Circulating proangiogenic cytokines and angiogenesis inhibitor endostatin in untreated patients with chronic lymphocytic leukemia

The serum concentration of two pro-angiogenic cytokines: basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF-beta1), and anti-angiogenic factor endostatin in the serum of 80 never treated B-cell chronic lymphocytic leukemia (CLL) patients and 27 healthy volunteers was measured using an enzyme linked immunosorbent assay. The serum levels of both bFGF and TGF-beta1 were found to be significantly higher in the CLL group (median 40.5 pg/ml and 38.6 ng/ml respectively) when compared to the control group (median 9.4 pg/ml and 18.9 ng/ml, respectively) (p<0.001). The levels of endostatin were not significantly different in CLL and control groups (median 12.3 ng/ml and 8.4 ng/ml, respectively) (p=0.09). In the group of CLL patients the level of bFGF was significantly higher in patients with progressive disease as compared with patients with stable disease (median 90.5 pg/ml and 40.5 pg/ml respectively) (p<0.001). Patients in Rai stage III and IV also had significantly higher levels of bFGF than patients in Rai stage 0-II (median 100.1 pg/ml and 29.3 pg/ml respectively) (p<0.001). The levels of both TGF-beta1 and endostatin were lower in patients in Rai stage III and IV (median 28.9 ng/ml and 9.1 ng/ml respectively) than in patients in Rai stage 0-II (42.8 ng/ml and 13.1 ng/ml respectively) (p<0.001 and p=0.002 respectively). The level of endostatin was also lower in the group of CLL patients with progressive disease (median 10.0 ng/ml) as compared to patients with stable disease (median 20.5 ng/ml) (p=0.008). In conclusion, the disturbance in the balance between pro- and anti-angiogenic factors may have an important influence on the course of CLL.     

Serum levels of IL-6 type cytokines and soluble IL-6 receptors in active B-cell chronic lymphocytic leukemia and in cladribine induced remission

We have investigated the serum concentrations of interleukin-6 (IL-6) and two IL-6 family cytokines-oncostatin M (OSM) and leukemia inhibitory factor (LIF)-in 63 patients with B-cell chronic lymphocytic leukemia (B-CLL) and 17 healthy controls using the enzyme-linked immunosorbent assay (ELISA) method. Simultaneously, we measured the serum levels of the soluble forms of two subunits of the IL-6 receptor complex-ligand binding glycoprotein 80 (sIL-6R) and glycoprotein 130 (sgp130). The cytokines and receptors were evaluated in 25 untreated patients and 38 patients treated with cladribine (2-CdA), as well as in 17 healthy controls. We have correlated the serum levels of these proteins with Rai's clinical stage of the disease, the response to 2-CdA treatment and some hematological parameters. We have also evaluated the correlation of the IL-6 serum level with the concentration of OSM and IL-6 soluble receptors. IL-6 was measurable in 62/63 (98.4%), OSM in 20/25 (80%) of untreated and 14/38 (37.8%) of the treated patients. sIL-6R and sgp130 were detectable in all 63 patients and LIF in none of the CLL patients. IL-6 serum level in untreated patients was not significantly different as compared to its concentration in the control group (P>0.05). However, in the patients treated with 2-CdA the IL-6 level was significantly lower (P<0.02), and the lowest concentration was found in the patients with complete remission (CR; median 1.4pg/ml; P<0.02). The concentration of sIL-6R was significantly higher in untreated (median 61.8 ng/ml) and treated (median 50.1 ng/ml) CLL patients when compared to normal persons (median 41.2 ng/ml; P=0.04; P<0.001, respectively). There was no difference between the sIL-6R levels in the patients with CR and the healthy controls. In non-responders sIL-6R concentration was the highest and similar to its level in the untreated patients. OSM level was higher in the untreated patients (median 1.8pg/ml) than in the normal controls (median 0.0pg/ml; P<0.001) and in the CR patients (median 0.0pg/ml; P<0.03). The serum concentration of sgp130 was similar in the untreated (median 480 pg/ml) and treated (median 470 pg/ml) patients, as well as in the healthy persons (median 420 pg/ml; P>0.05). We have found significant positive correlation between the levels of sIL-6R and the lymphocytes count in CLL patients (p=0.423; P<0.001). In addition, sIL-6R and OSM serum concentrations correlated also with CLL Rai stage. In conclusion, the serum level of IL-6, OSM and sIL-6R, but not LIF and sgp130, are useful indicators of CLL activity.     

