[Vascular endothelial growth factor in ovarian cancer patients]. / Cévní endotelový rustový faktor u pacientek s karcinomem ovaria.

OBJECTIVE: To compare plasma VEGF (vascular endothelial growth factor) levels in ovarian cancer patients, in patients with benign ovarian tumors and healthy women. DESIGN: Prospective study. SETTING: Department of Gynecology and Obstetrics, Medical Faculty Charles University, Prague and University Hospital, Hradec Králové. Department of Immunology and Alergology, Medical Faculty Charles University, Prague and University Hospital, Hradec Králové. METHODS: VEGF was estimated by ELISA (R&D Systems). RESULTS: We found that plasma VEGF levels were associated with the International Federation of Gynecology and Obstetrics (FIGO) stage (FIGO I+II, n=8) Med = 425,53 pg/ml (range 142,30-982,40 pg/ml), (FIGO III+IV, n=29) Med = 941,48 pg/ml (range202,10-2857,80 pg/ml) (p=0,03). Patients with primary ovarian cancer (n=37) had a significantly higher plasma VEGF level Med = 829,93 pg/ml (range142,30-2857,80 pg/ml), compared with patients with benign ovarian tumors (n=15) Med = 426,28 pg/ml (range 32,00-922,20 pg/ml) and healthy women (n=21) Med = 283,13 pg/ml (range 80,50-735,20 pg/ml) (p=0,0003). VEGF levels were lower in plasma (n=79) Med = 575,49 pg/ml (range 55,80-2185,00 pg/ml) compared with VEGF levels in ascitic fluid (n=37) Med = 745,74 pg/ml (range 142,30-2185,00 pg/ml) (p=0,04) in ovarian cancer patients. CONCLUSION: Plasma VEGF assay before primary treatment and the changes during the other treatment should contribute to better understanding of angiogenesis in ovarian cancer patients. Plasma VEGF correlates with the stage of primary ovarian cancer.

Quantification of ZAP-70 expression in chronic lymphocytic leukemia: T/B-cell ratio of mean fluorescence intensity provides stronger prognostic value than percentage of positive cells.

Expression of ZAP-70 measured by flow cytometry belongs to the most powerful prognostic parameters in chronic lymphocytic leukemia (CLL). However, many technical factors such as setting of the positivity threshold may significantly influence results.. Quantification using mean fluorescent intensity (MFI) may eliminate the subjective error which is inevitable in the isotype control method. The aim of the present project was therefore to assess the prognostic significance of ZAP-70 using three different methods. Between 2005 and 2010 we measured ZAP-70 expression in 157 patients with CLL (108 males, 49 females, median age 60 years [range, 31-82]; low/intermediate/high Rai risk in 41/48/11%). Expression of ZAP-70 was determined by flow cytometry using phycoerythrin (PE)-conjugated monoclonal antibody, clone 1E7.2. Evaluation was performed by 1) percentage of positive cells compared to isotype control (cut-off 20%), 2) MFI ratio of T-cells/CLL cells (cut-off 3.0); 3) MFI ratio of ZAP-70/isotype control on CLL cells (cut-off 2.5). MFI method with T-cells/CLL cells ratio was the best in the identification of patients with unfavourable outcome: ZAP-70 positive patients had significantly shorter time to treatment (TTT, median 24 vs. 55 months, p=0.0001) and overall survival (OS, median 97 vs 174 months, p=0.0074). The differences in TTT a OS were not significant with the use of isotype percentage and MFI isotype methods. Combined analysis of ZAP-70 with CD38 expression or IgVH mutation status lead to identification of a subgroup with the longest TTT and OS (ZAP-70 and CD38 negative, p<0.0001 and p=0.012; ZAP-70 negative and mutated IgVH genes, p<0.0001 and p=0.0019). In conclusion, our results suggest that measurement of ZAP-70 expression in CLL by MFI using T-cells/CLL cells ratio might be the optimal method for accurate prediction of clinical course. Combined analysis of ZAP-70 with CD38 or IgVH mutation status further refined individual patient´s prognosis.

[Prognostic markers in chronic lymphocytic leukemia]. / Prognostické faktory u chronické lymfocytární leukemie.

