Prognostic relevance of angiopoietin-2, fibroblast growth factor-2 and endoglin mRNA expressions in chronic lymphocytic leukemia.

Elevated levels of circulating angiogenic cytokines and increased expression of genes encoding angiogenic factors have been reported in recent years in patients with chronic lymphocytic leukemia (CLL) but data regarding prognostic and predictive significance are still limited. Therefore, in the present study based upon our prior pilot results, we measured mRNA expressions of angiopoietin-2 (Ang-2), fibroblast growth factor-2 (FGF-2) and endoglin (CD105) by reverse transcription quantitative PCR in purified CD19+ cells from 70 untreated CLL patients (median age, 63 years; males, 64%; Rai III/IV stages, 29 %; unmutated IgVH genes, 60 %) and evaluated their possible association with established prognostic factors and clinical course of the disease. Higher expression of Ang-2 was significantly associated with unmutated IgVH genes (n = 55, p = 0.003). Higher CD105 expression was significantly associated with unmutated IgVH genes (n = 55, p < 0.001), high CD38 expression (n = 66, p = 0.022), high ZAP-70 expression (n = 66, p = 0.010), Rai stage I-IV (n = 70, p < 0.001), progressive clinical course of CLL (n = 70, p = 0.001) and shorter time to treatment (n = 70; p < 0.001). Expression of FGF-2 was not significantly associated with any of the prognostic markers. These results indicate that elevated expression of Ang-2 and in particular CD105 by CLL cells is associated with unfavorable prognostic features and clinical outcome; thus, both cytokines appear to play an important role in biology and progression of CLL and warrant further investigation.

[Assessment of selected markers of apoptosis and angiogenesis in chronic lymphocytic leukemia]. / Hodnocení vybraných ukazatelu apoptózy a angiogeneze u chronické lymfocytární leukemie.

INTRODUCTION: Search for new prognostic markers in order to improve prognostic accuracy and predict clinical outcome at the time of dia-gnosis has recently become one of the major trends in chronic lymphocytic leukemia (CLL). PATIENTS AND METHODS, AIM OF STUDY: The aim of our study was assessment of selected markers of apoptosis and angiogenesis and their potential as new prognostic factors. We evaluated serum levels of tumor necrosis factor α (TNFα) and transforming growth factor ß 1 (TGFß1) using commercially available enzyme linked immunosorbent assay; furthermore, we quantified expression of type II receptor for transforming growth factor beta (TGFßRII) and type 2 receptor for fibroblast growth factor 2 (FGFR2) on CLL cells using flow cytometry analysis in 75 previously untreated patients with CLL (47 males and 28 females, median age, 65 years, range 38- 82) and healthy donors. RESULTS: We found significantly elevated TNFα in patients with CLL compared to the control group (p < 0.0001); high expression of TNFα was associated with unfavourable prognosis: significantly higher concentrations were found in patients with Rai highrisk group compared to low and intermediate-risk group (p = 0.0008 and p = 0.0097), with high serum ß2- microglobulin (p = 0.045), massive lymphadenopathy (p = 0.0083), unmutated genes for variable region of immunoglobulin heavy chain (IgVH) (p = 0.041) and unfavourable cytogenetic aberrations (p = 0.0014). In addition, patients with progressive CLL had significantly higher TNFα than those with stable clinical course (p = 0.0009); time to treatment was significantly shorter in patients with higher TNFα (p = 0.0049). Higher TGFß1 concentrations were associated with favourable subgroups: with Rai low  risk group compared to high risk group (p = 0.011), patients without massive lymphadenopathy (p = 0.041), patients with mutated IgVH (p = 0.012) and ZAP 70 negativity (zeta associated protein of 70 kilodaltons) (p = 0.044). Patients with progressive CLL had significantly lower TGFß1 levels than those with stable course (p = 0.0014) and time to treatment was significantly longer in patients with higher TGFß1 (p = 0.016). Patients with Rai high risk group had significantly lower TGFßRII expression than those with low  risk group (p = 0.022). The prognostic significance of FGFR2 was not found. Significant and independent prognostic factors for overall survival were high serum concentrations of TNFα and massive lymphadenopathy (p = 0.036, resp. p = 0.047). CONCLUSION: Based on our results, TNFα and TGFß1 possess prognostic significance in CLL; further research in this direction may also be important therapeutically, because these signal pathways could serve as possible treatment targets.

[Our experience with the treatment of primary lymphomas of the central nervous system]. / Nase zkusenosti s lécbou primárních lymfomu centrálního nervového systému.

