Tight junction protein claudin-1 is differentially expressed in craniopharyngioma subtypes and indicates invasive tumor growth

Background

Claudins are tight junction proteins expressed in epithelial tissues that play important roles in cell polarity and adhesion. Altered distribution of claudin-1(CLDN1) affects cell mobility and tumor invasiveness. Craniopharyngiomas (CPs) represent epithelial tumors of the sellar region, consisting of adamantinomatous (adaCP) and papillary (papCP) variants. Their tendency to infiltrate surrounding brain structures complicates successful surgery. Reliable markers are required to predict tumor behavior and to establish individualized treatment protocols.

Methods

We describe the distribution pattern of CLDN1 in a large cohort of 66 adaCPs, 21 papCPs, and 24 Rathke`s cleft cyst (RCC) cases using immunohistochemistry. CLDN1 mRNA levels were analyzed with qRT-PCR in 33 CP samples. The impact on the migration potential was studied in primary adaCP cell cultures (n = 11) treated with small interfering RNA (siRNA) for CLDN1. Furthermore, CLDN1 distribution patterns and expression levels were compared between invasive (n = 16) and noninvasive (n = 17) tumor groups.

Results

PapCPs and RCCs exhibited a distinct homogenous and membranous expression pattern, whereas CLDN1 immunoreactivity appeared weaker and more heterogeneous in adaCPs. In the latter cases, whirl-like cell clusters showed complete absence of CLDN1. mRNA analysis confirmed reduced CLDN1 levels in adaCPs versus papCPs. Interestingly, invasive tumors exhibited significantly lower CLDN1 expression compared with noninvasive counterparts regardless of CP subtype. Accordingly, siRNA treatment for CLDN1 altered tumor cell migration in vitro.

Conclusion

CLDN1 represents a novel marker in the differential diagnosis of CP variants and RCCs. Low CLDN1 expression levels correlate with an invasive CP growth pattern and may serve as a prognostic marker.     

Emerging Histopathological and Genetic Parameters of Pituitary Adenomas: Clinical Impact and Recommendation for Future WHO Classification

The review assesses immunohistochemical findings of somatostatin receptors and of metalloproteinases in different pituitary adenoma types and the significance of molecular genetic data. Current evidence does not support routine immunohistochemical assessment of somatostatin or dopamine receptor subtype expression on hormone-secreting or nonfunctioning pituitary adenomas. Further prospective studies are needed to define its role for clinical decision making. Until then we suggest to restrict membrane receptor profiling to individual cases or for study purposes. The problems of adenoma expansion and invasion are discussed. Despite partially contradictory publications, proteases clearly play a major role in permission of infiltrative growth of pituitary adenomas. Therefore, detection of at least MMP-2, MMP-9, TIMP-2, and uPA seems to be justified. Molecular characterization is important for familial adenomas, adenomas in MEN, Carney complex, and McCune-Albright syndrome and can gain insight into pathogenesis of sporadic adenomas.     

Neural expression profile of alpha-synuclein in developing human cortex.

Alpha-synuclein is a predominantly neuronal presynaptic protein that may play an important role during synaptogenesis and CNS development. In order to elucidate the human developmental expression profile, we used a polyclonal antiserum against the NAC domain of alpha-synuclein. In normal fetal cortex neuroectodermal precursor cells elicited staining in the soma, whereas, in adult cortex, we observed a staining pattern compatible with synaptic function. The same developmental intraneuronal redistribution was found in neurodegenerative disorders, i.e. somatic staining in neuroectodermal precursors in fetal (trisomy 21) and a synaptic pattern in adult (Down's syndrome, Alzheimer's disease) brains. RT-PCR and Western blot analysis revealed expression at all time points studied (4-7.5 months) during human brain development.     

Gliomas: histopathologic evaluation of changes in directionality and magnitude of water diffusion at diffusion-tensor MR imaging

