Modification of immunocytochemical ZAP-70 assay for potential clinical application in B-cell chronic lymphocytic leukemia

The ZAP-70 protein is a member of the Syk/ZAP protein tyrosine kinase family, normally expressed in T cells and NK cells but not found in normal, mature B cells. The protein plays a critical role in the initiation of T-cell signaling. Leukemic cells from patients with B-cell chronic lymphocytic leukemia (B-CLL) that expressed nonmutated immunoglobulin V genes were found to express levels of ZAP-70 protein that were comparable to those detected in T cells of healthy adults. The ZAP-70 protein expression can be evaluated by flow cytometry and may be used as a prognostic marker in B-CLL patients. We modified the method of immunocytochemical assessment of ZAP-70 expression. The traditional two-step method with monoclonal anti-ZAP-70 antibody in the first step followed by FITC-conjugated goat anti-mouse IgG was changed for one-step method with monoclonal anti-ZAP-70 antibody labeled by Zenon Alexa Fluor 488. The method is simple and fast. The major advantage of Zenon labeling technique is its compatibility with simultaneous staining of surface antigens. The cells may be earlier immunostained for CD3, CD19 and/or CD5 to compare of the ZAP-70 kinase expression in B and T cells

Przezskórne zamknięcie uszka lewego przedsionka jako leczenie z wyboru u pacjentów z migotaniem przedsionków oraz przeciwwskazaniami do leczenia przeciwkrzepliwego — przypadek kliniczny

The use of anticoagulants in the management of cardiac arrhythmias, such as atrial fibrillation, is associated with increased risk of bleeding, especially gastrointestinal. According to guidelines of the European Society of Cardiology, for patients in whom oral anticoagulation is contraindicated, left atrial appendage closure should be considered. A case of 78-year-old male patient presents both thromboembolic (stroke) and haemorrhagic complications, which were the indications to surgical treatment.Leczenie przeciwkrzepliwe stosowane w zaburzeniach rytmu serca, takich jak migotanie przedsionków wiąże się z ryzykiem istotnego krwawienia, głównie do przewodu pokarmowego. Według zaleceń Europejskiego Towarzystwa Kardiologicznego, u pacjentów z przeciwwskazaniami do doustnej antykoagulacji należy rozważyć zabieg przezskórnego zamknięcia uszka lewego przedsionka. Opisany przypadek 78-letniego mężczyzny to przykład występowania powikłań zakrzepowo-zatorowych (udar mózgu) oraz powikłań krwotocznych, dlatego też pacjenta zakwalifikowano do leczenia zabiegowego

Zatorowość płucna czy ostry zespół wieńcowy — trudności diagnostyczne

Zatorowość płucna (PE) to zagadnienie, które mimo dostępności badań obrazowych oraz laboratoryjnych pozostaje trudnym problemem diagnostycznym. Wystąpienie choroby wiąże się ze zwiększonym ryzykiem wczesnego zgonu, dlatego istotne jest wczesne zastosowanie odpowiedniego leczenia. W przebiegu PE pojawiają się typowe zmiany w zapisie elektrokardiograficznym, jednak część z nich budzi wątpliwości diagnostyczne i wymaga różnicowania z innymi stanami ostrymi, takimi jak zawał serca. Przedstawiony przypadek to przykład występowania objawów sugerujących początkowo ostry zespół wieńcowy, jednak dalsza analiza doprowadziła do rozpoznania PE jako przyczyny dolegliwości

