Emerging role of linker histone variant H1x as a biomarker with prognostic value in astrocytic gliomas. A multivariate analysis including trimethylation of H3K9 and H4K20.

Although epigenetic alterations play an essential role in gliomagenesis, the relevance of aberrant histone modifications and the respective enzymes has not been clarified. Experimental data implicates histone H3 lysine (K) methyltransferases SETDB1 and SUV39H1 into glioma pathobiology, whereas linker histone variant H1.0 and H4K20me3 reportedly affect prognosis. We investigated the expression of H3K9me3 and its methyltransferases along with H4K20me3 and H1x in 101 astrocytic tumors with regard to clinicopathological characteristics and survival. The effect of SUV39H1 inhibition by chaetocin on the proliferation, colony formation and migration of T98G cells was also examined. SETDB1 and cytoplasmic SUV39H1 levels increased from normal brain through low-grade to high-grade tumors, nuclear SUV39H1 correlating inversely with grade. H3K9me3 immunoreactivity was higher in normal brain showing no association with grade, whereas H1x and H4K20me3 expression was higher in grade 2 than in normal brain or high grades. These expression patterns of H1x, H4K20me3 and H3K9me3 were verified by Western immunoblotting. Chaetocin treatment significantly reduced proliferation, clonogenic potential and migratory ability of T98G cells. H1x was an independent favorable prognosticator in glioblastomas, this effect being validated in an independent set of 66 patients. Diminished nuclear SUV39H1 expression adversely affected survival in univariate analysis. In conclusion, H4K20me3 and H3K9 methyltransferases are differentially implicated in astroglial tumor progression. Deregulation of H1x emerges as a prognostic biomarker.

Complex interactions between the components of the PI3K/AKT/mTOR pathway, and with components of MAPK, JAK/STAT and Notch-1 pathways, indicate their involvement in meningioma development.

We investigated the significance of PI3K/AKT/mTOR pathway and its interactions with MAPK, JAK/STAT and Notch pathways in meningioma progression. Paraffin-embedded tissue from 108 meningioma patients was analysed for the presence of mutations in PIK3CA and AKT1. These were correlated with the expression status of components of the PI3K/AKT/mTOR pathway, including p85α and p110γ subunits of PI3K, phosphorylated (p)-AKT, p-mTOR, p-p70S6K and p-4E-BP1, as well as of p-ERK1/2, p-STAT3 and Notch-1, clinicopathological data and patient survival. A mutation in PIK3CA or AKT1 was found in around 9 % of the cases. Higher grade meningiomas displayed higher nuclear expression of p-p70S6K; higher nuclear and cytoplasmic expression of p-4E-BP1 and of Notch-1; lower cytoplasmic expression of p85αPI3K, p-p70S6K and p-ERK1/2; and lower PTEN Histo-scores (H-scores). PTEN H-score was inversely correlated with recurrence probability. In univariate survival analysis, nuclear expression of p-4E-BP1 and absence of p-ERK1/2 expression portended adverse prognosis, whereas in multivariate survival analysis, p-ERK1/2 expression emerged as an independent favourable prognostic factor. Treatment of the human meningioma cell line HBL-52 with the PI3K inhibitor LY294002 resulted in reduction of p-AKT, p-p70S6K and p-ERK1/2 protein levels. The complex interactions established between components of the PI3K/AKT/mTOR pathway, or with components of the MAPK, JAK/STAT and Notch-1 pathways, appear to be essential for facilitating and fuelling meningioma progression.

Phosphorylated 4E-binding protein 1 (p-4E-BP1): a novel prognostic marker in human astrocytomas.

