Metastatic Clear Cell Renal Carcinoma Without Evidence of a Primary Renal Tumour Mimicking Advanced Stage of Malignant Lung Tumour.

BACKGROUND: Clear cell renal carcinoma (ccRC) accounts for 65-70% of renal carcinomas with peak occurrence at the 6th and 7th age decade, predominantly in males. At the time of diagnosis, especially pulmonary metastases can be found in one-third of patients. There have also been described as late metastases for several decades after nephrectomy. In our case report, clinical course indicated primary lung tumour. Histological differential diagnosis included malignant pleural mesothelioma, lung adenocarcinoma and squamous cell carcinoma with clear cell differentiation or primary clear cell adenocarcinoma of the lung. However, using immunohistochemistry, all these possible diagnoses were excluded. CASE REPORT: We present a case of 62-year old man with 3 months history of progressive dyspnea accompanied with a cough and recurrent pleural effusions. PET/CT scan revealed metastatic tumour spread with right-sided pleural thickening, multiple pulmonary tumour foci, mediastinal, cervical, abdominal para-aortic and pelvic lymph node involvement and skeletal metastasis. The patient died one day after administration of palliative chemotherapy. The autopsy showed the majority of changes in the right hemithorax, was caused by a diffuse yellowish, extremely tough tumour infiltrating parietal and visceral pleura with adhesions and obliteration of truncus pulmonalis. In left lung and both renal cortices we could see scant nodules, mimicking primary lung tumour metastasis. In close proximity to the left renal hilum we found unusual homogeneous white round to oval tissue of 80 × 86 × 72mm in diameter, with identical histological pattern. Extensive immunohistochemical profile (positivity of CK18, PAX8, vimentin, androgen receptor, napsin A; negativity of mesothelial markers, TTF-1, CK7, CK20, CDX-2, CD10, PSA, CK34B12 and PAS-D) was compatible with metastatic ccRC. CONCLUSION: We present an extremely rare case of morphologically verified metastatic ccRC without evidence of primary lesion in the kidneys. There is speculated the possibility of spontaneous regression of primary tumour. In our case, however, we cannot exclude the possibility of generalized primary tumour of ectopic kidney. This hypothesis is based on the finding of isolated tumour mass adjacent to left renal hilum.

Infiltration of Prostate Cancer by CD204+ and CD3+ Cells Correlates with ERG Expression and TMPRSS2-ERG Gene Fusion.

BACKGROUND: The aim of the study was to detect CD204 + and CD3 + cells in the infiltrate of benign prostatic hyperplasia, prostatic intraepithelial neoplasia and prostatic cancer in prostate specimens after radical prostatectomy. Another goal was to determine correlation of the intensity of the infiltration with ERG oncoprotein expression as well as with the presence of activat-ing translocation TMPRSS2-ERG. MATERIALS AND METHODS: To confirm the translocation, we used fluorescence in situ hybridization. Imunohistochemistry was used to detect the presence of ERG oncoprotein and for assesment of the number of CD204+ and CD3+ infiltrat-ing cells. We determined the capability to infiltrate malignant structures accord-ing to differences in infiltration of benign and malignant prostate structures. RESULTS: Biometric analysis confirmed that the number of CD204+ macrophages in the malignant structure was significantly higher than in the benign prostatic hyperplasia regardless of the fusion pattern. Increased infiltration by CD3+ cells was only detected in malignant structures of the prostate in a group with normal signal pattern and in a group with TMPRSS2-ERG fusion. Expression of ERG positively correlated with CD204+ and CD3+ cells infiltration of malignant structures only in cases where the TMPRSS2-ERG fusion was found. In the group with a break in the TMPRSS2 gene, a positive correlation was only found between ERG expression and CD204+ macrophages infiltration. In cases with a normal signal pattern, no correlation was found. In the group with TMPRSS2-ERG fusion we observed significantly more cases with a good capability of CD204+ cells to infiltrate malignant structures, unlike the group with a normal signal pattern, where there were more cases with the weak reactivity of CD204 + cells to infiltrate the malignant structures. The same was observed for CD3+ cells. CD204+ macrophages and CD3+ T-lymphocytes in the group with TMPRSS2-ERG gene fusion, infiltrated the malignant prostate structures more intensely, but their effect on malignant transformation may be different. CONCLUSIONS: The association between the presence of the TMPRSS2-ERG fusion and the different capability of inflammatory cells to infiltrate malignant structures has not been reported so far. The results confirm the important role of the activated ERG gene, due to TMPRSS2-ERG fusion, in the development of inflammation of the prostate as well as the effect of inflammatory cells on the course of neoplastic process. This leads to considerations about introduc-ing immunomodulatory modalities into prostate cancer therapeutic protocols. Key words: prostate cancer -  TMPRSS2-ERG gene fusion -  ERG -  immune response -  CD204+ macrophages -  CD3+ T-lymphocytes.