Zatorowość płucna czy ostry zespół wieńcowy — trudności diagnostyczne

Despite the availability of imaging and laboratory tests, pulmonary embolism remains a difficult diagnostic problem. The disease is associated with an increased risk of an early death and for this reason, it requires prompt treatment. There are some ECG changes that are typical for pulmonary embolism, but some of them need to be differentiated with other acute conditions, such as myocardial infarction. This case report presents a patient with symptoms suggesting acute coronary syndrome, finally diagnosed with pulmonary embolism.Zatorowość płucna (PE) to zagadnienie, które mimo dostępności badań obrazowych oraz laboratoryjnych pozostaje trudnym problemem diagnostycznym. Wystąpienie choroby wiąże się ze zwiększonym ryzykiem wczesnego zgonu, dlatego istotne jest wczesne zastosowanie odpowiedniego leczenia. W przebiegu PE pojawiają się typowe zmiany w zapisie elektrokardiograficznym, jednak część z nich budzi wątpliwości diagnostyczne i wymaga różnicowania z innymi stanami ostrymi, takimi jak zawał serca. Przedstawiony przypadek to przykład występowania objawów sugerujących początkowo ostry zespół wieńcowy, jednak dalsza analiza doprowadziła do rozpoznania PE jako przyczyny dolegliwości

Expression of HLA-G in patients with B-cell chronic lymphocytic leukemia (B-CLL).

The expression of HLA-G was reported in certain malignancies and its role in escaping from immunosurveillance in cancers was proposed since HLA-G is a nonconventional HLA class I molecule that protects fetus from immunorecognition during pregnancy. Recent studies proposed HLA-G as novel prognostic marker for patients with B-CLL. HLA-G was showed to bear even better prognostic information compared to Zeta-chain associated protein of 70kDa (ZAP-70) and CD38 although some other authors did not find HLA-G expression in CLL. Therefore in this study we characterized the expression of HLA-G on both RNA and protein level. In most of 20 B-CLL patients we were able to detect signal from HLA-G using flow cytometry analysis. The expression of HLA-G was confirmed on messenger level by real-time RT-PCR experiments. No correlation between HLA-G expression and expression of well established prognostic factors such as ZAP-70 and CD38 was detected. These results confirm that HLA-G is expressed on CLL leukemic cells. Furthermore the expression of HLA-G on CLL cells suggests that this molecule might be involved in escaping of CLL cells from immunosurveillance

Upper Respiratory Tract Colonization by Gram-Negative Rods in Patients with Chronic Lymphocytic Leukemia: Analysis of Risk Factors

The aim of the study was to assess the frequency and predisposing factors of colonization of upper respiratory tract by Gram-negative rods (GNRs) in chronic lymphocytic leukemia (CLL) patients. Antimicrobial susceptibility of the isolated strains was determined. A significantly higher frequency of GNR colonization in CLL patients was observed (36.7%) in comparison to healthy volunteers (8.3%). GNR isolates mainly belonged to the Enterobacteriaceae family. Three isolates of GNR demonstrating presence of AmpC β-lactamases and one ESBL-producing strain were obtained from CLL patients. GNR colonization rate was higher among CLL patients with lower level of IgG in serum (P = 0.017), with higher number of neutrophils (P = 0.039) or higher number of lymphocytes in serum (P = 0.053). The longer the time elapsed since diagnosis, the higher the frequency of GNR colonization observed. Multivariate analysis showed importance of the Rai stage, number, and type of infections as independent predictors of GNR colonization in CLL patients

ZAP-70 and CD38 expression are independent prognostic factors in patients with B-cell chronic lymphocytic leukaemia and combined analysis improves their predictive value.

Recently identified biological risk factors in B-cell chronic lymphocytic leukemia (B-CLL) include ZAP-70 and CD38 expression. The present study was conducted to clarify whether a combined analysis could improve predictive impact of these two parameters. We examined the expression of ZAP-70 and CD38 by flow cytometry method in 217 newly diagnosed, consecutive, unselected and well characterized B-CLL patients in relation to laboratory parameters and clinical outcome. We confirmed that both ZAP-70 as well as CD38 were independent of prognostic factors. There was a significant correlation between the percentage of leukemic cells positive for ZAP-70 and the percentage of CD38+CD19+ cells (R=0.629; p=0.000001). Combined analysis of ZAP-70 and CD38 showed concordant results in 158/217 patients (72.8%), while in 59 patients the results were discordant (27.2%). A mean treatment free survival (TFS) was the longest in ZAP-70-CD38-patients (45.6 months, comparing to 13.6 months in ZAP-70+CD38+ group). Also a mean overall survival was the longest in ZAP-70-CD38- patients (224.7 months compared to 77.9 months in ZAP-70+CD38+ patients)