AIMS: To investigate the significance of the mammalian target of rapamycin (mTOR) pathway in astrocytic tumours, published information in this context being limited, especially regarding phosphorylated 4E-binding protein (p-4E-BP) 1. METHODS AND RESULTS: Paraffin-embedded tissue from 111 patients with astroglial tumours (grades II-IV) was investigated for the association of phosphorylated mTOR (p-mTOR) signalling components with phosphorylated extracellular signal-related kinase 1/2 (p-ERK1/2) and phosphorylated AKT (p-AKT) expression, clinicopathological features, angiogenesis, isocitrate dehydrogenase 1 (IDH1)-R132H, and survival. Expression was also quantified by western blot analysis in 12 cases and in three primary glioma cell cultures following rapamycin treatment. p-mTOR expression correlated with p-4E-BP1 expression and marginally with p-p70S6K expression. p-4E-BP1 expression increased with tumour grade. Rapamycin induced a decline in phosphorylation levels of all three proteins. Nuclear p-AKT and cytoplasmic p-ERK1/2 immunoexpression correlated with p-4E-BP1 expression, whereas cytoplasmic p-AKT expression correlated with p-p70S6K expression. All three proteins were associated with increased angiogenesis but not with IDH1-R132H expression status. p-mTOR adversely affected overall and disease-free survival in univariate analysis. In multivariate survival analysis, the presence of p-4E-BP1 predicted shortened overall survival in the entire cohort and glioblastomas. CONCLUSIONS: mTOR signalling components are differentially involved in the acquisition of a more aggressive and angiogenic phenotype in astrocytic tumours. Moreover, p-4E-BP1 emerges as a novel prognostic marker, which might aid in the selection of patients who are more likely to benefit from therapy with mTOR inhibitors.

Expression of interleukin-8 receptor CXCR2 and suppressor of cytokine signaling-3 in astrocytic tumors.

The aim was to expand recently published information regarding the significance of the interleukin (IL)-8/p-STAT-3 (signal transducer and activator of transcription) pathway in astrocytomas, focusing on the IL-8 receptor, chemokine (C-X-C motif) receptor 2 (CXCR2), and the STAT-3 inhibitor SOCS-3 (suppressors of cytokine signaling). A total of 91 paraffin-embedded human astrocytoma tissues (grades II-IV) were investigated for the association of SOCS-3 and CXCR2 expression with clinicopathologic and morphometric microvascular characteristics, vascular endothelial growth factor (VEGF), IL-8 and p-STAT-3 expression and patient survival. Peripheral IL-8 secretion levels were assessed by enzyme-linked immunosorbent spot (ELISPOT). SOCS-3, p-STAT-3 and CXCR2 protein levels were also quantified by Western immunoblotting in six cases, and the protein levels of SOCS-3 and CXCR2 were correlated with the immunohistochemical expression of the respective proteins. All CXCR2-positive cases by Western immunoblotting displayed increased peripheral IL-8 secretion levels. Treatment of primary glioblastoma cell cultures with exogenous IL-8 enhanced proliferation, and this effect was inhibited by treatment with a neutralizing anti-CXCR2 antibody. SOCS-3 and CXCR2 were expressed by neoplastic astrocytes in 92.4% and 48.78% of cases, respectively, with their levels increasing with histological grade and extent of necrosis. VEGF expression and microvessel density, CXCR2 and IL-8 levels were interrelated. SOCS-3 and p-STAT-3 were co-expressed in 85.7% of cases, although they were not interrelated. In univariate survival analysis, increased SOCS-3 expression and the presence of CXCR2 adversely affected survival, whereas in multivariate analysis, only CXCR2 remained significant. The prognostic significance of CXCR2 was validated in an independent set of 63 patients. Our data implicate IL-8/CXCR2 signaling pathway in the progression and regulation of angiogenesis in astrocytomas and provide a rationale for CXCR2 therapeutic exploitation in these tumors.

Prognostic significance of IL-8-STAT-3 pathway in astrocytomas: correlation with IL-6, VEGF and microvessel morphometry.