[Can Analysis of Cellular Lipidome Contribute to Discrimination of Tumour and Non-tumour Colon Cells?] / Muze analýza bunecného lipidomu prispet k rozlisení nádorových a nenádorových bunek tlustého streva?

BACKGROUNDS: Colon cancer development is often characterized by abnormalities in lipid synthesis and metabolism, which may influence energetic balance, structure and function of biological membranes, or production of specific mediators and cell signalling. The changes in lipid profile and metabolism (lipidome) may significantly affect cell behaviour and response to therapy. Permanent epithelial cell lines at various stages of cancer development are used for better understanding of this topic on cellular and molecular levels. In our study, we hypothesized that detailed analyses of colon cancer cell line lipidomes may help to identify major alterations in the amount and profile of specific lipid classes/species, which can contribute to their different response to various stimuli. MATERIAL AND METHODS: Cellular lipids were isolated from six human epithelial cell lines derived from tissues at various stages of tumour development. Liquid chromatography coupled with tandem mass spectometry analyses were performed in order to determine amount and mass profiles of all phospholipid (PL), lysophospholipid (lysoPL) and sphingolipid classes. The data was statistically evaluated (cluster and discrimination analyses) with respect to mutual comparison of cell lines and to significantly discriminating lipid types. RESULTS: The results of cluster analysis arranged cell lines in order corresponding to their level of transformation (normal cells, adenoma, carcinoma and lymph node metastasis). The results of discrimination analyses revealed the most discriminating lipid types and distinction in PL: lysoPL ratios. Particularly, significant correlation of the amount and profiles of both specific lysoPL and sphingolipid classes with cell transformation level were observed. Similar approaches are now applied to compare lipidomes of colon epithelial cells isolated from tumour vs. non-tumour samples of colon cancer patients. CONCLUSION: Our results indicate that a) selected cancer cell lines are suitable model for lipidomic studies that can serve as a basis for subsequent clinical research, b) cellular lipidome analyses may help to discriminate tumour and non-tumour cells in clinical samples, where specific types of lipids could serve as biomarkers.Key words: colon cancer - cell lines - liquid chromatography - mass spektrometry - phospholipids - sphingolipids - bioinformatics The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. This work was supported by Czech Health Research Council, grant No. AZV 15-30585A.Submitted: 19. 3. 2018Accepted: 18. 4. 2018.

MicroRNAs in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a molecular subtype of breast cancer with one of the worst prognoses. Current treatment is based on chemo- and/or radiotherapy and surgery. New targets, however, offering other therapeutic approaches, have been identified. These involve poly (ADP-ribose) polymerase (PARP), vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR), androgen receptor (AR), long non-coding RNAs (lncRNAs) and microRNAs (miRs). The latter are non-coding RNAs which control the expression of more than 50% of human genes via regulation of basic cellular processes at post-transcriptional level and dysregulation of miRs is found in many types of tumors. The role of dysregulated miRs in carcinogenesis lies in their acting as tumor suppressors or oncogenes, and in resistance to treatment (chemotherapy, hormonal and targeted therapy or radiotherapy). Circulating miRs are also promising prognostic and predictive biomarkers in patients with breast cancer. The aim of this review is to analyze recently published data on miRs and therapeutic targets potentially influenced by miRs in TNBC.

