Angiogenesis in acute myeloid leukemia and myelodysplastic syndrome

Increased angiogenesis is important in the pathophysiology of solid tumors. Recent studies show that angiogenesis and angiogenic factors play an important role in hematological malignancies. Both acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are associated with a substantial increase in vascularity in the bone marrow as well as increased levels of various angiogenic factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor, angiogenin, angiopoietin-1, platelet-derived growth factor, hepatocyte growth factor, epidermal growth factor, tumor necrosis factor-alpha, and transforming growth factor-alpha and transforming growth factor-beta. Most of these angiogenic factors appear to be secreted by the neoplastic hematopoietic cells and appear to promote the growth and proliferation of the leukemic cells in an autocrine fashion. More importantly, angiogenic factors play a role in the clinical behavior and outcome of both AML and MDS. Despite significant overlap between MDS and AML in many aspects, higher levels of cellular VEGF and lower levels KDR are seen in MDS than in AML. Antiangiogenic therapy may play a role in AML and MDS and some differences in response may exist between MDS and AML.     

The JAK2V617F activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia, but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia

Activating mutations in tyrosine kinases have been identified in hematopoietic and nonhematopoietic malignancies. Recently, we and others identified a single recurrent somatic activating mutation (JAK2V617F) in the Janus kinase 2 (JAK2) tyrosine kinase in the myeloproliferative disorders (MPDs) polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. We used direct sequence analysis to determine if the JAK2V617F mutation was present in acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML)/atypical chronic myelogenous leukemia (aCML), myelodysplastic syndrome (MDS), B-lineage acute lymphoblastic leukemia (ALL), T-cell ALL, and chronic lymphocytic leukemia (CLL). Analysis of 222 patients with AML identified JAK2V617F mutations in 4 patients with AML, 3 of whom had a preceding MPD. JAK2V617F mutations were identified in 9 (7.8%) of 116 CMML/a CML samples, and in 2 (4.2%) of 48 MDS samples. We did not identify the JAK2V617F disease allele in B-lineage ALL (n = 83), T-cell ALL (n = 93), or CLL (n = 45). These data indicate that the JAK2V617F allele is present in acute and chronic myeloid malignancies but not in lymphoid malignancies.     

Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) are elevated in peripheral blood plasma of patients with chronic lymphocytic leukemia and decrease after intensive fludarabine-based treatment

Chronic lymphocytic leukemia (CLL) is characterized by extraordinary heterogeneity in terms of clinical course with overall survival ranging from several months to dozens of years. It is currently not possible to accurately predict the future clinical course in an individual patient. Angiogenesis has been recently reported as a potential prognostic factor in various hematological malignancies including CLL. The objective of the present study was to quantify plasma levels of key angiogenic activators vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in patients with CLL and determine their potential change after intensive fludarabine-based treatment. Peripheral blood EDTA plasma concentrations of bFGF and VEGF were measured using comercially available enzyme-linked immunosorbent assay in 73 patients with untreated CLL (43 males, 30 females, median age, 65 years, range 31-88) and 80 healthy donors serving as control group. We found statistically significant increase in concentrations in patients with chronic lymphocytic leukemia compared to the control group (p < 0.0001 for both cytokines). No differences in angiogenic factors were noted between subgroups with low vs. intermediate vs. high-risk stage according to modified Rai staging or males vs. females. In twelve patients who achieved at least partial response after intensive fludarabine-based treatment, levels of bFGF as well as VEGF decreased significantly (bFGF, p = 0.0005; VEGF, p = 0.0068); in addition, they were no more significantly different from controls (bFGF, p = 0.524; VEGF, p = 0.728). Our data showed that key angiogenic activators bFGF and VEGF were elevated in plasma ofCLL patients. Furthemore, treatment with intensive fludarabine-containing regimens resulted in significant decrease of both cytokines. These data suggest that angiogenic cytokines may indeed play a significant role in CLL biology and that treatment with combination of fludarabine, cyclophosphamide +/- rituximab may exhibit antiangiogenic properties. Further studies with longer follow-up are necessary for evaluation of a possible association between angiogenic markers and progression-free survival or overall survival.     