Malignant astrocytomas are highly vascular neoplasms characterized by a potent angiogenic and immunosuppressive phenotype. Th2-cytokines (IL-6/IL-8) are implicated as major regulators of glioma cell growth and invasiveness. STAT-3, a downstream transducer of cytokine signaling is positively associated with tumor angiogenesis. The present study aimed to investigate the expression of IL-8 and p-STAT-3 in 97 diffusely infiltrating astrocytomas of various grades, in relation to IL-6, VEGF, clinicopathological features, microvascular characteristics and patients' survival. IL-8 expression was localized in neoplastic cells, being associated with p-STAT-3 (p = 0.0013), IL-6 (p = 0.0004) and VEGF (p < 0.0001) around areas of necrosis as well as in perivascular inflammatory and endothelial cells. All the molecules under study correlated with tumor grade and degree of necrosis (p < 0.05, respectively). p-STAT-3, IL-8 and VEGF expression was positively associated with microvessel density (p = 0.0491, p < 0.0001 and p = 0.0118, respectively). Univariate analysis indicated that overexpression of IL-8 and IL-6 adversely affected survival in the entire cohort whereas increased p-STAT-3 expression was predictive of improved survival in high grade (III/IV) astrocytomas (p = 0.0032). In multivariate analysis only IL-8 expression (p = 0.043) retained its significance. The prognostic significance of IL-8 expression and its correlation with p-STAT-3 and VEGF implicates this novel signaling pathway in astroglial tumors progression providing new targets for effective immunotherapy.

Expression of pERK and pAKT in human astrocytomas: correlation with IDH1-R132H presence, vascular endothelial growth factor, microvascular characteristics and clinical outcome.

Although pERK and pAKT are reportedly activated in various neoplasms, little information is available about their significance in astrocytomas. Paraffin-embedded tissue from 82 patients with diffuse infiltrating astrocytomas (grades II to IV) was investigated for the association of pERK and pAKT activation with clinicopathological features, vascular endothelial growth factor (VEGF), isocitrate dehydrogenase 1 and microvascular parameters. Nuclear pERK labelling index (LI) increased with increasing cytoplasmic pERK LI and nuclear and cytoplasmic pAKT LI (p = 0.0019, p = 0.0260 and p = 0.0012, respectively). Accordingly, cytoplasmic pERK increased with increasing levels of nuclear (p = 0.0001) and marginally with cytoplasmic pAKT LI (p = 0.0526). Nuclear and cytoplasmic pERK LI and nuclear pAKT LI were positively correlated with tumour histological grade (p = 0.0040, p = 0.0238 for pERK and p = 0.0004 for pAKT, respectively). VEGF expression was correlated with nuclear pERK (p = 0.0099) and nuclear pAKT LI (p = 0.0002). Interestingly, pERK cytoplasmic LI increased with microvessel calibre (p = 0.0287), whereas pAKT nuclear LI was marginally related to microvessel density (p = 0.0685). The presence of IDH1-R132H was related only to histological grade and lower microvessel calibre. Multivariate survival analysis in the entire cohort selected cytoplasmic pAKT LI (p = 0.045), histological grade, microvessel calibre (p = 0.028), patients' age, gender and surgical excision as independent predictors of survival. Moreover, in glioblastomas, pERK nuclear LI emerged as a favourable prognosticator in the presence of IDH1-R132H. pERK and pAKT in astrocytomas are interrelated and associated with tumour grade and angiogenesis. Moreover, the importance of cytoplasmic pAKT immunoexpression in patients' prognosis and nuclear pERK immunoexpression in glioblastomas is confirmed.

Synchronous well differentiated neuroendocrine tumour and gastrointestinal stromal tumour of the stomach: a case report.