Assessment of the pathway of apoptosis involving PAR-4, DAXX and ZIPK proteins in CLL patients and its relationship with the principal prognostic factors

Par-4 (prostate apoptosis response-4) protein was originally found upregulated in prostate tumor cells undergoing apoptosis. Then it was further identified as a proapoptotic protein upregulated both in normal and leukemic lymphocytes. The aim of our study was to assess PAR-4 protein expression in the B cells of CLL patients and to examine its relationship with the expression of other proteins involved in the apoptosis process, such as DAXX, ZIPK and BCL-2. We found a positive relationship between PAR-4 and BCL-2 protein expression. Additionally, there was a positive correlation between PAR-4 and both DAXX and ZIPK protein expression. The results of our research were also analyzed in association with the principal CLL prognostic factors. There was a positive correlation between the expression of PAR-4 protein and the lactate dehydrogenase (LDH) serum concentration (p < 0.005). The expression of PAR-4 protein in B cells correlated positively with the percentage of CD38+ cells (p < 0.05), as well as with CD38+/ZAP-70+ cells (p < 0.05). Moreover, we found a close relationship between LPL protein expression or LPL/ADAM29 MFI ratio and PAR-4 protein expression. Our results confirm the significance of apoptosis deregulation in CLL, and suggest a possible relationship between PAR-4 expression and the clinical course of the disease. This however requires further investigation. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 1, pp. 98&#8211;103

CD3+/CD16+CD56+ cell numbers in peripheral blood are correlated with higher tumor burden in patients with diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma is the commonest histological type of malignant lymphoma, andremains incurable in many cases. Developing more efficient immunotherapy strategies will require betterunderstanding of the disorders of immune responses in cancer patients. NKT (natural killer-like T) cells wereoriginally described as a unique population of T cells with the co-expression of NK cell markers. Apart fromtheir role in protecting against microbial pathogens and controlling autoimmune diseases, NKT cells havebeen recently revealed as one of the key players in the immune responses against tumors. The objective of thisstudy was to evaluate the frequency of CD3+/CD16+CD56+ cells in the peripheral blood of 28 diffuse largeB-cell lymphoma (DLBCL) patients in correlation with clinical and laboratory parameters. Median percentagesof CD3+/CD16+CD56+ were significantly lower in patients with DLBCL compared to healthy donors(7.37% vs. 9.01%, p = 0.01; 4.60% vs. 5.81%, p = 0.03), although there were no differences in absolute counts.The frequency and the absolute numbers of CD3+/CD16+CD56+ cells were lower in advanced clinical stagesthan in earlier ones. The median percentage of CD3+/CD16+CD56+ cells in patients in Ann Arbor stages 1–2 was5.55% vs. 3.15% in stages 3–4 (p = 0.02), with median absolute counts respectively 0.26 G/L vs. 0.41 G/L (p == 0.02). The percentage and absolute numbers of CD3+/CD16+CD56+ cells were significantly higher in DL-BCL patients without B-symptoms compared to the patients with B-symptoms, (5.51% vs. 2.46%, p = 0.04;0.21 G/L vs. 0.44 G/L, p = 0.04). The percentage of CD3+/CD16+CD56+ cells correlated adversely with serumlactate dehydrogenase (R= –445; p < 0.05) which might influence NKT count. These figures suggest a relationshipbetween higher tumor burden and more aggressive disease and decreased NKT numbers. But it remains tobe explained whether low NKT cell counts in the peripheral blood of patients with DLBCL are the result of theirsuppression by the tumor cells, or their migration to affected lymph nodes or organs