tumours with known recurrence compared with a recurrence-free control group. Additional matched pair analysis in individual cases showed no significant differences between primary and recurrent tumours. No significant correlation between EpoR expression and WHO grade, age, sex or brain invasion was detected. Using specific primer pairs for RT-PCR, we were able to detect all three known isoforms of EpoR: the full-length isoform EpoR-F, the truncated isoform EpoR-T and the soluble isoform EpoR-S. CONCLUSIONS: Our results demonstrate the expression of EpoR in meningiomas. Lower EpoR mean levels might be a useful marker for a higher recurrence risk, but further studies are needed to clarify the influence of EpoR on recurrences and the role of the different isoforms.

Diagnostic value of WT1 in neuroepithelial tumours.

AIMS: Currently, clinical trials using WT1 (Wilms tumour gene) peptide vaccines are conducted in haematopoietic malignancies and solid cancers. Single reports showed that the Wilms tumour gene product WT1 is also expressed in astrocytic neoplasms. Our aim was to investigate WT1 expression in a large cohort of various neuroepithelial tumours of different World Health Organization (WHO) grades and in normal central nervous system (CNS) tissue specimens to test its potential value as a diagnostic marker. METHODS: Specimens were assessed by RT-PCR, Western blotting and immunohistochemistry. The samples investigated in our study consisted of 334 human neuroepithelial tumours, among those 33 oligodendrogliomas, 219 astrocytomas (including 105 glioblastomas) and 47 ependymomas. RESULTS: Our results showed a de novo WT1 expression in neuroepithelial tumours. In diffuse astrocytomas and ependymomas, WT1 expression increased significantly with the grade of malignancy. In contrast, no significant difference was seen between WHO grade-II and -III oligodendrogliomas. Controlling for WHO grade, the comparison of oligodendrogliomas with ependymal and astrocytic tumours showed higher expression values for the latter. CONCLUSIONS: Our study shows that WT1 is expressed de novo in numerous neuroepithelial tumours and increases with the grade of malignancy. These results suggest an important role of WT1 in tumourigenesis and progression in human brain tumours.

Cushing's disease associated with both pituitary microadenoma and corticotroph hyperplasia.

Herein, we present the case of a 63-year old female patient with initial symptoms of myopathy, hypokaliemia, glucosuria and psychotic symptoms. Laboratory analysis demonstrated elevated plasma levels of ACTH and cortisol. Additionally, urine cortisol excretion was increased approximately 60-fold. MRI imaging revealed a possible pituitary microadenoma. To confirm the diagnosis a bilateral inferior petrosal sinus sampling was performed presenting higher ACTH levels on the right side. However, after surgery cortisol levels did not return to normal range. Histological examination of the tumor revealed a microadenoma. Six days postoperatively, the patient developed several pneumonic infiltrations and fever therefore antibiotic and antifungal therapy was started immediately. In addition aspergillus antigen was elevated. During this septic condition, cortisol levels further increased. The patient died despite optimal intensive care under septical conditions 8 days after surgery. Microbiological analysis identified Aspergillus fumigatus in broncho-alveolar lavage and several organ systems including the heart and brain. Neuropathological autopsy revealed nodular proliferations of corticotropic cells in the pituitary gland that are assumed to be morphological entities between diffuse hyperplasias and adenomas, termed as tumorlets. In single reports, multiple pituitary lesions in patients with Cushing's disease have been demonstrated, but to our knowledge none of these cases presented the combination of an ACTH-producing microadenoma and corticotroph cell hyperplasia in the same patient. Therefore, even after resection of a pituitary microadenoma one should be aware of the possibility of continuously elevated ACTH level being due to multifocal nodular corticotroph hyperplasia which is invisible by neuroradiological examination.