BACKGROUND: Well differentiated neuroendocrine tumours (carcinoids), arising from cells of the diffuse neuroendocrine system, represent the most commonly encountered gastric endocrine tumours. Gastrointestinal stromal tumours (GISTs), which stem from interstitial Cajal cells located within the wall of the gastrointestinal tract and have a characteristic immunoreactivity for CD117 (c-kit protein), account for the majority of gastrointestinal mesenchymal neoplasms. Simultaneous occurrence of a GIST with a well differentiated neuroendocrine tumour in the stomach is very rare. METHODS: Clinical history, endoscopy and histopathological findings were utilized for our diagnostic considerations. RESULTS: We report the coexistence of a high risk GIST with a well differentiated neuroendocrine tumour of benign clinical behavior, both located in the stomach, in a 62-year-old man previously operated for a gastric well differentiated neuroendocrine tumour with uncertain malignant behaviour. CONCLUSIONS: Even single well differentiated, sporadic, NETs of small size may coexist with GISTs. An appropriate initial therapeutic approach combined with a scrupulous follow-up seems to play a significant role in terms of preventing a metastatic disease.

Chronic bacterial and parasitic infections and cancer: a review.

BACKGROUND: A relatively underestimated facet of infectious diseases is the association of chronic bacterial and parasitic infections with cancer development. Therefore, we sought to evaluate the evidence regarding the association of such infections with the development of malignancy, excluding the overwhelming evidence of the association of Helicobacter pylori and cancer. METHODOLOGY: We searched Pubmed, Cochrane, and Scopus without time limits for relevant articles. RESULTS: There is evidence that some bacterial and parasitic infections are associated with cancer development. The level of evidence of this association varies from high to low; in any case, a long time interval is mandatory for the development of cancer. A high level of evidence exists for the association of Salmonella Typhi with gallbladder and hepatobiliary carcinoma; Opisthorchis viverrini and Clonorchis sinensis with cholangiocarcinoma; Schistosoma hematobium with bladder cancer; chronic osteomyelitis with squamous cell carcinoma of the skin; and hidradenitis suppurativa with squamous cell carcinoma of the skin. In contrast, the level of evidence regarding the association of Chlamydia spp. with cancer is low. Mycobacterium tuberculosis is associated with lung cancer, albeit probably not etiopathogenetically. CONCLUSIONS: A considerable number of bacterial infections and parasitic infections are associated with the development of cancer. Further research into recognizing additional associations of bacterial and parasitic infections with cancer is mandatory.

Comparative immunohistochemical analysis of aurora-A and aurora-B expression in human glioblastomas. Associations with proliferative activity and clinicopathological features.

In the present study, we carried out a comparative immunohistochemical analysis of aurora-A and aurora-B expression in 40 patients with primary glioblastomas, and attempted to identify any associations with Ki-67 index and the patients' clinical features. The impact of various treatment modalities and proliferative activity on patient outcome was also assessed. Immunohistochemistry was carried out using formalin-fixed and paraffin-embedded tissue sections. Aurora-A expression was higher in tumors with high Ki-67 expression (p=0.01) and was positively, though marginally, related to aurora-B expression (p=0.085). Aurora-B expression was not linked to Ki-67 expression (p=0.182). Lower aurora-A immunohistochemical expression, chemotherapy administration, and tumor localization in one lobe of the brain implied a greater probability of patient survival in univariate analysis (p=0.044, p=0.008, p=0.041, respectively). Ki-67 and aurora-B immunoreactivities were not associated with patient survival (p=0.918 and p=0.539, respectively). To our knowledge, for the first time, the association between aurora-A and aurora-B expression, the correlation of aurora-A with Ki-67 index, and the prognostic impact of aurora-A expression were assessed in glioblastomas. Although we addressed a prognostic connotation of aurora-A, we presume that aurora-A and aurora-B play a complicated role within glioblastomas. Further examinations of larger series are required, so that definite conclusions can be drawn.

Analysis of interleukin (IL)-8 expression in human astrocytomas: associations with IL-6, cyclooxygenase-2, vascular endothelial growth factor, and microvessel morphometry.