Vascular endothelial cell growth factor is an autocrine promoter of abnormal localized immature myeloid precursors and leukemia progenitor formation in myelodysplastic syndromes

Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of hematopoietic stem cell development, extracellular matrix remodeling, and inflammatory cytokine generation. To delineate the potential role of VEGF in patients with myelodysplastic syndrome (MDS), VEGF protein and receptor expression and its functional significance in MDS bone marrow (BM) were evaluated. In BM clot sections from normal donors, low-intensity cytoplasmic VEGF expression was detected infrequently in isolated myeloid elements. However, monocytoid precursors in chronic myelomonocytic leukemia (CMML) expressed VEGF in an intense cytoplasmic pattern with membranous co-expression of the Flt-1 or KDR receptors, or both. In situ hybridization confirmed the presence of VEGF mRNA in the neoplastic monocytes. In acute myelogenous leukemia (AML) and other MDS subtypes, intense co-expression of VEGF and one or both receptors was detected in myeloblasts and immature myeloid elements, whereas erythroid precursors and lymphoid cells lacked VEGF and receptor expression. Foci of abnormal localized immature myeloid precursors (ALIP) co-expressed VEGF and Flt-1 receptor, suggesting autocrine cytokine interaction. Antibody neutralization of VEGF inhibited colony-forming unit (CFU)-leukemia formation in 9 of 15 CMML and RAEB-t patient specimens, whereas VEGF stimulated leukemia colony formation in 12 patients. Neutralization of VEGF activity suppressed the generation of tumor necrosis factor-alpha and interleukin-1beta from MDS BM-mononuclear cells and BM-stroma and promoted the formation of CFU-GEMM and burst-forming unit-erythroid in methylcellulose cultures. These findings indicate that autocrine production of VEGF may contribute to leukemia progenitor self-renewal and inflammatory cytokine elaboration in CMML and MDS and thus provide a biologic rationale for ALIP and its adverse prognostic relevance in high-risk MDS.     

The incidence and outcome of myeloid malignancies in 2,112 adult patients in southeast England

There is a paucity of epidemiological data on chronic myeloproliferative disorders and myelodysplastic syndromes (MDS), while subtypes of acute myeloid leukemia (AML) are rarely defined. We identified 2,112 adult myeloid malignancies in the South Thames area between 1999 and 2000. The incidence (European standard population) of AML was 3.00/100,000, that of MDS 3.47/100,000, chronic myelomonocytic leukemia (CMML) 0.46/100,000, idiopathic myelofibrosis (IMF) 0.37/100,000, polycythemia vera (PV) 1.08/100,000, primary thrombocythemia (PT) 1.65/100,000 and chronic myeloid leukemia (CML) 1.09/100,000. The 3-year survival for AML was 15%, MDS 45%, CMML 29%, IMF 48%, PV 80%, PT 81% and CML 50% We believe this study reflects the true incidence and outcome of myeloid malignancies in South East England.     

Survivin expression, apoptosis and proliferation in chronic myelomonocytic leukemia

We analyzed the expression of the inhibitor of apoptosis survivin by immunocytochemistry in bone marrow cells from patients with chronic myelomonocytic leukemia (CMML) to evaluate possible abnormalities in comparison with other myelodysplastic (MDS) and myeloproliferative syndromes, and to investigate a possible correlation between survivin expression and altered apoptosis or proliferation, or relevant laboratory and clinical findings. Thirty-four patients with CMML [18 MDS-CMML and 16 myeloproliferative disorder (MPD)-CMML], 90 with MDS, 41 with acute myeloid leukemia (AML), 19 with chronic MPD and 25 control subjects were studied. In normal samples survivin was never detectable. In CMML survivin levels higher than in MDS and AML (P < 0.0001), but similar to those found in MPD were observed. In CMML and MDS apoptosis was significantly higher compared to normal controls and all other subtypes of leukemias (P < 0.0001). Proliferation did not differ significantly in normal controls, MDS and CMML; the lowest levels were observed in AML and MPD (P < 0.0001). In CMML there was no correlation between survivin expression and blast cell percentage, apoptosis or proliferation, FAB or WHO subgroup. Proliferation was higher in MDS-CMML and tended to correlate with overall survival. CMML-2 cases with higher survivin expression showed higher evolution rate and shorter survival. In conclusion, CMML is characterized by high proliferation and apoptosis. Survivin overexpression, by disrupting the balance between cell proliferation/differentiation and apoptosis, may play an important role in its pathophysiology. The detection of survivin-deregulated expression may provide a useful tool for diagnosis, prognosis and a possible target for experimental treatments.     