Serum vascular endothelial growth factor and its receptor level in patients with chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a disorder which encompasses not only morphological changes in parenchyma, central and peripheral airways but also in structural and functional changes of pulmonary vessels. The role of angiogenic factors leading to abnormal pulmonary vessel remodeling remains unclear. We have investigated a cytokine vascular endothelial growth factor (VEGF) known to be involved in angiogenesis, and its soluble receptors (sVEGF R1, sVEGF R2) in the serum of 20 patients with mild COPD and 10 patients with very severe COPD, using sensitive enzyme-linked immunoassays. The control group consisted of 10 healthy volunteers. We found that the concentration of VEGF in the serum of patients with mild COPD was significantly higher (665.31 +/- 102.20 pg/mL) in comparison to the control group (318.94 +/- 51.40 pg/mL; p < 0.05), and there was a strong negative correlation with FEV1 (r = -0.859; p < 0.001). Additionally, the level of sVEGF R1 in the serum of patients with very severe COPD was also significantly higher (96.60 +/- 26.85 pg/mL) than in the control (36.01 +/- 3.29 pg/mL; p < 0.05) and a positive correlation between the serum level of sVEGF R1 and FEV1 was found (r = 0.748; p < 0.01). Moreover, we observed an insignificant increase of sVEGF R2 in the serum of patients with mild COPD and those with very severe COPD. These results suggest that VEGF and sVEGF R1 receptor are involved in the development of abnormal pulmonary vascular remodelling in patients with COPD.     

Circulating angiogenic cytokines in multiple myeloma and related disorders

We investigated the serum concentrations of selected angiogenic cytokines including: vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), transforming growth factor beta 1 (TGF-beta1) and basic fibroblast growth factor (bFGF) in 162 patients with multiple myeloma (MM), 5 patients with Waldenstr m's macroglobulinaemia (WM), and 31 healthy controls. Among the MM patients there were 2 cases of primary plasma cell leukemia (PCL) and one case of extramedullary plasmacytoma. The levels of measured cytokines were correlated with the phase and stage of the disease as well as the most important clinical and laboratory parameters associated with disease activity (haemoglobin, creatinine, albumins, calcium, M-component, CRP,beta2m, LDH and bone involvement). We have found correlations between serum levels of angiogenic cytokines and some parameters depicting the disease activity and advancement. The serum level of VEGF in MM patients (median 244.5 pg/mL) correlated with serum concentrations of beta-2-microglobulin (beta2m) greater than 2.5 mg/L (p = 0.0005) and abnormal values of lactate dehydrogenase (> 425 U/L, median 329.0 pg/mL and < 210 U/L, median 426.6 pg/mL, p = 0.004 and p = 0.04 respectively). MM patients in stage III had higher serum levels of HGF (median 1 411.3 pg/mL) than those in stage I (median 1 219 pg/mL) (p = 0.01) according to Durie and Salmon staging, and those in phase I (at diagnosis) (median 1 555.6 pg/mL) and phase III (in progression) (median 1 309.7 pg/mL) had higher levels than those in phase II (plateau phase) (median 1 047.9 pg/mL) (p = 0.002 and p = 0.02 respectively). Significantly elevated values of HGF were found in MM patients with anaemia (median 1 962.0 pg/mL) and hypercalcaemia (median 2 085.6 pg/mL) (p = 0.00001 and 0.04 respectively). TGF-beta1 (median 33.9 ng/mL) correlated positively with highbeta2m values (> 2.5 mg/L) (p = 0.04) and was significantly higher in phase I (median 40.1 ng/mL) than in phase II (median 30.9 ng/mL) (p = 0.03) of the disease. The concentration of bFGF was significantly higher in stage III of MM (median 3.1 pg/mL) than in stage I (median 1.2 pg/mL) (p = 0.04). We found that the survival probability was statistically higher for newly diagnosed MM patients with a concentration of VEGF lower than the median value for this cytokine. The concentrations of the cytokines analyzed in patients with Waldenstr m's macroglobulinaemia (WM), primary plasma cell leukaemia (PCL) and non-secretory (NS) myeloma were not distinguishable from those found in MM patients. We also studied the relationship between the levels of cytokines analyzed and found positive correlations between bFGF and TGF-beta1 (rh? = 0.183, p < 0.02), as well as VEGF and TGF-beta 1 (rh? = 0.537, p < 0.001) and VEGF and bFGF (rh? = 0.197, p < 0.02). In conclusion, our data indicate a strong relationship between angiogenic cytokine serum levels and clinical course as well as selected laboratory parameters of patients with MM.     