Prognostic significance of miR-23b in combination with P-gp, MRP and LRP/MVP expression in non-small cell lung cancer.

Recently, miR-23b has emerged as a promising new cancer biomarker but its role in lung cancer has not been established yet. Patients still do not respond well to available treatments, probably due to expression of multidrug resistance (MDR) proteins, such as P-gp, MRP and LRP/MVP. The aim of this study was to determine the role of miR-23b in non-small cell lung cancer (NSCLC) and its relationship to the patient outcome together with MDR transporter proteins. We immunohistochemically evaluated expression of P-gp, MRP and LRP/MVP and quantified the relative levels of miR-23b in 62 NSCLC patients´ samples. The prognostic significance of miR-23b and MDR proteins was tested by Kaplan-Meier and Cox-regression analysis. Our results showed that miR-23b is mostly downregulated in NSCLC samples (57/62) and that its upregulation in tumors is connected with longer progression-free survival (PFS; P = 0.065) and overall survival (OS; P = 0.048). The Cox proportional hazard model revealed that the risk of death or relapse in NSCLC patients with miR-23b downregulation increases together with LRP/MVP expression and both risks decrease with miR-23b upregulation (HRPFS = 4.342, PPFS = 0.022; HROS = 4.408, POS = 0.015). Our findings indicate that miR-23b, especially in combination with LRP/MVP expression, might serve as a suitable prognostic biomarker for NSCLC patients.

Overexpression of filamin-A protein is associated with aggressive phenotype and poor survival outcomes in NSCLC patients treated with platinum-based combination chemotherapy.

An actin-binding protein filamin A connects the actin filament network to cell membrane receptors, and acts as a scaffold for various signaling pathways related to cancer growth and progression. Recently, it has been reported that filamin A is required for efficient regulation of early stages of DNA repair process. Moreover, some in vitro studies showed that the overexpression of filamin A determines resistance to various cytotoxic drugs, including cisplatin. We aimed to analyse the expression of filamin A protein in resected NSCLC (Non Small Cell Lung Cancer) specimens, to investigate the association of the level of filamin A protein expression and other clinicopathological features, and possible relationship between the expression of filamin A and survival outcome in NSCLC patients, treated with platinum-based combination chemotherapy. We performed filamin A protein immunohistochemistry on formalin-fixed and paraffin-embedded (FFPE) tissue sections from 135 NSCLC patients, using EP2405Y antibody against C-terminus of filamin A. Cytoplasmic, membranous and nuclear positivity of filamin A was evaluated semi-quantitatively and correlated with available clinicopathological data. Patients were divided into two groups for survival analysis (I group - patients treated with adjuvant platinum-based chemotherapy, II group - patients with surgical treatment only). We found significant positive correlation between filamin A protein expression and NSCLC stage (r=0.249; p<0,05), presence of lymph node (N)(r=0.205; p<0,05) and distant metastases (M) (r=0.332; P<0.01). Increased filamin A protein expression was significantly related with poor survival outcomes in patients with adjuvant platinum-based chemotherapy: OS (HR=1.005, 95%CI[1.000;1.010], p=0.037), DFS (HR=1.004, 95%CI [1.001:1.008], p=0,017). Multivariate Cox proportional hazards regression analysis also showed that overexpression of filamin A represents an independent risk factor for disease relapse, in addition to tumor size, stage, and metastases status (HR=1.723, 95%CI [1.021:2.909], p<0.05). Thus, filamin A expression might be a new prognostic marker in patients with NSCLC.

Androgenetic alopecia and polymorphism of the androgen receptor gene (SNP rs6152) in patients with benign prostate hyperplasia or prostate cancer.