Concentrations of hepatocyte growth factor, basic fibroblast growth factor, and vascular endothelial growth factor in pericardial fluid and plasma

Some angiogenic factors, including hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF), have been reported to promote angiogenesis and improve myocardial perfusion in experimental models of ischemic heart disease. These factors are produced in various tissues, including myocardium. We measured the concentrations of HGF, bFGF, and VEGF by enzyme-linked immunosorbent assay in plasma and in pericardial fluid sampled during open heart surgery (12 patients with ischemic heart disease and 17 with nonischemic heart disease). HGF levels were significantly higher in plasma than in pericardial fluid (12.0 +/- 1.8 versus 0.26 +/- 0.04 ng/mL, P < 0.0001). On the other hand, bFGF levels were significantly higher in pericardial fluid than in plasma (243.5 +/- 50.9 versus 49.6 +/- 7.8 pg/mL, P = 0.009). VEGF levels were not significantly different between pericardial fluid and plasma (47.2 +/- 17.6 versus 24.5 +/- 3.6 pg/mL, P = 0.23). Concentrations of angiogenic factors in pericardial fluid and in plasma were not significantly different between patients with ischemic and nonischemic heart disease. These results suggest that the production, secretion, and kinetics of HGF, bFGF, and VEGF are different. These angiogenic factors may have different pathophysiologic roles.     

Regulation of angiogenesis in the bone marrow of myelodysplastic syndromes transforming to overt leukaemia

To investigate the regulatory mechanisms of angiogenesis in the development of myelodysplastic syndromes (MDS) and its progression to overt leukaemia (OL), bone marrow samples from control, paired samples from MDS patients before and after transformation to OL (MDS --> OL) and de novo acute myeloid leukaemia (AML) were analysed. Immunohistochemical staining showed a significant increase of bone marrow microvascular density (MVD) in MDS and de novo AML compared with controls. Surprisingly, in MDS, MVD significantly decreased upon transformation to OL, which was also significantly lower than the MVD of de novo AML. This evidence was strengthened by the pattern of angiogenic mediator gene expression, confirming the importance of various angiogenic mediators including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumour necrosis factor alpha (TNFalpha), hepatocyte growth factor (HGF) and the angiopoietin family of mediators (Ang-1 and Ang-2) as well as the receptors for angiogenic mediators, such as VEGF receptor 2 (VEGFR2) and the tyrosine kinase receptor, TIE2. By contrast, the anti-angiogenic mediator, transforming growth factor-beta (TGFbeta) exhibited significantly higher expression in the bone marrow of MDS --> OL, indicating the importance of this cytokine as the suppressive factor of angiogenesis in MDS. These findings indicate that the bone marrow microenvironment in MDS --> OL and de novo AML differs remarkably, suggesting the different efficacy of anti-angiogenic therapy between de novo AML and leukaemia secondary to MDS.     

Angiogenesis in chronic myeloproliferative diseases

Increased angiogenic activity has been demonstrated in myelofibrosis with myeloid metaplasia (MMM), chronic myeloid leukemia (CML), and essential thrombocythemia (ET) by both bone marrow microvessel density evaluation and measurement of circulating angiogenic factors. MMM is probably the disease with the more pronounced angiogenesis among myeloproliferative disorders but the significance of this finding remains speculative since the angiogenic activity is not correlated with any of the clinical and laboratory features of the disease. Circulating serum levels of angiogenic factors such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were found increased in MMM, CML and ET but the frequent thrombocytosis that accompanies these diseases could limit the interpretation of these data since platelets and megakaryocytes may be considered a major source at least for VEGF. However, CML patients treated with interferon were found to have lower VEGF and HGF levels than untreated or hydroxyurea-treated patients, thus suggesting a possible antiangiogenic mechanism of this drug. In addition, preliminary experiences with the antiangiogenic drug thalidomide have shown therapeutic activity in some myeloproliferative disorders.     