Circulating proangiogenic molecules PIGF, SDF-1 and sVCAM-1 in patients with systemic lupus erythematosus

Serum concentrations of three angiogenic cytokines: vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1) and placental growth factor (PIGF) and soluble vascular cell adhesion molecule 1 (sVCAM-1), were investigated in the serum of 61 patients with systemic lupus erythematosus (SLE) and 20 healthy subjects. The possible association between serum levels of these proteins and SLE activity, as well as correlation between the concentrations of cytokines were also analysed. All of these factors were detectable in all SLE patients and the healthy control group. The median concentration of VEGF was higher in active SLE (386 pg/mL) than in inactive disease (327 pg/mL) or in the control group (212 pg/mL, p<0.004). The median serum level of SDF-1 was higher in SLE patients (1,814 pg/mL) than in the control group (1,507 pg/mL, p<0.02). The median concentration of PIGF was higher (14 pg/mL) in SLE patients than in the control group (12 pg/mL, p=0.03), and particularly in active disease (17 pg/mL) as compared to the inactive phase (13 pg/mL, p=0.01). The correlations between the levels of cytokines examined and clinical features, laboratory abnormalities and the type of treatment were also analysed. We found a positive correlation between serum concentrations of PIGF and SLE activity according to SLAM score (p=0.33, p=0.13).     

Identification of a global gene expression signature of B-chronic lymphocytic leukemia

B-chronic lymphocytic leukemia (B-CLL) is an adult-onset leukemia characterized by significant accumulation of apoptosis-resistant monoclonal B lymphocytes. In this study, we performed gene expression profiling on B cells obtained from 10 healthy age-matched individuals and CLL B cells from 38 B-CLL patients to identify key genetic differences between CLL and normal B cells. In addition, we leveraged recent independent studies to assess the reproducibility of our molecular B-CLL signature. We used a novel combination of several methods of data analysis including our own software and identified 70 previously unreported genes that differentiate leukemic cells from normal B cells, as well as confirmed recently reported B-CLL specific expression levels of an additional 10 genes. Importantly, many of these genes have previously been linked with other cancers, thus lending further support to their importance as candidate genes leading to B-CLL pathogenesis. We have also validated a subset of these genes using independent methodologies. Moreover, we show that our genes can be used to create a diagnostics signature that performs with perfect sensitivity and specificity in an independent cohort of 21 B-CLL and 20 normal subjects, thus strongly validating the informative nature of our set of genes. Finally, we identified a group of 31 genes that distinguish between low (Rai stage 0) and high (Rai stage 4) risk patients, suggesting that there may also be a gene expression signature that associates with disease progression.     

The effect of venous thromboembolism on survival of cancer patients and its relationship with serum levels of factor VIII and vascular endothelial growth factor: a prospective matched-paired study

BACKGROUND: Venous thromboembolism (VT) increases mortality and morbidity in cancer patients. The primary aim of this study was to evaluate the effect of VT on the survival of cancer patients and its relationship with serum vascular endothelial growth factor (VEGF) and plasma factor VIII levels. PATIENTS AND METHODS: Eighty-two patients with locally advanced or metastatic cancer were included in this study between September 2001 and March 2004, and 31 of them had VT. Fifty-one matched-paired cancer patients without VT were prospectively selected as a control group in the same period. Criteria for the selection of control group patients were having the same malignancy, stage, metastatic site, performance status and age (+/-5 years) as patients in the VT group. RESULTS: Plasma factor VIII and serum D-dimer levels in the VT group were significantly higher than those in the control group (p=0.030 and p=0.016, respectively). However, mean serum VEGF levels were similar in both groups (p=0.199). In the VT group, the median survival of patients who had higher serum VEGF levels (>150 pg/mL) was significantly shorter than that of patients in the same group with lower serum VEGF levels (p=0.005). The median survival of the VT group was 14 months, whereas it was 25 months in the control group (p=0.199). CONCLUSION: There was a worse prognostic trend for cancer patients with VT. Nevertheless, the difference in survival was not statistically significant between the groups. Plasma factor VIII and serum D-dimer levels might have prognostic value in cancer patients with VT. Cancer patients with VT and higher serum VEGF levels had a significantly poorer prognosis.     