BACKGROUND: Both androgenetic alopecia (AGA) and carcinoma of the prostate (CaP) or benign prostatic hyperplasia (BPH) are androgen-dependent disorders. OBJECTIVE: To investigate the relationships between male androgenetic alopecia, androgen receptor (AR) gene polymorphism (SNP rs6152) and clinical characteristics of BPH and prostate cancer. METHODS: Overall, 309 male subjects with prostate disease (BPH or CaP) were examined. We evaluated the standard grades of AGA (I-VII) by Hamilton-Norwood classification and 195 patients were also assessed by phototrichogram. Prostate-specific antigen (PSA) and testosterone levels were also measured. Polymorphism rs6152 of the AR was evaluated from blood samples by PCR-RFLP. Data were statistically evaluated. RESULTS: The expected positive correlation between age and AGA grade and the expected negative correlation between hair density and age and between anagen/telogen and AGA were found. A statistically significant difference between patients with A and G alleles in terms of AGA grade was found. The predominant G allele was more frequent in patients with higher grade of alopecia and in patients with significantly higher PSA. There was no correlation between diagnosis (BPH or CaP) and polymorphism. Patients with prostate inflammation had a statistically significant higher grade of AGA, together with higher PSA. CONCLUSIONS: We confirmed that the AR gene polymorphism (SNP rs6152 G>A) is associated with the development of AGA and higher PSA levels in patients with BPH but not cancer. A novel finding of our study is that BPH patients with prostate inflammation had a significantly higher grade of AGA together with significantly higher PSA levels.

Relation of ETS transcription factor family member ERG, androgen receptor and topoisomerase 2ß expression to TMPRSS2-ERG fusion status in prostate cancer.

Fusion of TMPRSS2 with ERG in prostate cells is determined by double-strand DNA breaks induced by androgen signaling and transcription stress. The enzyme topoisomerase 2ß (TOP2B) mediating DNA processing, plays an important role in DNA cleavage. The aim of this study was to analyse expression of AR, TOP2B and ERG in relation to TMPRSS2-ERG gene rearrangement and relevant clinicopathological characteristics in prostate cancer (CaP). Immunohistochemical staining and FISH were used for investigation. ERG expression in prostate cell lesions positively correlated with levels of TMPRSS2-ERG fusion gene (p<0.0001). The most significant co-expression of ERG was found with AR in CaP (p=0.001). Significantly more frequent co-expression of ERG was also revealed with TOP2B (p=0.028). ERG protein expression did not correlate with CaP differentiation status as we found no significant differences in ERG expression for different Gleason categories. We demonstrated a statistically significant positive correlation between the percentage of cells with fusion gene TMPRSS2-ERG in CaP and metastatic potential of tumors (p=0.011). Besides these positive corelations of AR with ERG (p=0.001) and TOP2B with ERG (p=0.028), we also demonstrated a significant co-expression of AR with TOP2B (p=0.007) in CaP. There was a statistically significant increase in the TOP2B H-index in locally advanced CaP in comparison with localized tumors (p=0.046). ERG expression correlates with occurrence of TMPRSS2-ERG fusion and with AR-driven malignant transformation. The results indicate that detection of the TMPRSS2-ERG fusion gene and parallel immunohistochemical examination of AR, TOP2B and ERG has diagnostic significance and may be useful in assessing the biological character of the prostate cancer as well as selecting the best treatment.

[Biomarkers in the detection of minimal systemic dissemination in lung cancer patients]. / Biomarkery detekce minimální systémové diseminace u nemocných s karcinomem plic.