Angiogenic factors in multiple myeloma: higher levels in bone marrow than in peripheral blood

BACKGROUND AND OBJECTIVES: To study the role of some soluble factors in the process of angiogenesis that accompanies multiple myeloma (MM). DESIGN AND METHODS: The concentrations of three well-known angiogenic peptides, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF) were evaluated by an ELISA method. All of these factors were measured in the plasma obtained from peripheral blood (PB) and bone marrow (BM) aspirates of 34 patients affected by plasma cell disorders. This series included one patient with a solitary extramedullary plasmacytoma, 17 patients with MM at diagnosis, and 16 with previously treated MM. RESULTS: In all the patients, the concentration of each angiogenic factor was higher in bone marrow than in peripheral blood. Mean values of the three angiogenic factors in BM or in PB were lower in stage I than stage II-III. One patient with extramedullary solitary myeloma had high levels of VEGF and bFGF but this increase was not found in the other 6 patients with extramedullary disease when compared with patients without extramedullary disease. VEGF and bFGF did not correlate with each other while HGF showed a weak correlation with VEGF and a stronger one with bFGF. Moreover, VEGF correlated with features of disease activity, such as C-reactive protein, and 2-microglobulin, while both bFGF and HGF showed an inverse correlation with albumin level. No correlation was found between VEGF, bFGF and HGF levels and age, M protein level, osteolytic lesions, or percentage of BM plasma cells. Since angiogenic factors may be released by normal cells in response to hypoxia, we also evaluated erythropoietin (EPO) levels (which correlate with the hypoxic stimulus) both in PB and BM plasma of these patients but none of the measured angiogenic factors correlated with EPO levels. Interpretation and Conclusions. Several soluble factors may play a role in the angiogenic activity described in MM but their contribution to the progression of disease may be different. The finding of higher levels of these factors in BM than in PB might indicate that the bone marrow environment is their major source. Concentrations of angiogenic factors parallel the activity of disease and are independent of the hypoxic stimulus.     

Levels of angiogenic proteins in plasma and platelets are not different between patients with hepatitis B/C-related cirrhosis and patients with cirrhosis and hepatocellular carcinoma

Increasing evidence suggests that levels of angiogenic proteins within blood platelets change at the earliest stages of cancer development and may thus provide a promising diagnostic and prognostic tool. Patients with cirrhosis have increased risk of developing hepatocellular carcinoma (HCC). We aimed to study whether development of HCC in hepatitis-related cirrhosis results in changes in platelet levels of angiogenic proteins. We studied the intraplatelet levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), endostatin, platelet factor 4 (PF4) and thrombospondin type 1 (TSP-1) in 38 consecutive patients with hepatitis B- or C-related liver cirrhosis with or without HCC in addition to plasma levels of the same proteins. Twenty healthy volunteers were included to establish reference values for the various tests. Intraplatelet levels of VEGF, bFGF, HGF and endostatin were significantly higher in patients compared to controls. Intraplatelet levels of PDGF, PF4 and TSP-1 were comparable between patients and controls. Plasma levels of VEGF, bFGF and endostatin were comparable between patients and controls. Plasma levels of PDGF, PF4 and TSP-1 were decreased in patients, but this difference disappeared when levels were corrected for platelet count. Intraplatelet and plasma levels of all proteins assessed were comparable between patients with and without HCC. In conclusion, the intraplatelet levels of some angiogenic proteins are elevated in cirrhosis, but do not discriminate between patients with and without HCC. Thus, intraplatelet levels of angiogenic proteins do not seem useful as diagnostic or prognostic biomarker of HCC in cirrhotic patients.     