Serum levels of angiogenic cytokines in systemic lupus erythematosus and their correlation with disease activity

We investigated the serum concentration of hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF-beta1) using an enzyme-linked immunosorbent assay (ELISA) in a group of 60 patients with systemic lupus erythematosus (SLE), and 20 healthy controls. We also examined the possible association between the serum concentrations of these factors and certain clinical, laboratory parameters and SLE activity. HGF, VEGF and TGF-beta1 were detectable in all patients with SLE, and in all normal individuals. bFGF was measurable in 70% of the patients with SLE and in 65% of the healthy controls. The HGF level was higher in active SLE (median 1,019.5pg/ml) than in inactive SLE (median 787.8 pg/ml) (p < 0.005) or in the control group (median 847.0 pg/ml) (p < 0.009). The level of VEGF in active SLE was also higher (203.5 pg/ml) than in inactive disease (116.1 pg/ml) (p < 0.05) or in healthy persons (133.5 pg/ml) (p < 0.04). The levels of bFGF and TGF-beta1 were similar for both the active and inactive SLE, and the control group (p > 0.05). We found a significant, positive correlation between the levels of HGF and bFGF (r = 0.268, p < 0.04), HGF and TGF-beta1 (r = 0.365, p < 0.005) and HGF and VEGF (r = 0.327, p < 0.02) as well as VEGF and TGF-beta1 (r = 0.543, p < 0.001). We found a positive correlation between VEGF serum levels and platelet counts (r = 0.272, p < 0.04), and the TGF-beta1 concentration and platelet count (r = 0.313; p < 0.02). There was also a positive correlation between HGF serum concentration and the SLE activity score (r = 0.435, p < 0.001), as well as between the level of VEGF and SLE activity (r = 0.252, p = 0.05). In conclusion, serum levels of the angiogenic factors HGF and VEGF may be relevant in SLE pathogenesis. Their concentrations seem to be markers of SLE activity.     

Vascular endothelial growth factor and its soluble receptors VEGFR-1 and VEGFR-2 in the serum of patients with systemic lupus erythematosus

We investigated the serum concentration of vascular endothelial growth factor (VEGF) and its two soluble receptors, sVEGFR-1 and sVEGFR-2, in a group of 60 patients with systemic lupus erythematosus (SLE), and 20 healthy controls, using an enzyme-linked immunosorbent assay. We examined a possible association between serum levels of these proteins and certain clinical and laboratory parameters as well as SLE activity. VEGF, sVEGFR-1 and sVEGFR-2 were detectable in all patients with SLE and in all normal individuals. The VEGF level was higher in active SLE (mean, 300.8 pg/ml) than in inactive SLE (mean, 165.9 pg/ml) (p < 0.05) or in the control group (mean, 124.7 pg/ml) (p < 0.04). The highest sVEGFR-1 concentrations were also detected in active SLE patients (mean, 42.2 pg/ml) and the lowest in inactive disease (mean, 32.0 pg/ml) (p < 0.01). In contrast, the levels of sVEGFR-2 were lower in SLE (mean, 12557.6 pg/ml) than in the control group (mean, 15025.3 pg/ml) (p < 0.05). We found a positive correlation between sVEGFR-1 concentration and the SLE activity score p = 0.375 (p < 0.004) and a negative, but statistically insignificant correlation between sVEGFR-2 and SLE activity (p = -0.190, p > 0.05). Treatment with steroids and cytotoxic agents did not influence VEGF or its soluble receptors levels. In conclusion, in SLE patients the levels of VEGF and sVEGFR-1 are higher in patients with active SLE than in inactive disease or healthy persons. In contrast, the level of sVEGFR-2 is lower in active SLE than in inactive disease. The imbalance between VEGF and its soluble receptors may be important in SLE pathogenesis.     