INTRODUCTION: The aim of this study was to assess treatment efficiency, overall survival (OS) and identify risk factors with the influence on patients prognosis in patients with primary central nervous system lymphomas (PCNSL) who were treated with intensive chemotherapy based on high-dose methotrexate and cytosin-arabinoside followed by whole-brain radiotherapy (MPV regimen). PATIENTS AND METHODS: From January 1998 to February 2011, 39 patients with PCNSL were diagnosed on our department. The median from the first clinical symptomatology to histological diagnosis was 4 weeks (range, 2-19). Thirty-seven patients were treated with MPV regimen. RESULTS: The therapeutic response was evaluated in 35 patients (2 patients died early during treatment). The overall response/complete remission rate was 63/60%. At the time of analysis (november 2011), the median of follow-up was 16,5 months; 31 patients died (the most often causes of death were poor treatment effect and treatment complications). The 2-year OS was 30% and median PFS and OS were 9 and 12 months. Patients with WHO performance status 0-1 and those with normal serum lactate dehydrogenase serum had significantly longer OS (p = 0.0495 and p = 0.0232). CONCLUSION: The treatment results of our patients appear to be inferior than data from literature. The reason is probably high occurrence of negative prognostic factors. Early diagnosis and intensive treatment are crucial for improvement of prognosis.

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.

Antibodies against Pneumococcal Capsular Polysaccharides and Natural Anti-Galactosyl (Alpha-Gal) in Patients with Humoral Immunodeficiencies.

Humoral deficiencies represent a broad group of disorders. The aim of the study was to compare the levels of antibodies against pneumococcal capsular polysaccharides (anti-PCP) and natural anti-galactosyl (anti-Gal) antibodies in (1) patients with chronic lymphocytic leukaemia (CLL), (2) patients with common variable immunodeficiency (CVID), and (3) a healthy population and to explore their diagnostic and prognostic potential. Serum immunoglobulin levels and levels of anti-Gal IgG, IgA, and IgM and anti-PCP IgG and IgG2 were determined in 59 CLL patients, 30 CVID patients, and 67 healthy controls. Levels of IgG, IgA, IgM, anti-Gal IgA, anti-Gal IgM, and anti-PCP IgA were lower in CLL and CVID patients than in healthy controls (p value for all parameters < 0.0001). Decrease in the levels of IgA, IgM, anti-Gal IgA, and anti-PCP IgA was less pronounced in the CLL group than in the CVID group. IgA decline, anti-Gal IgA, anti-PCP IgA, and anti-PCP IgG2 were negatively correlated with CLL stage. We devise the evaluation of anti-Gal antibodies to be a routine test in humoral immunodeficiency diagnostics, even in cases of immunoglobulin substitution therapy. Significant reductions, mainly in anti-Gal IgA, IgM, and anti-PCP IgA levels, may have prognostic importance in CLL patients.

Venous thromboembolism in patients with chronic lymphocytic leukemia.

INTRODUCTION: Venous thromboembolism (VTE) is a major cause of morbidity and mortality in patients (pts) with malignant tumors. Increased risk of VTE is well described in a variety of hematologic malignancies; however, data regarding VTE in chronic lymphocytic leukemia (CLL) is very limited. PATIENTS AND METHODS: We retrospectively analyzed clinical and laboratory data of 346 consecutive pts with CLL followed up at 4th Department of Internal Medicine - Hematology, University Hospital, Hradec Kralove, Czech Republic, diagnosed between 1999 and 2011 (males, 64%; median age, 64 years; low/intermediate/high Rai modified risk in 41/47/12%). RESULTS: After a median follow-up of 72 months (range, 26-138), at least one episode of VTE occurred in 38 patients (11%). VTE developed after a median of 34 months from CLL diagnosis. Incidence of VTE was 1.67% per patient year of follow-up. There was a high proportion of unfavourable prognostic factors (advanced Rai stages, unmutated IgVH genes, unfavourable cytogenetics) in pts with VTE. The presence of 0/1/2/3 additional risk factors for VTE was identified in 2/16/14/6 patients. The most common risk factors for VTE besides age (n=24) were corticosteroid therapy (n=13), other malignancies (n=9) and obesity (n=7). Recurrence of VTE was diagnosed in 7 pts. Performance status ≥ 2 and inherited thrombophilia were significant risk factors for VTE development in univariate and multivariate analysis. VTE was not associated with shorter overall survival. CONCLUSION: Based on our results, VTE is a relatively frequent complication in patients with CLL. Although most patients had other known risk factors for VTE including CLL treatment, 29% had no risk factors or only age ≥ 60 years. These findings demonstrate the possible role of CLL in the development of VTE.