Chronic lymphocytic leukemia is the most common leukemia type in Western countries. Even incidence of chronic lymphocytic leukemia is high, this disease remained beyond interest for a very long time. However, in the last few years the view of this disease fundamentally changed and due to intensive study, new knowledge especially on pathogenesis, prognostic factors and therapy based on intensive therapeutic procedures were made. Today we know that usage of classical prognostic factors is insufficient for prognosis evaluation in the individuals. However modern (IgVH mutation status, cytogenetic abberations) and new markers (LPL/ADAM29 ratio, microRNA, markers of angiogenesis etc) have potential to distinguish patients in early stages to groups with significantly different prognosis and predict clinical course of the disease.

Determination of angiogenic factors in serum by protein array in patients with renal cell carcinoma.

Using the protein array method we determined the serum levels of a number of angiogenic factors. We identified serum levels of angiogenin, PDGF and MCP-1 (CCL2 chemokine) in serum of 32 patients with RCC, and 14 healthy volunteers by means of antibody array analysis. The patients were divided into three groups according to their disease stages (I+II, III, and IV). We found significant differences between the controls and patients with RCC both pre-operatively and post-operatively in angiogenin, PDGF and MCP-1 serum levels. The increase in angiogenin, PDGF and MCP-1 lasted in patients with RCC stages I-III even without metastases eight weeks post-operatively. The patients with stage IV RCC showed disturbed production of PDGF and MCP-1. Protein array analysis is a powerful tool for the identification of large numbers of trace proteins. Multiplex antibody array is able to provide data more precisely reflecting the nature of pathological processes.

[Monitoring of anti-tumour cell-mediated response in patients with renal cell carcinoma, disturbance of T cell proliferation]. / Sledování protinádorové bunecné imunitní odezvy u nemocných s renálním karcinomem, porucha proliferace T-lymfocytu.

INTRODUCTION: When checking tumour growth, a number of observations indicate that the immune system plays a significant role in patients with renal cell carcinoma (,,RCC"). Infiltration by lymphocytes (tumour infiltrating lymphocytes, "TILs") is more prevalent in RCC than any other tumours. T lymphocytes are the dominant population of TIL cells. Views concerning the role ofT lymphocytic subpopulations, B lymphocytes and NK cells in an anti-tumour response are not established. AIM: The aim is to determine the phenotype and activation of lymphocytic cells and to compare their representation in tumour stroma (TIL), peripheral blood (PBL) and renal vein blood in patients with RCC. PATIENTS AND METHODS: The samples of peripheral blood taken from the cubital and renal veins and tumour stroma cells were obtained from 60 patients in the course of their surgeries carried out due to primary RCC. TILs were isolated from mechanically disintegrated tumour tissue. Immunophenotype multiparametric analysis of PBL and TILs was carried out. Their surface and activation characteristics were determined by means of flow cytometer. RESULTS: CD3+ T lymphocytes (70.4%) were the main population of TILs. The number of CD3+/CD8+ T lymphocytes was significantly higher in TILs, 39.7% (p < 0.01), while CD4+ T lymphocytes were the majority population in peripheral blood, 41.35% (p < 0.001). The representation of CD3+/69+ T lymphocytes was significantly higher in TILs, 32.05%, compared to PBL (p < 0.001). On the contrary, the numbers of CD3+/CD25+, CD8+/57+ and CD4+/RA+ (naive CD4+ T lymphocytes) were higher in PBL (p < 0.001). The differences in representation of (CD3+/16+ 56+) NK cells and CD3+/DR+ T cells in TILs and PBL were not significant. CONCLUSION: The above-mentioned results prove that the characteristics and intensity of anti-tumour responses are different in compared compartments (tumour/PBL). CD3+/CD8+ T lymphocytes are the dominant lymphocytic population of TILs. The knowledge of phenotype and functions ofeffector cells, which are responsible for anti-tumour response, are the basic precondition for understanding the anti-tumour immune response and the cause of its failure.

[Late complications of chronic respiratory infections in patients with common variable immunodeficiency]. / Pozdní komplikace chronických zánetu respiracního traktu u nemocných s beznou variabilní imunodeficiencí.