Circulating VEGF and its soluble receptors sVEGFR-1 and sVEGFR-2 in patients with acute leukemia

Angiogenesis plays an important role in the pathogenesis of acute leukemia, and vascular endothelial growth factor (VEGF) is a crucial, positive regulator of this process. The biological activity of VEGF is mediated by two different receptor tyrosine kinases: VEGFR-2 and VEGFR-1. The soluble form of VEGFR-1 is likely to be a negative regulator of VEGF availability, but the physiological role of sVEGFR-2 is still unclear. The plasma levels of sVEGFR-1 and sVEGFR-2 in patients with acute leukemia have not been investigated. We measured the plasma concentrations of VEGF and its two soluble receptors in 39 AML and 15 ALL patients as well as in the control group, using the ELISA assay. We also correlated the plasma levels of these proteins with disease status and known prognostic factors. The sVEGFR-1 level was significantly higher in patients with AML and ALL than in the healthy subjects (p < 0.002 and p < 0.03 respectively). The sVEGFR-2 level was significantly higher in AML patients compared with the control group (p < 0.03). The VEGF levels in AML and ALL patients and in healthy subjects did not differ significantly. The sVEGFR-1 level was higher in AML patients with > 50% of blasts in the bone marrow (BM), WBC > 20 G/L and elevated LDH level, than in the group with BM blasts < 50% (p < 0.01), WBC < 20 G/L (p < 0.02) and a normal LDH level (p < 0.05). Positive correlations between sVEGFR-1 level and WBC (p < 0.02),% of BM blasts (p < 0.05), the absolute blast count in peripheral blood (ABC) (p < 0.009) and LDH (p < 0.000001) were found. The sVEGFR-1/VEGF ratio (R1) was calculated, and a positive correlation between R1 and ABC in AML (p < 0.03) was determined. A higher (above median) sVEGFR-1/VEGF ratio correlated with a lower CR rate and a shorter survival (p < 0.03 and p = 0.0007 respectively). In conclusion, the plasma concentration of sVEGFR-1 is higher in leukemia patients than in healthy subjects and correlates with tumour burden and poor prognosis. The sVEGFR-1/VEGF ratio may be of greater prognostic value than VEGF alone. Further investigation is recommended to better determine their function.     

Identification of VEGF receptor-2 tyrosine phosphorylation sites involved in VEGF-mediated endothelial platelet-activating factor synthesis

Vascular endothelial growth factor (VEGF)-mediated inflammation requires the synthesis of acute platelet-activating factor (PAF) by endothelial cells (ECs). We previously reported that VEGF-mediated PAF synthesis involves the activation of the homodimeric tyrosine kinase receptor VEGFR-2/R-2, leading to the recruitment of p38 and p42/p44 mitogen-activated protein kinases (MAPKs) and activation of secreted group V phospholipase A? (sPLA?-V). We have also reported that VEGF-A???-mediated prostacyclin (PGI?) synthesis requires VEGFR-1/R-2 heterodimeric receptor activation. Selective activation of VEGF receptors can coordinate the synthesis of pro-PAF and anti-PGI? inflammatory factors. It is unknown which VEGFR-2 tyrosine phosphorylation site(s) contribute(s) to PAF synthesis. Bovine aortic endothelial cells (BAECs) were transfected with pcDNA vectors encoding for native VEGF receptor-2 (VEGFR-2) cDNA or VEGFR-2 cDNA containing tyrosine phosphorylation sites mutated into phenylalanine residues (Y801F, Y1059F, Y1175F, Y1214F); an empty pcDNA vector was used as a negative control. Treatment of pcDNA-transfected BAECs with VEGF (10?? mol/L) for 15 min increased PAF synthesis by 180%. In BAECs transfected with pcDNA vectors encoding mutated Y801F, Y1059F, Y1175F, or Y1214F VEGFR-2 cDNA, we observed a marked reduction of VEGF-mediated PAF synthesis by 38%, 46%, 69%, and 31%, respectively, compared with BAECs transfected with pcDNA vector encoding VEGFR-2 cDNA. Our data provide a novel insight as to the mechanisms by which VEGF promotes PAF synthesis.     