PURPOSE: To retrospectively correlate changes in fractional anisotropy (FA) and mean diffusivity in gliomas at diffusion-tensor magnetic resonance (MR) imaging with the degree of tumor cell infiltration determined histologically. MATERIALS AND METHODS: The institutional review board required neither ethics committee approval nor patient informed consent for this study. Twenty patients (eight women, 12 men; age range, 18-53 years) with glioma (seven World Health Organization grade II and 13 grade III tumors) underwent diffusion-tensor MR imaging at 1.5 T. Diffusion-tensor data were obtained with an echo-planar imaging sequence with six diffusion directions (b = 1000 sec/mm(2)), isotropic 1.9-mm voxels, and five averages. FA and mean diffusivity values were calculated from diffusion-tensor data. Coregistration with a three-dimensional MR imaging data set (used for stereotactic brain biopsies) enabled correlation of FA and mean diffusivity values with the histopathologic findings total cell number (CN), tumor CN, and percentage tumor infiltration (TI) by using linear, exponential, and logarithmic models. Student t and Mann-Whitney U tests were performed. RESULTS: Histopathologic findings of 77 MR image-guided stereotactic biopsies in all 20 patients were correlated with FA and mean diffusivity values at the biopsy locus. For FA and mean diffusivity, a logarithmic model showed strongest correlation with tumor CN and total CN; a linear model showed strongest correlation with percentage TI. For FA there were negative logarithmic (R = -0.802, P < .001) and linear (R = -0.796, P < .001) correlations with tumor CN and percentage TI, respectively. For mean diffusivity there were positive logarithmic (R = 0.557, P < .001) and linear correlations (R = 0.521, P < .001) with tumor CN and percentage TI, respectively. Differences between correlations for FA and mean diffusivity versus tumor CN (P < .001) and percentage TI (P < .001) were significant. CONCLUSION: FA is better than mean diffusivity for assessment and delineation of different degrees of pathologic changes (ie, TI) in glioma.     

Acquired temozolomide resistance in human glioblastoma cell line U251 is caused by mismatch repair deficiency and can be overcome by lomustine

Purpose

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. While the alkylating agent temozolomide (TMZ) has prolonged overall survival, resistance evolution represents an important clinical problem. Therefore, we studied the effectiveness of radiotherapy and CCNU in an in vitro model of acquired TMZ resistance.

Methods

We studied the MGMT-methylated GBM cell line U251 and its in vitro derived TMZ-resistant subline, U251/TMZ-R. Cytotoxicity of TMZ, CCNU, and radiation was tested. Both cell lines were analyzed for MGMT promotor status and expression of mismatch repair genes (MMR). The influence of MMR inhibition by cadmium chloride (CdCl₂) on the effects of both drugs was evaluated.

Results

During the resistance evolution process in vitro, U251/TMZ-R developed MMR deficiency, but MGMT status did not change. U251/TMZ-R cells were more resistant to TMZ than parental U251 cells (cell viability: 92.0% in U251/TMZ-R/69.2% in U251; p = 0.032) yet more sensitive to CCNU (56.4%/80.8%; p = 0.023). The effectiveness of radiotherapy was not reduced in the TMZ-resistant cell line. Combination of CCNU and TMZ showed promising results for both cell lines and overcame resistance. CdCl₂-induced MMR deficiency increased cytotoxicity of CCNU.

Conclusion

Our results confirm MMR deficiency as a crucial process for resistance evolution to TMZ. MMR-deficient TMZ-resistant GBM cells were particularly sensitive to CCNU and to combined CCNU/TMZ. Effectiveness of radiotherapy was preserved in TMZ-resistant cells. Consequently, CCNU might be preferentially considered as a treatment option for recurrent MGMT-methylated GBM and may even be suitable for prevention of resistance evolution in primary treatment.

     

Insights into the Infiltrative Behavior of Adamantinomatous Craniopharyngioma in a New Xenotransplant Mouse Model

Adamantinomatous craniopharyngiomas (adaCP) cause hypothalamic pituitary dysfunction. Elucidation of pathomechanisms underlying tumor progression is essential for the development of targeted chemotherapeutic treatment options. In order to study the mechanisms of tumor outgrowth, we implanted human primary adaCP tissue from three different surgical specimens stereotactically into the brain of immunodeficient mice (n = 20). Three months after tumor inoculation, magnetic resonance imaging and histology confirmed tumor engraftment in all 20 mice (100%) that obtained tissue transplants. The lesions invaded adjoining brain tissue with micro finger‐shaped protrusions. Immunohistochemical comparison of the primary tumor and xenotransplants revealed a similar amount of proliferation (Mib‐1) and cytokeratin expression pattern (KL‐1). Whole tumor reconstruction using serial sections confirmed whirl‐like cell clusters with nuclear β‐catenin accumulations at the tumor brain border. These whirls were surrounded by a belt of Claudin‐1 expressing cells, showed an activated epidermal growth factor receptor (EGFR) and distinct CD133 as well as p21^WAF1/Cip1 positivity, indicating a tumor stem cell phenotype. Consistent with our previous in vitro studies, intracranial xenotransplants of adaCP confirmed cells with nuclear β‐catenin and activated EGFR being the driving force of tumor outgrowth. This model provides the possibility to study in vivo tumor cell migration and to test novel treatment regimens targeting this tumor stem cell niche.