INTRODUCTION: Minimal systemic disease (MSD) means the presence of circulating or disseminated tumour cells in mesenchymal compartments of a patientts' body (lymphatic nodes, blood or bone marrow). The aim of our pilot study was to identify sensitive and specific markers for MSD detection in 50 lung cancer patients, who underwent curative surgery in the I. Department of Surgery, Faculty of Medicine and Dentistry, Palacky University and Faculty Hospital Olomouc in 2009 and 2010. MATERIAL AND METHODS: Absolute gene expression of carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR1), lung-specific X protein (LUNX) and hepatocyte growth factor receptor (c-met) was determined in peripheral blood, bone marrow and pulmonary blood of 50 lung cancer patients using real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR). RESULTS: (1) The LUNX marker is specific and sensitive for MSD detection in lung cancer patients. (2) The CEA positivity for MSD in the bone marrow correlated significantly with histopathological grading (GI-GIII). (3) Higher expression of CEA and c-met was found in pulmonary blood of patients with hilar or mediastinal lymphadenopathy. (4) Higher expression of MSD markers (CEA in bone marrow, c-met in peripheral blood and LUNX in pulmonary blood) correlated with higher pTNM classification. CONCLUSION: Minimal systemic disease detection in lung cancer patients is technically feasible using sufficiently sensitive and specific markers for RT-PCR. Minimal systemic disease detection can be used to guide further systemic treatment. This theory must be validated in a larger group of patients and correlated with clinical data, especially with survival data.

Quadriplex model enhances urine-based detection of prostate cancer.

BACKGROUND: The major advantages of urine-based assays are their non-invasive character and ability to monitor prostate cancer (CaP) with heterogeneous foci. While the test for the prostate cancer antigen 3 (PCA3) is commercially available, the aim of our research was to test other putative urine markers in multiplex settings (AMACR (α-methylacyl-CoA racemase), EZH2 (enhancer of zeste homolog 2), GOLM1 (golgi membrane protein 1), MSMB (microseminoprotein, ß), SPINK1 (serine peptidase inhibitor) and TRPM8 (transient receptor potential cation channel, subfamily M, member 8)). METHODS: Expression of the candidate biomarkers was studied in sedimented urine using quantitative reverse transcriptase polymerase chain reaction in two sets of patients with and without restriction on serum PSA levels. RESULTS: We confirmed that PCA3 is an independent predictor of cancer in the patients without restriction of serum PSA values (set 1, n=176, PSA=0.1-587 ng ml(-1)). However, AMACR was the only parameter that differentiated CaP from non-CaP patients with serum PSA between 3 and 15 ng ml(-1) (set 2, n=104). The area under curve (AUC) for this gene was 0.645 with both sensitivity and specificity at 65%. Further improvement was achieved by multivariate logistic regression analysis, which identified novel duplex (TRPM8 and MSMB), triplex (plus AMACR) and quadriplex (plus PCA3) models for the detection of early CaPs (AUC=0.665, 0.726 and 0.741, respectively). CONCLUSIONS: Novel quadriplex test could be implemented as an adjunct to serum PSA or urine PCA3 and this could improve decision making for diagnostics in the case of 'PSA dilemma' patients.

Immunohistochemical localization and analysis of kallikrein-related peptidase 7 and 11 expression in paired cancer and benign foci in prostate cancer patients.

Kallikrein-related peptidases 7 and 11 (KLK7/KLK11) share a high degree of structural similarity with PSA (KLK3) and other KLKs. The aim of this study was to evaluate differences in KLK7/ KLK11 expression in paired cancer/benign prostate foci and to determine possible associations with clinicopathological parameters. Seventy archived paraffin-embedded tissue samples obtained from radical prostatectomy were stained for KLK7, KLK11, PSA and PSMA and expression was evaluated semiquantitatively. The results showed statistically significant differences for all studied proteins between BPH and CaP foci. Both KLK7 (P=0.026) and KLK11 (P<0.001) expressions were decreased in prostate cancer cells compared to normal/benign prostate cells. Positive correlations were found for both KLK7 (Rs=0.74, P<0.001) and KLK11 (Rs=0.35, P=0.003) between CaP and BPH. We found a statistically significant upregulation of KLK11 in advanced cases compared to localized ones (P=0.026). For the first time, we report lower expression of KLK11 in CaP compared to BPH and slight upregulation of KLK11 in advanced tumors compared to localized ones. Our observations support the diagnostic potential of KLK7/KLK11 for early prostate cancers but further studies on larger cohorts are needed in order to validate the clinical value of these biomarkers and clarify their biological role in prostate development and tumorigenesis.

Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas.