Clinical relevance of serum angiogenic activity in patients with transitional cell carcinoma of the bladder

Angiogenesis is essential for tumor growth and progression and is mediated by positive and negative regulators of vessel growth. Since angiogenic mediators found in patient serum have been postulated to reflect the angiogenic potential of a malignant tumor, we investigated the angiogenic activity in the serum of patients with transitional cell carcinoma (TCC). The data were correlated to tumor characteristics and the clinical course of the patients. Eighty-one patients with transitional cell carcinoma and 53 control persons were included in the study. Preoperative serum samples were collected and both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were quantified by ELISA. Additionally, the serum evoked proliferative activity on human umbilical vein endothelial cells (HUVEC) was evaluated. Data were compared to the clinical course of the patients. Serum of tumor patients significantly enhanced the proliferative capacity of HUVEC, compared to cells grown in standard culture medium (p = 0.0032), but not when compared to serum from control persons. Serum from patients with superficial TCC and well differentiated tumors induced a significantly higher angiogenic response (ANG(hi)) than serum from patients with poorly differentiated and invasive carcinomas (ANG(lo); p = 0.037). VEGF level of ANG(hi) serum was 384.22 +/- 247.76 pg/ml (n = 37) which significantly differed from mean VEGF level detected in ANG(lo) serum (247.72 +/- 211.93 pg/ml, n = 42; p = 0.019). Similarly, mean bFGF levels were 9.58 +/- 5.91 pg/ml in ANG(hi) serum versus 5.74 +/- 3.52 pg/ml) in ANG(lo) serum (p = 0.0043). A negative correlation was established between VEGF/bFGF serum concentration and patient prognosis. The experiments demonstrate a positive correlation between VEGF and bFGF serum level and endothelial proliferation in vitro. The inverse relationship between angiogenic activity and tumor stage might disclose information about angiogenesis and tumor progression in TCC.     

Alterations in TP53, cyclin D2, c-Myc, p21WAF1/CIP1 and p27KIP1 expression associated with progression in B-CLL

B-cell chronic lymphocytic leukaemia (B-CLL) originates from B lymphocytes that may differ in the activation level, maturation state or cellular subgroups in peripheral blood. Tumour progression in CLL B cells seems to result in gradual accumulation of the clone of resting B lymphocytes in the early phases (G0/G1) of the cell cycle. The G1 phase is impaired in B-CLL. We investigated the gene expression of five key cell cycle regulators: TP 53, c-Myc, cyclin D2, p21WAF1/CIP1 and p27KIP1, which primarily regulate the G1 phase of the cell cycle, or S-phase entry and ultimately control the proliferation and cell growth as well as their role in B-CLL progression. The study was conducted in peripheral blood CLL lymphocytes of 40 previously untreated patients. Statistical analysis of correlations of TP53, cyclin D2, c-Myc, p21WAF1/CIP1 and p27KIP1 expressions in B-CLL patients with different Rai stages demonstrated that the progression of disease was accompanied by increases in p53, cyclin D2 and c-Myc mRNA expression. The expression of p27KIP1 was nearly statistically significant whereas that of p21 WAF1/CIP1 showed no such correlation. Moreover, high expression levels of TP53 and c-Myc genes were found to be closely associated with more aggressive forms of the disease requiring earlier therapy.     

Dysregulated angiogenesis in B-chronic lymphocytic leukemia: Morphologic, immunohistochemical, and flow cytometric evidence

Background

The extent of enhanced bone marrow angiogenesis in chronic lymphocytic leukemia (CLL) and relationship to proangiogenic factors and prognostic indicators is largely unexplored.

Methods

To further investigate the role of angiogenesis in CLL by evaluating the topography and extent of angiogenesis in a group of CLL bone marrow biopsies, to study the expression of pro and antiangiogenic vascular factors in CLL cells to more precisely document the cell types producing these factors, and to evaluate the role, if any, of localized hypoxia in upregulation of angiogenesis in CLL We used immunohistochemistry (IHC) (n = 21 pts) with antibodies to CD3 and CD20, proangiogenic (VEGF, HIF-1a) and antiangiogenic (TSP-1) factors, and VEGF receptors -1 and -2 to examine pattern/extent of CLL marrow involvement, microvessel density (MVD), and angiogenic characteristics; flow cytometry (FC) was performed on 21 additional cases for VEGF and TSP-1.