Malignant astrocytomas are highly vascular neoplasms with potent angiogenic activity. The present study aimed to investigate peripheral and local expression of interleukin (IL)-8 in astrocytomas with possible associations to IL-6, cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF) expression, and microvessel morphometry. IL-6- and IL-8-secreting peripheral blood monocytes (PBMCs) were evaluated in 17 glioblastoma (WHO grade IV), 5 anaplastic astrocytoma (WHO grade III), and 6 diffuse astrocytoma patients (WHO grade II), in parallel with 23 healthy controls using enzyme-linked immunosorbent spot (ELISPOT) assay. The IL-8 expression was assessed immunohistochemically in patients' tumor tissue sections and correlated with the expression of COX-2, VEGF, IL-6, and microvessel morphometry (assessed using CD34 antibody). Eighteen cases were also stained for CD31 and used as an additional vessel marker to validate our results regarding microvessel morphometry. IL-6 and IL-8 were highly secreted in the PBMCs of glioma patients compared with controls (p = 0.0001, p < 0.0001, respectively), with a positive correlation between IL-8 expression and secretion levels (p = 0.001). IL-8 immunoreactivity was detected in malignant cells or macrophages in perivascular areas and in pseudopalisading cells around necrosis and was positively correlated with histological grade (p = 0.0175) and tumor necrosis (p = 0.0793). IL-6 and IL-8 expression levels were positively correlated (p = 0.0036) and associated with COX-2 and VEGF expression (IL-6: p = 0.0133, p = 0.065; IL-8: p = 0.0139, p = 0.0101), but not with microvessel morphometry, by either CD31 or CD34. The coordinate expression and topographical relationship of IL-6, IL-8, COX-2, and VEGF in the same tumor areas (e.g., perinecrotic areas) attest to their intimate liaison in terms of cancer-induced angiogenesis, which is probably secondary to the induction of multiple interdependent molecular pathways. Moreover, our study seems to be the first attempt to link IL-8 expression by tumor cells with histological grade, implicating its potent role in gliomagenesis.

Is there any potential link among caspase-8, p-p38 MAPK and bcl-2 in clear cell renal cell carcinomas? A comparative immunohistochemical analysis with clinical connotations.

BACKGROUND: Clear cell renal cell carcinomas (ccRCCs) constitute the most common renal carcinomas, characterized by a relatively aggressive clinical course. Thus, scientific research is targeting towards the identification of immunohistochemical and molecular markers that could be useful regarding diagnosis, appropriate therapy and prediction of prognosis. In the present study we assessed and correlated the expression of caspase-8, phosphorylated p38 mitogen-activated protein kinase (p-p38) and bcl-2 protein with histopathological features and clinical outcome of 27 patients with ccRCCs. METHOD: Immunohistochemistry in formalin-fixed and paraffin-embedded tissue sections was performed. The associations among various features were assessed utilizing statistical analysis. RESULTS: We found that increased expression of cytoplasmic caspase-8 and bcl-2 protein was strongly associated with low Fuhrman's grade of carcinomas (p = 0.019 and p = 0.041, respectively). On the other hand, increased p-p38 expression was significantly related to high Fuhrman's grade (p = 0.006). Moreover, high bcl-2 expression was correlated with low pathological stage of ccRCCs (p = 0.026). Increased expression of cytoplasmic caspase-8 as well as low-grade tumors (grade 1 and 2) implied a greater probability of patients' survival, in univariate statistical analysis (p = 0.037 and p = 0.019, respectively). Neither p-p38 nor bcl-2 expression was significantly linked to patients' survival. There were not emerged statistically significant associations among caspase-8, p-p38 kinase and bcl-2 protein. CONCLUSION: For the first time the prognostic impact of caspase-8 and p-p38 was studied in a series of ccRCCs, using immunohistochemistry in formalin-fixed and paraffin-embedded tissue sections. The suggestive relationship of caspase-8 with patients' clinical outcome, as well as the role of p-p38 within different grade categories, mandates further studies in larger cohorts of RCCs.

The effect of leptin on the tonic secretion of gonadotropins in the female rats.