Malignant gliomas, the deadliest of brain neoplasms, show rampant genetic instability and resistance to genotoxic therapies, implicating potentially aberrant DNA damage response (DDR) in glioma pathogenesis and treatment failure. Here, we report on gross, aberrant constitutive activation of DNA damage signalling in low- and high-grade human gliomas, and analyze the sources of such endogenous genotoxic stress. Based on analyses of human glioblastoma multiforme (GBM) cell lines, normal astrocytes and clinical specimens from grade II astrocytomas (n=41) and grade IV GBM (n=60), we conclude that the DDR machinery is constitutively activated in gliomas, as documented by phosphorylated histone H2AX (gammaH2AX), activation of the ATM-Chk2-p53 pathway, 53BP1 foci and other markers. Oxidative DNA damage (8-oxoguanine) was high in some GBM cell lines and many GBM tumors, while it was low in normal brain and grade II astrocytomas, despite the degree of DDR activation was higher in grade II tumors. Markers indicative of ongoing DNA replication stress (Chk1 activation, Rad17 phosphorylation, replication protein A foci and single-stranded DNA) were present in GBM cells under high- or low-oxygen culture conditions and in clinical specimens of both low- and high-grade tumors. The observed global checkpoint signaling, in contrast to only focal areas of overabundant p53 (indicative of p53 mutation) in grade II astrocytomas, are consistent with DDR activation being an early event in gliomagenesis, initially limiting cell proliferation (low Ki-67 index) and selecting for mutations of p53 and likely other genes that allow escape (higher Ki-67 index) from the checkpoint and facilitate tumor progression. Overall, these results support the potential role of the DDR machinery as a barrier to gliomagenesis and indicate that replication stress, rather than oxidative stress, fuels the DNA damage signalling in early stages of astrocytoma development.

Binding of AR to SMRT/N-CoR complex and its co-operation with PSA promoter in prostate cancer cells treated with natural histone deacetylase inhibitor NaB.

Signaling through the androgen receptor (AR) plays a critical role in prostate cancer progression. The AR is a classical nuclear receptor (NR) providing a link between signaling molecule and transcription response. Histone deacetylase inhibitors- (HDACI) have antiproliferative and proapoptotic effects on prostate cancer cells and their implication in silence AR signaling may have potential therapeutic use. We aimed to study the inhibitory effects of the corepressor SMRT (Silencing Mediator for Retinoid and Thyroid -hormone receptors) which forms a complex together with nuclear receptor corepressor (N-CoR) and with histone deacetylase 3 (HDAC3) on AR activity.The androgen-sensitive prostate cancer cell line LNCaP and androgen-insensitive prostate cancer cell line C4-2 both AR-positive, and androgen-insensitive DU145 and PC3 prostate cancer cell lines were treated with two HDACIs, sodium butyrate (NaB) and/or trichostatin A (TSA). We amplified immunoprecipitated DNA by conventional PCR and in the -following step we used the chromatin immunoprecipitation (ChIP) analysis coupled with quantitative PCR for monitoring NaB induced formation of AR-SMRT/N-CoR complex binding on the PSA promoter. The co-immunoprecipitation assay revealed increase in AR-SMRT formation in NaB treated cells. Simultaneously, the Western blot analysis showed a significant decrease in AR protein expression. In conclusion, the inhibitory effect of NaB on AR gene expression seems to be specific and unique for prostate cancer AR-positive cell lines and corresponds with its ability to stimulate AR-SMRT complex formation. We suggest that AR and SMRT/N-CoR corepressors may form a stable complex in vitro and NaB may facilitate the interaction between AR nuclear steroid receptor and SMRT corepressor prote.

Urine markers in monitoring for prostate cancer.