Results

CLL patients had higher MVD (23.8 vs 14.6, p~0.0002) compared to controls (n = 10). MVD was highest at the periphery of focal infiltrates, was not enhanced in proliferation centers, and was increased irrespective of the presence or absence of cytogenetic/immunophenotypic markers of aggressivity. By IHC, CLL cells were VEGF(+), HIF-1a (+), TSP-1(-), VEGFR-1(+), and VEGFR-2(+). By FC, CLL cells were 1.4–2.0-fold brighter for VEGF than T cells and were TSP-1(-).

Conclusion

CLL demonstrates enhanced angiogenesis, with increased MVD, upregulated VEGF and downregulated TSP-1. Upregulation of HIF-1a in all CLL cases suggests localized tissue hypoxia as an important stimulant of microvessel proliferation. The presence of VEGF receptors on CLL cells implies an autocrine effect for VEGF. Differences in MVD did not correlate with traditional genetic/immunophenotypic markers of aggressiveness.     

Interleukin-6 and interleukin-6 receptor secretion by chronic lymphatic leukaemia and normal B lymphocytes: effect of PMA and PWM

Interleukin-6 (IL-6) and soluble interleukin-6 receptor (sIL-6R) were detected in supernatants of cultures of B chronic lymphatic leukaemia (CLL) lymphocytes. Phorbol-12-myristate 13 acetate (PMA) caused a decrease in the levels of IL-6 in 14 out of 16 cultures and an increase in levels of sIL6R in all 15 cases. The effect of pokeweed mitogen (PWM) was variable and not significant. The levels of IL-6 were below the detection limit (60 pg/ml) in sera of 13 CLL patients whereas sIL-6R was detected (13 ng/ml to 97 ng/ml) in the 13 sera. IL6 was not detected in cultures of unstimulated or stimulated with PMA or PWM normal human B cells. Levels of sIL-6R were minimal in cultures of normal B lymphocytes and were increased in PMA stimulated cultures. The results are consistent with the view that B-CLL cells produce spontaneously IL-6 which could act in an autocrine fashion to cause shedding of surface IL-6R and account for the correlation found between serum levels of sIL-6R and B-CLL lymphocyte numbers. The fall in levels of IL-6 in PMA stimulated CLL cultures might express masking or degradation of IL-6 after combination with the receptor.     

Elevated IL-10 plasma levels correlate with poor prognosis in diffuse large B-cell lymphoma

The aim of the study was to confirm whether plasma levels of interleukin-10 (IL-10) correlate with the prognosis in diffuse, large B-cell lymphoma (DLBCL) patients. Plasma IL-10 levels were determined at the time of diagnosis in a group of 157 consecutively treated, DLBCL patients. Of those, 122 patients (78%) had IL-10 plasma levels below the detection limit (< 5 pg/mL) and 35 (22%) above this value. The median value for patients with detectable IL-10 levels was 35 pg/mL (range, 5 to 2480 pg/mL). Detectable plasma IL-10 levels were significantly associated with age > 60 years, ECOG performance status > or = 2, Ann Arbor advanced disease stage, bulky tumor mass, elevated serum levels of LDH and beta2-microglobulin, presence of anemia and low serum albumin levels as well as the presence of B symptoms. The patients with detectable IL-10 levels had lower probability of CR achievement (OR = 0.23, 95% CI 0.1-0.5, p = 0.0003). In addition, detectable IL-10 levels were significantly associated with shorter PFS (OR = 2.5, 95% CI 1.5-4.4, p = 0.001) and OS (OR = 3.0, 95% CI 1.7-5.2, p = 0.0001). In conclusion, we confirmed in this large group of DLBCL patients that elevated plasma IL-10 levels correlated with adverse disease features and poor prognosis. The plasma concentration of IL-10 may be a useful marker for evaluation of disease activity.     

Assessment of pro- and antiangiogenic factors blood serum concentrations in patients with hormonal inactive adrenal tumors