Common variable immunodeficiency (CVID) is the most frequent serious humoral deficiency manifested in adulthood in the form of acute and chronic respiratory infections which in most patients lead to respiratory failure. Retrospective analysis of 28 CVID patients was made. Mean age at time of diagnosis was 38.6 +/- 18.6 years. The time which elapsed from the first symptoms to determining the diagnosis was three times as long as that indicated in other studies, i.e., 14.1 +/- 10.2 years. Twenty-three patients (82.1%) had respiratory complications. Chronic obstructive pulmonary diseases, n=16 (57.1%), and bronchiectasis, n=10 (35.7%), were the most frequent types of lung damage. In addition, two patients (7.1%) displayed evidence of interstitial lung process. Morbidity associated with CVID can be reduced by early diagnosis and adequate dosage of immunoglobulins to minimise the occurrence and progression of lung damage.

[Tumor angiogenesis]. / Nádorová angiogeneze.

Angiogenesis have shown a major role in tumor growth and metastasis formation. For tumor growth beyond the size 1-2 mm3, angiogenesis must be started to form vascular supply of tumor cells. Angiogenesis is a complex process, involving degradation of the basement membrane of preexisting vessel, proliferation of endothelial cells towards the angiogenetic stimulus, maturation of endothelial cells with formation of luminized capillary, and finally formation of a functional vessel, surrounded by basement membrane and pericytes. Angiogenesis is regulated by numerous angiogenic and anti-angiogenic factors. Hypoxia is a significant stimulus for angiogenesis. For many cancers the extent of vascularisation is a negative prognostic indicator signifying aggressive disease and increased potential for metastasis.

Pitfalls and limitations of ZAP-70 detection in chronic lymphocytic leukemia.

Zeta-associated protein of 70 kDa (ZAP-70) is a tyrosine kinase that plays a role in signal transduction from the T-cell receptor. ZAP-70 is expressed in normal T-cells and NK-cells. Increased expression of ZAP-70 has been identified in chronic lymphocytic leukemia (CLL). CLL patients with increased ZAP-70 expression have significantly worse prognosis in terms of both progression-free survival and overall survival. There are several methods to quantify ZAP-70: polymerase chain reaction (PCR), immunoblotting, immunohistochemistry, and flow cytometry. Use of flow cytometry for ZAP-70 detection seems to be advantageous as this technique enables us to assess the presence of ZAP-70 separately on CLL clone, T-cells, and NK-cells. On the other hand, detection of ZAP-70 by flow cytometry is substantially influenced by many variables. The principal drawback of flow cytometry is the absence of consensus regarding selection of optimal anti-ZAP-70 antibody, fluorochrome conjugate, the most reliable staining technique, and optimal positivity threshold. This article summarizes pitfalls of flow cytometric analysis of ZAP-70 in CLL.

Angiopoietin-2 mRNA expression is increased in chronic lymphocytic leukemia patients with poor prognostic features.

Several studies have demonstrated the potential prognostic importance of angiogenesis in chronic lymphocytic leukemia (CLL). Elevated expression of angiopoietin-2 (Ang-2), an angiogenic cytokine, was recently reported in CLL. However, data regarding prognostic significance of Ang-2 in CLL are limited. Therefore, we quantitated Ang-2 mRNA in purified mononuclear cells of 33 untreated CLL patients and compared the transcript levels to traditional as well as modern prognostic factors in patients with CLL (clinical stage, disease course, IgVH mutation status, CD38, and ZAP-70 expression). Elevated Ang-2 mRNA concentrations were detected in 12 cases; 21 patients had very low or undetectable levels of Ang-2 transcript. There was significant association between high Ang-2 mRNA levels and unmutated IgVH genes (n=27, P=0.010) and with CD38 expression (n=32, P=0.011), but not with ZAP-70 expression (n=32, P=0.784), Rai stage (n=33, P=0.305) or stable versus progressive clinical course (n=33, P=0.443). There was a trend towards shorter progression-free survival in patients with high Ang-2 expression; however, it did not reach statistical significance (P=0.090). Our pilot data show that Ang-2 mRNA is differentially expressed in patients with CLL and its increased expression appears to be associated with poor prognostic features. Further studies are needed to confirm the results in a larger patient cohort.