Frequent clonal loss of heterozygosity but scarcity of microsatellite instability at chromosomal breakpoint cluster regions in adult leukemias

Microsatellites are important highly polymorphic genetic markers dispersed in the human genome. Using a panel of 22 (CA)n repeat microsatellite markers mapped to recurrent breakpoint cluster regions specifically involved in leukemia, we investigated 114 adult leukemias (25 acute lymphocytic leukemia [ALL], 32 acute myeloid leukemia [AML], 36 chronic lymphocytic leukemia [CLL], and 21 chronic myeloid leukemia [CML] in chronic phase) for somatic mutations at these loci. In each patient, DNA from fresh leukemia samples was analyzed alongside normal constitutive DNA from buccal epithelium. We detected loss of heterozygosity (LOH) in 81 of 114 patients (ALL 16/25, AML 25/32, CLL 30/36, CML 10/21). Deletions were most often seen in ALL at 11q23 and 19p13; in AML at 8q22 and 11q23; in CLL at 13q14.3, 11q13, and 11q23; and in CML at 3q26. Only six deletions were reported in 74 karyotypes analyzed, whereas in these same cases, 91 LOH events were detected by microsatellites. Of 26 leukemias with a normal karyotype, 16 nevertheless showed at least one LOH by microsatellite analysis. Replication errors were found in 10 of 114 patients (8.8%). Thus, microsatellite instability is rare in leukemia in contrast to many solid tumors. Our findings suggest that in adult leukemia, LOH may be an important genetic event in addition to typical chromosomal translocations. LOH may point to the existence of tumor suppressor genes involved in leukemogenesis to a degree that has hitherto been underestimated.     

Marked decrease of plasma VEGF after implantation of autologous bone marrow mononuclear cells in a patient with critical limb ischemia--a case report

The authors performed autologous bone marrow mononuclear cells implantation (BMI) in a 79-year-old man with critical limb ischemia. After BMI, the resting pain of the ischemic leg improved gradually. They measured the plasma concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and serum hepatocyte growth factor (HGF) in the blood from bilateral femoral veins. Before BMI, the plasma VEGF and bFGF concentrations were much greater in the ischemic leg than in the other lower limb, but decreased to the same concentrations as those in the contralateral lower extremity after BMI. The large concentrations of the angiogenic factors VEGF and bFGF in plasma indicate the severity and extent of the leg ischemia. BMI resulted in lower levels of VEGF and bFGF, and this fall is the hallmark of the effectiveness of BMI in the treatment of peripheral artery disease.     

Current status of vaccination therapy for leukemias

Therapeutic vaccination has challenged immunologists and hematologists for more than two decades and has developed from early animal studies to phase III trials in some human malignancies. Acute and chronic leukemias are common diseases in the clinical practice but few vaccination protocols have found their way to phase I trials in leukemias. Therapeutic vaccination protocols share the goal of inducing or augmenting leukemia-specific immune responses in the tumor-bearing host in order to potentially achieve therapeutical benefit in these otherwise fatal diseases. Major interest has been drawn to the use of dendritic cell (DC-based immunotherapy protocols relying on the unique properties of these most powerful antigen-presenting cells. With the bcr-abl oncogene a target of specific immunotherapy has been determined in chronic myelogenous leukemia (CML), while there is a limited information on leukemia-specific tumor antigens in acute myelogenous and lymphoblastic leukemias. This review will focus on immunotherapy development in acute and chronic leukemias and will discuss published clinical trials in acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and CML.     

Phenotyping of malignant hematopoietic cells

Summary 1255 cases of leukemia-lymphoma were tested between 1972 and 1984 by multiple marker analysis. Routine leukemia phenotyping was performed using standard morphological and cytochemical techniques in combination with clinical and histo-pathological information; the main emphasis was put on immunological surface marker analysis using erythrocyte rosette assays, TdT and a large panel of poly- and monoclonal antibody tests. The 1255 cases were divided into these major types and subtypes: 349 cases of ALL and related immature T- and Burkitt-lymphomas (cALL, pre B-ALL, B-ALL and Burkitt-lymphomas, T-ALL and immature, mostly leukemic T-lymphomas, Null-ALL), 454 cases of mature T- and B-cell malignancies (T-CLL, mycosis fungoides, Sezary-syndrome, T-lymphomas, B-CLL, hairy cell leukemia, multiple myeloma, B-lymphomas), 263 cases of acute myeloid leukemias (AML, AMMoL/AMoL), 182 cases of chronic myeloid leukemias (CML in chronic phase, CMoL, CML in blast crisis), 6 cases of erythroleukemia and 1 case of megakaryoblastic leukemia. A simplified classification scheme which has been used in our laboratories is presented. Phenotyping is of diagnostic, prognostic and therapeutic relevance, most evidently for patients with ALL. Routine leukemia phenotyping should be performed with highly standardized techniques and reagents and by combining information from several fields in the multiple marker analysis. New areas of leukemia research might become very useful for the routine procedure of phenotyping.Abbreviations ALL acute lymphoblastic leukemia - AML acute myeloblastic leukemia - AMMoL acute myelomonoblastic leukemia - AMoL acute monoblastic leukemia - cALL common ALL - CLL chronic lymphocytic leukemia - CML chronic myelocytic leukemia - CML-BC CML in blastic crisis - CMoL chronic monocytic leukemia     