OBJECTIVES: This study is an attempt to determine, the in vivo action of leptin on this hypophysiotropic hypothalamic area, by evaluating the concentrations of luteinizing hormone (LH) and follicular stimulating hormone (FSH) in the serum. METHODS: The experiments were done by stereotaxic injection of recombinant rat leptin (rrleptin) into the third cerebral ventricle (V3) of adult female Wistar rats. Subjects were divided into five groups. Group A included normal intact animals. In Group B, the rats were stereotaxically administered with rrleptin in the V3. The rats, in Groups C, D and E, were subjected to electrolytic lesion of the arcuate nucleus (ARC), of the ventromedial nucleus (VMH) and of both of these hypothalamic nuclei, respectively. Immediately after the electric lesion, they were intracranially injected with rrleptin. Blood samplings for serum LH and FSH levels estimation were performed three times: 1) just before any stereotaxic procedure, 2) six hours, and 3) twenty-four hours after leptin administration. RESULTS: The results showed that serum LH levels increased dramatically in group B, six hours after leptin administration. The LH levels in Groups C, D and E presented the same pattern with a lower peak. The FSH levels were doubled six hours after leptin administration in all groups without any exception. Both LH and FSH serum levels reverted to the initial basic levels after 24 hours. DISCUSSION: The significant conclusion derived from this study is that ARC and VMH, which are responsible for controlling the tonic secretion of gonadotropins, respond in a different way for the FSH and LH secretion. This also suggests that some other mechanism(s) or factor(s) may additionally participate in the control of the tonic component of FSH secretion.

Simultaneous occurrence of cerebellar medulloblastoma and pituitary adenoma: A case report.

PURPOSE: We present the unusual occurrence of two distinct neoplasms in a 42-year-old woman with an operated pituitary adenoma 18 years ago. METHODS: Clinical history, magnetic resonance imaging studies and histopathological findings were utilized for our diagnostic considerations. RESULTS: Concomitant presence of a cerebellar medulloblastoma secondary disseminated within the spinal canal and a pituitary macroadenoma, was identified. CONCLUSION: To the best of our knowledge, this is the first reported case in which these two neoplasms are simultaneously occurred in the same individual. A short review of the literature is performed.

Application of the ELISPOT method for comparative analysis of interleukin (IL)-6 and IL-10 secretion in peripheral blood of patients with astroglial tumors.

Glioblastoma, (grade IV astrocytoma), is characterized by rapid growth and resistance to treatment. Identification of markers of aggressiveness in this tumor could represent new therapeutic targets. Interleukins (IL)-6 and IL-10 may be considered as possible candidates, regulating cell growth, resistance to chemotherapy and angiogenesis. ELISPOT method provides a useful tool for the determination of the exact cell number of peripheral lymphocytes secreting a specific cytokine. IL-6 and IL-10 secretion levels were determined using ELISPOT methodology in peripheral blood mononuclear cells of 18 patients with astrocytic neoplasms (3 grade II and 15 grade IV), in parallel with 18 healthy controls. Additionally, immunohistochemical expression of these two cytokines was performed in paraffin-embedded neoplastic tissue in 12 of these patients. The secretion of IL-6 from peripheral monocytes was significantly higher in glioma patients compared to controls (P = 0.0003). In addition, IL-10 secretion from peripheral mononuclear and tumor cells of glioma patients was also higher as compared to healthy controls (P = 0.0002). Based on immunohistochemical staining, IL-6 expression was localized in tumor cells and macrophages as well as in areas of large ischemic necrosis, while the major source of IL-10 expression in glioblastomas was the microglia/macrophage cells. It is suggested that IL-10 contributes to the progression of astrocytomas by suppressing the patient's immune response, whereas IL-6 provides an additional growth advantage. This study demonstrates for the first time the usefulness of ELISPOT in estimating the secretion of IL-6 and IL-10 from peripheral blood and the correlation of their expression in neoplastic cells.