The major advantages of urine-based assays are their noninvasive character and ability to monitor prostate cancer with heterogeneous foci. Almost all urine-detectable prostate-specific markers have been recently reviewed. For this reason, we focus here on only a few promising markers which have been independently evaluated (in particular PCA3, fusion genes, TERT, AMACR, GSTP1, MMP9 and VEGF) and very recent ones (ANXA3 and sarcosine). The emphasis is also on multiplex biomarker analysis and on microarray-based analysis of fusion genes. A combination of multiple urine biomarkers may be valuable in the case of men with persistently elevated serum prostate-specific antigen and a history of negative biopsies. The emerging urine tests should help in both early diagnosis of prostate cancer and identifying aggressive tumors for radical treatment.

Analysis of VEGF, Flt-1, Flk-1, nestin and MMP-9 in relation to astrocytoma pathogenesis and progression.

Astrocytomas, particularly high grade astrocytoma, are brain tumors with potent angiogenic activity. Our immnunohistochemical study assessed vascular endothelial growth factor (VEGF), VEGF receptors (Flk-1, and Flt-1), the intermediate filamental protein nestin which plays a role in central nervous system development, and MMP-9, which belongs the family of matrix metalloproteinases implicated in tumor invasion and angiogenesis regulation. We investigated the expression of VEGF, its receptors, nestin and MMP-9 in astrocytomas and their correlation with tumor grade. We used paraffin-embedded samples from 66 patients, 29 with low grade (WHO-grade II) and 37 with high grade (WHO-grade III and IV) astrocytomas. Antibodies against VEGF, Flk-1, Flt1, nestin, CD34 and MMP-9 were used, followed by standard indirect immunohistochemical methods. Expression of Flt-1 and Flk-1 showed no significant differences between low and high grade tumor groups. Expression of VEGF and MMP-9 was increased in the high grade group (p equal to or less than 0.026 and 0.024). Nestin expression in tumor astrocytes and endothelial cells increased in high grade group (p same 0.007 and 0.003). Higher expression of VEGF in high grade astrocytomas may subsequently lead to activation of survival, angiogenesis and migration. Expression of nestin and MMP-9 also suggest their likely role in astrocytoma vascular development and proliferation.

Expression of STATs and their inhibitors SOCS and PIAS in brain tumors. In vitro and in vivo study.

Proteins of STAT family belongs to the transcription factors. Through their binding to the DNA specific sites and consequent regulation of transcription of various genes, these signaling proteins play an important role in many cell functions. Recent studies demonstrated persistent activation of STATs and loss of their natural inhibitors SOCS and PIAS in various human cancers. There is also evidence that experimental pharmacologic or genetic modulation of their function mignt by a new approach in anticancer treatment. The aim of this study was in vitro assesment and analysis of expression of STATs, SOCS and PIAS in glioblastoma cell lines undergoing treatment by PPARgamma agonists/antagonists because PPARgamma and STATs are tightly regulated by an overlapping set of nuclear regulatory proteins. We further analysed immunohistochemical expression of these proteins in vivo, with its correlation to grading in various brain tumors. The results of in vitro study showed decreased expression of phosphorylated form of STAT3 and increase of its inhibitors SOCS3 and PIAS3 in glioblastoma cell lines after treatment with IC50 of PPARgamma agonist ciglitazone. In vivo study failed to reveal changes in STAT3 and SOCS3 expression in either low and high grade astrocytomas, however we detect lower expression of STAT2 in low grade astrocytomas when comparing with high grade astrocytomas and lower expression of STAT3 in ependymomas when comparing with anaplastic ones. The results showed existing relationship between STAT and PPARgamma signaling in glial tumors and further suppport expected important role of STATs in regulation of growth and differentiation in these tumors.

Re-expression of nestin in the myocardium of postinfarcted patients.