p16INK4A and p15INK4B gene deletions in primary leukemias

The 9p21 locus has been deleted at a high frequency in a wide variety of tumors. Recently, two genes, p16INK4A and p15INK4B (also called MTS1 and MTS2), have been localized in close proximity at the 9p21 locus, encoding cyclin-dependent kinases 4/6 inhibitors of relative molecular mass 16 kD and 15 kD, respectively and also found to be deleted at a high frequency in tumor cell lines. We analyzed p16INK4A and p15INK4B genes in 178 cases of primary leukemias including 81 cases of chronic lymphocytic leukemia (CLL), seven of hairy cell leukemia (HCL), seven of chronic myelogenous leukemia (CML), 43 of acute myelogenous leukemia (AML), 27 of acute lymphoblastic leukemia (ALL), and 13 of myelodysplastic syndrome (MDS) by Southern blot analyses. The ALL cases showed a relatively high frequency of homozygous deletions (22%, 6 of 27) at the p16INK4A gene locus. Interestingly, of the six cases with p16INK4A homozygous deletions, only three showed homozygous deletions at the p15INK4B gene. In 81 CLL patients, we detected one homozygous and five heterozygous deletions at both the p16INK4A and p15INK4B genes and two heterozygous deletions at the p16INK4A gene alone. Deletion of these two genes in AML cases is relatively low (9%). We did not detect deletions in any of the MDS, HCL, and CML cases examined. Sequence analyses of p16INK4A gene of six CLL cases with heterozygous deletion at this locus showed a 27-bp deletion at the splice acceptor site of intron 1 in one case and changes in the coding sequence in three other cases. The data presented in this report showed that (1) p16INK4A and p15INK4B genes are preferentially deleted homozygously in ALL and heterozygously in CLL cases with frequent mutation in the second allele, and (2) p16INK4A gene appears to be more frequently deleted than p15INK4B gene.     

Mutation analysis of the FAS and TNFR apoptotic cascade genes in hematological malignancies

OBJECTIVE: The existence of properly functioning apoptotic pathways is of utmost importance in the maintenance of a normal cell count. Several groups have searched for mutations in the FAS receptor, a well-characterized apoptotic protein carrying a death domain, and reported the existence of rare mutations in multiple myeloma, T-acute lymphoblastic leukemia (T-ALL), and adult T-cell leukemia. Our aim was to expand these searches by looking for mutations in the death domains of FAS, FADD, TNFR, TRADD, and RIP, in the promoter region of FAS, and in the protease domain of caspase 10, in a larger variety of hematological malignancies, some of which express an apoptosis-resistant phenotype. METHODS: We extracted RNA and DNA samples from 92 hematological malignancies: chronic lymphocytic leukemia (CLL; 31 cases), chronic myelogenous leukemia (CML; 28 cases), essential thrombocythemia (ET; 8 cases), acute lymphocytic leukemia (ALL; 6 cases), acute myeloblastic leukemia (AML; 6 cases), hairy-cell leukemia (HCL; 3 cases), Burkitt's lymphoma (3 cases), polycythemia vera (PV; 3 cases), myelofibrosis (2 cases), and chronic myelomonocytic leukemia (CMML; 2 cases) and performed PCR-SSCP and sequence analysis on these samples. RESULTS: Five polymorphic patterns were found: three in the death domain of the FAS gene in CML patients, one in the promoter of this gene in a CLL patient, and the fifth in the death domain of the TRADD gene in a CML patient. No mutations, altering amino acids, were found in these genes in any of the aforementioned malignancies. CONCLUSIONS: These observations imply that mutations in the death domains of FAS, FADD, TNFR, TRADD, and RIP and in the protease domain of caspase 10 are not a major cause for failure of apoptosis in hematological malignancies, mainly CML and CLL. Regulatory and epigenetic abnormalities in these apoptotic cascade members and aberrations in other components of all death machinery should be looked for.     