INTRODUCTION: The growth and persistence of solid tumors and their metastases is connected with angiogenesis. This process is determined by activity of pro- and antyangiogenic factors. VEGF is the one of the most important factors having a stimulant effect on angiogenesis. Soluble forms of VEGF receptors are inhibitors of angiogenesis. The soluble forms of VEGF receptors containing extra cellular part of receptor, which binds ligand, seem to be real inhibitors of VEGF. THE AIM OF THE STUDY: Evaluation the value of serum VEGF and soluble forms of VEGF receptors concentration as a marker of malignancy in patients with hormonal inactive adrenal tumors. MATERIAL AND METHODS: Twenty seven patients (18 female, 9 male; mean age 48+/-4.3 years) with adrenocortical carcinoma (N=8), adrenal metastases (N=4) and adrenocortical adenoma (N=15) were included in this study. Age- and gender-matched control samples were acquired from healthy volunteers (N=10). Serum VEGF and sVEGFR levels were determinated by means of ELISA assay. Statistical analysis was performed using the Student-t test, the Pearson's test and the series test. RESULTS: In healthy controls mean VEGF level was 197.2 pg/ml, sVEGFR-1 43.5 pg/ml and sVEGFR-2 8976.3 pg/ml. Patients with adrenocortical carcinoma had the levels of VEGF (1263.8 pg/ml) significantly higher and of sVEGFR-2 (5893.7 pg/ml) significantly lower in comparison to control group (p<0.05). On the other hand the mean VEGF (334.2 pg/ml) concentration in patients with benign adrenocortical adenoma wasn't significant different than in control group (p>0.05) but mean sVEGFR-1 (21.7 pg/ml) and sVEGFR-2 (7106.4 pg/ml) concentrations were significantly lower than in the control (p<0.05). In metastases group mean VEGF (485.9 pg/ml) level was higher and sVEGFR-2 (5455.2 pg/ml) was lower than in control group (p<0.05). CONCLUSION: These data suggest that determination of VEGF and sVEGFR concentration in the serum of patients with hormonal inactive adrenal tumors may be applied as an additional marker of malignancy.     

Flow cytometric analysis of T-lymphocyte subpopulations in B-cell chronic lymphocytic leukemia: correlation with clinical stage

Several authors have studied the T-lymphocyte subpopulations in B-cell chronic lymphocytic leukemia (B-CLL), but previous studies were performed after preceding enrichment procedures, which are known to cause selective losses of certain subpopulations. To correct for this deficiency we used flow cytometric analysis, which enabled us to measure subpopulations directly on total blood samples. We studied T-lymphocyte subsets with OKT monoclonal antibodies in 45 patients with B-CLL. Serum levels of IgG, IgA and IgM were assayed simultaneously and findings were correlated with clinical stage (Rai classification). The absolute number of CD4-positive cells decreased in more advanced Rai stages, while the absolute number of CD8-positive cells increased, resulting in a progressive reduction in CD4/8 ratio. Results from patients in stages with equal prognosis (Rai I and II, Rai III and IV) were similar and when these results were grouped the observed differences were highly significant and clearly correlated with all prognostic groups.     

Vascular endothelial growth factor levels in serum and plasma following esophageal cancer resection--relationship to platelet count

In patients with cancer circulating vascular endothelial growth factor (VEGF) may be tumor-derived and have prognostic significance. Activated platelets may also be a source of VEGF, releasing it in serum formation. Debate exists as to whether serum or plasma VEGF (S-VEGF, P-VEGF) is the most appropriate surrogate marker of tumor angiogenesis. As healing wounds produce VEGF that can spill over into the circulation, we aimed to investigate the potential confounding effects of cancer surgery on both perioperative S-VEGF and P-VEGF levels and to evaluate their relationship with platelet count. S-VEGF, P-VEGF and platelet counts were measured in 23 patients undergoing esophageal cancer resection. Samples were taken preoperatively and six weeks following surgery. Seven patients were also sampled on postoperative days 1, 5 and 10. VEGF was assayed using a commercial enzyme linked immunosorbent assay. S-VEGF and P-VEGF both rose after surgery (S-VEGF; day 5: 1017 [446-1224] pg/mL and day 10: 1231 [626-2046] pg/mL versus pre-op: 329 [189-599] pg/mL. P-VEGF; day 1: 55 [46-104] pg/mL and day 10: 58 [20-154] pg/mL versus pre-op: 23 [13-46] pg/mL), falling towards preoperative levels by six weeks. Platelet count correlated with S-VEGF (rho=0.281; p<0.05, Spearman's rank) and P-VEGF (rho=0.330; p<0.01, Spearman's rank). Platelets may contribute to VEGF levels in plasma as well as in serum. The effects of surgery on S-VEGF or P-VEGF levels are mainly transient. Care must be exercised when interpreting circulating VEGF levels in the early postoperative period.