Intact cardiac muscle cells in the adult heart do not express intermediate filament nestin. In this study, we report on widespread expression of intermediate filament nestin in human myocardium of patients who died from the myocardial infarction. Nestin was detected in cardiomyocytes, endothelial cells, and few interstitial cells. Elevated levels of nestin were observed in cardiac muscle cells in all specimens, although the intensity of immunoreactivity and distribution of the signal differed. The strongest immunoreactivity was observed from 4 days after myocardial infarction in the infarction border zone where nestin was distributed homogeneously in the entire sarcoplasm of cardiac muscle cells. Within the following week, nestin in immunoreactive cardiomyocytes was redistributed and restricted to small subsarcolemmal foci and to intercalated discs. Angiogenic capillaries that grew between vital nestin-positive cardiomyocytes and entered the necrotic area expressed also high levels of nestin. Nestin-positive endothelial cells were often observed in mutual interactions with nestin-positive cardiac muscle cells. These findings document a crucial role of nestin in remodeling cytoskeleton of cells in the human postinfarcted myocardium.

Topical isotopic exchange and compartmental analysis approach for probing solute behaviour at the soil/arsenate solution interface.

Isotopic exchange based approaches have for many years been applied in soil and solute research. However, acquiring and elaboration of experimental data were not always straightforward and complete. A strict and correct use of combined isotopic exchange-compartmental analysis may widen the knowledge database and provide information not available as yet. The experiments were carried out with arsenic (arsenate) from IAEA-SOIL-5 in contact with water or phosphate solution in dynamic equilibrium. After contacting the soil suspension for 28 days, the amount of arsenate released is 2.8 and 6.3 % of arsenic (solutes) in the soil, respectively. Addition of a radioactive arsenate (73)As(V)-spike and following the distribution of this radiotracer from the aqueous to the solid phase in time shows that the accessible fraction, i.e. available for exchange, is in both cases 12%. This implies that the remainder of the arsenic is enclosed in the lattice of minerals and for that reason unavailable for exchange, at least on the time scale of the experiment (weeks). From deconvolution of compartmental analysis results the distribution of accessible arsenate in the soil could be attributed to sorption onto external surfaces (2.6 and 2.0% of total arsenic present for the water and phosphate system, respectively) and sorption onto internal surfaces after diffusion through soil particle pores (6.5 and 4.2% of total arsenic present for the water and phosphate system, respectively). The mean residence time in two out of three compartments was in the order of minutes for the external surfaces and in the order of days for the diffusion-controlled internal surfaces.

The status and role of ErbB receptors in human cancer.

Changes in the expression of cellular receptors contribute to the progression of many types of solid tumors. In this review, we focus on the normal role of ErbB receptors as signal transducers and their contribution to carcinogenesis when there are abnormalities in ErbB signaling due to the overactivity of the receptors or the overexpression of ligands, which can lead to developmental defects and have been associated with many types of cancers.

Expression of E-cadherin and c-erbB-2/HER-2/neu oncoprotein in high-grade breast cancer.

E-cadherin (E-CD) is an epithelial-specific cell adhesion molecule, whose expression is lost in invasive lobular (ILC) but not in invasive ductal carcinoma (IDC) of the breast. This cell adhesion system can be disrupted by tyrosine kinase c-erbB-2/HER-2/neu. We examined 106 cases of high-grade invasive breast cancer, including 91 IDCs, 12 ILCs and 3 pleomorphic lobular carcinomas (PLCs). We determined Nottingham histological grade and performed immunohistochemistry for estrogen and progesterone receptors (ER/PR), Ki-67, E-CD and c-erbB-2/HER-2/neu with subsequent fluorescence in situ hybridization. Amplification of c-erbB-2/HER-2/neu gene was observed in 55/91 (60.4%) of IDCs, 3/12 (25%) of ILCs and 1/3 (33.3%) of PLCs, and associated with positive axillary lymph nodes. E-CD expression was lost in 14/91 (15.4%) of IDCs, 10/12 (83.3%) of ILCs and 2/3 (66.7%) of PLCs. The loss of E-CD immunoreactivity in IDCs appeared to be associated with c-erbB-2/HER-2/neu gene amplification, negative ER/PR status and positive lymph nodes, whereas E-CD-positive ILCs tended to be HER-2/neu-positive. The biological significance of E-CD expression seems to be different in high-grade IDC and ILC. Oncogenic pathway mediated by c-erbB-2/HER-2/neu may affect the E-CD expression in most invasive ductal breast carcinomas in vivo.