Angiogenic factors in chronic lymphocytic leukaemia (CLL): Where do we stand?

The role of angiogenesis in haematological malignancies such as chronic lymphocytic leukaemia (CLL) is difficult to envision, because leukaemia cells are not dependent on a network of blood vessels to support basic physiological requirements. Regardless, CLL cells secrete high levels of major angiogenic factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet derived growth factor (PDGF). Nonetheless, it remains unclear how most angiogenic factors regulate accumulation and delayed apoptosis of CLL cells. Angiogenic factors such as leptin, granulocyte colony-stimulating factor (G-CSF), follistatin, angiopoietin-1 (Ang1), angiogenin (ANG), midkine (MK), pleiotrophin (PTN), progranulin (PGRN), proliferin (PLF), placental growth factor (PIGF), and endothelial locus-1 (Del-1), represent novel therapeutic targets of future CLL research but have remained widely overlooked. This review aims to outline our current understanding of angiogenic growth factors and their relationship with CLL, a still uncured haematopoietic malignancy.     

Soluble angiogenic factors: implications for chronic myeloproliferative disorders

The role of angiogenesis for the progressive growth and metastatic process of tumours is well established. What is not clear, though, is the clinical prognostic significance of the angiogenic factors in malignant haematological diseases. In this study, we have assessed the plasma and serum levels of two major angiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) in 55 patients affected by chronic myeloproliferative disorders (CMD). This series included 25 patients with essential thrombocythemia (ET), 10 patients with chronic myelocytic leukaemia (CML), 14 patients with polycythemia vera (PV), and 6 patients with primary myelofibrosis (MF), and they were compared to 20 healthy control subjects. In all patients the plasma VEGF concentration was significantly increased to the healthy control group (P < 0.004). The highest concentrations were found in the patients with ET (178.25 +/- 125.22 pg/ml). The VEGF levels were significantly higher in CMD patients with vascular complications than those in CMD patients without complications (P < 0.01). The b-FGF serum levels also appeared to be significantly higher in almost all the CMD patients compared to the control group (P < 0.07). A significant correlation was found between the VEGF levels and the platelet count in the ET patients and the spleen index in the CML patients. VEGF level, in this study, is associated with increased risk of thrombotic complications. There is evidence of increased levels of soluble angiogenic factors in malignant haematological disorders, but their contribution to the progression of diseases is yet unclear.     

Role of the microenvironment in promoting angiogenesis in acute myeloid leukemia

Angiogenesis is a crucial event in the survival and progression of solid tumors. To determine whether angiogenesis in acute myeloid leukemia (AML) is an intrinsic property of leukemic cells, the vascularity of bone marrow biopsies was determined. Bone marrow vascularity in newly diagnosed or post-chemotherapy AML patients was increased 4-fold (P < 0.01) and 8.7-fold (P < 0.01), respectively, relative to controls. Vascular endothelial growth factor (VEGF) expression by AML blast cells was assessed by immunohistochemistry, and bone marrow cell supernatants were assayed for secretion of VEGF, fibroblast growth factor-2 (FGF-2), and endostatin by enzyme-linked immunosorbent assay. Diffuse cytoplasmic and strong extracellular VEGF immunoreactivity was seen in bone marrow aspirates from AML patients, but not controls. In contrast, there was no difference in the levels of VEGF, FGF-2, and endostatin secreted by mononuclear cells cultured from bone marrows of AML patients compared to normal controls following two days of culture in vitro. Total angiogenic potential of bone marrow cell supernatants was assessed by endothelial sprouting in vitro and by a chick chorioallantoic membrane assay. No differences were found between 2-day conditioned medium from normal and AML bone marrow mononuclear cells in either assay. Our data show a discrepancy between bone marrow vascularity and VEGF expression in vivo and VEGF expression and angiogenesis from 2-day conditioned medium ex vivo. This suggests that angiogenesis in AML likely represents a response to microenvironmental factors in vivo, rather than being an intrinsic property of leukemic cells.