George Tsirakis

Histological expression of angiogenic factors: VEGF, PDGFRα, and HIF-1α in Hodgkin lymphoma

Angiogenesis is a prerequisite for solid tumor growth, but there is relatively limited data regarding Hodgkin lymphoma. The purpose of this study was to examine the immunohistochemical expression of angiogenic and proliferation markers in Hodgkin biopsies in relation to clinical parameters. Immunostaining was performed on 65 Hodgkin biopsies with vascular endothelial growth factor (VEGF), hypoxia inducible factor-1 alpha (HIF-1alpha), platelet-derived growth factor receptor alpha (PDGFRalpha), Ki-67, and p53. Microvessel density (MVD) was determined by CD31 staining. In all cases, neoplastic cells and reactive background cells were evaluated. The neoplastic population expressed VEGF in 48% of the cases, HIF-1alpha in 54% of the cases, and PDGFRalpha in 95% of the cases. Both Ki-67 and p53 were positive in neoplastic cells in over 60% of the cases. The MVD had a median of 2.6/0.0625mm(2) which was not different from normal lymph nodes. VEGF in the non-neoplastic compartment showed increased staining in Ann Arbor stage I-II versus III-IV. In conclusion, VEGF, HIF-1alpha, and predominantly PDGFRalpha are expressed in neoplastic cells in the majority of Hodgkin lymphomas. As microvessel formation is not increased in Hodgkin, additional functions of these angiogenic molecules should be investigated.

Serum Evaluation of Angiogenic Cytokines Basic Fibroblast Growth Factor, Hepatocyte Growth Factor and TNF-ALPHA in Patients with Myelodysplastic Syndromes: Correlation with Bone Marrow Microvascular Density:

Recent studies have documented that angiogenesis plays a significant role in haematological malignancies, including mylodysplastic syndromes (MDS). Basic fibroblast growth factor (b-FGF), Hepatocyte growth factor (HGF) and Tumor necrosis factor-α (TNF-α) are multifunctional cytokines that potently stimulate angiogenesis. The aim of the present study was to evaluate the microvascular density (MVD) and the serum levels of these angiogenic factors in patients with myelodysplastic syndromes (MDS). In 61 patients with MDS, MVD was measured in bone marrow biopsies and b-FGF, HGF and TNF-α were determined in the serum of the same patients by enzyme-linked immunosorbent assay (ELISA). Serum levels of b-FGF, HGF and TNF-α as well as MVD in the bone marrow were increased in MDS patients compared to healthy controls (p<0.0001). Levels of b-FGF, HGF and TNF-α were also significantly higher in high-risk for leukemic transformation MDS than in low-risk (p<0.0001). Significant differences were also found regarding MVD in high and low risk patients (p<0.001). Both b-FGF and HGF levels were significant predictors of survival (p<0.0005, log-rank test). The present study showed that serum levels of b-FGF, HGF and TNF-α are significantly increased and dependent on the severity of MDS suggesting that the determination of these parameters may offer considerable information regarding disease progression and prognosis.

Systemic levels of interleukin-6 and matrix metalloproteinase-9 in patients with multiple myeloma may be useful as prognostic indexes of bone disease

Abstract Multiple myeloma is characterized by accelerated production of the proteolytic enzyme matrix metalloproteinase (MMP)-9. We hypothesized that myeloma-produced MMP-9 may influence the rate of bone turnover in a paracrine manner. Thus, we examined the correlations of MMP-9 levels, disease severity, and bone turnover rate as evaluated by markers of bone formation and resorption. Thirty-seven newly diagnosed multiple myeloma patients (nine of Durie-Salmon stage I, 12 of stage II and 16 of stage III) and 12 age-matched controls were studied. Serum MMP-9 levels were significantly higher at stage II compared to stage I (188.78±91.27 vs. 59.25±33.09 ng/mL, p<0.004). Additionally, free urine pyridinolines (F-Pyd), free urine deoxy-pyridinolines (F-Dpd) and urine N-telopeptide fragment (NTx) were elevated, their level correlating with disease stage (p<0.001, p<0.03, p<0.001, respectively), as were bone marrow infiltration and serum interleukin-6 (IL-6) levels (p<0.0001, p<0.01, respectively). MMP-9 levels were lower in patients compared with controls (p<0.001), whereas IL-6 and bone resorption marker levels were higher in patients than in controls (p<0.001 in all cases). Significant correlation was found between infiltration, MMP-9, free urine pyd, free urine dpd and NTx for each stage of the disease (p<0.03, p<0.003, p<0.002, p<0.003 and p<0.001, respectively). Levels of MMP-9 and of IL-6 in multiple myeloma correlate well with bone turnover rate and may be useful in disease evaluation.

Role of platelet-derived growth factor-AB in tumour growth and angiogenesis in relation with other angiogenic cytokines in multiple myeloma

Angiogenesis is a complex process essential for the growth, invasion, and metastasis of various malignant tumours, including multiple myeloma (MM). Various angiogenic cytokines have been implicated in the angiogenic process. Among them, platelet‐derived growth factor‐AB (PDGF‐AB) has been reported to be a potent stimulator of angiogenesis in many solid tumours and haematological malignancies, including MM. The aim of the study was to investigate the relationship between PDGF‐AB, microvascular density (MVD), and various angiogenic cytokines, such as basic fibroblast growth factor (b‐FGF), angiogenin (ANG), and interleukin‐6 (IL‐6), in MM patients. Forty‐seven MM patients before treatment, 22 of whom were in plateau phase, were studied. We determined the serum levels of the aforementioned cytokines and MVD in bone marrow biopsies before and after treatment. Mean serum values of PDGF‐AB, b‐FGF, ANG, and MVD were significantly higher in patients compared with controls and with increasing disease stage. Significant positive correlations were observed between serum PDGF‐AB, ANG, and IL‐6 levels and MVD. Furthermore, we found significant positive correlations between PDGF‐AB and b‐FGF, IL‐6, ANG, and β2 microglobulin. We also found that patients with high MVD had statistically significantly higher serum levels of PDGF‐AB when a median MVD value of 7.7 was used as the cutoff point. Furthermore, a significant difference was found in serum levels of PDGF‐AB between pre‐ and post‐treatment patients. Finally, survival time was significantly higher in the low MVD group versus the high MVD group (76 vs 51 months). Our results showed that there is a strong positive correlation between PDGF‐AB and the studied angiogenic cytokines and MVD. It seems that PDGF‐AB plays a role in the complex network of cytokines inducing bone marrow neovascularization in patients with MM. Copyright

Serum BAFF levels are related to angiogenesis and prognosis in patients with multiple myeloma

B-cell activating factor (BAFF) is a B-cell growth factor. We measured its serum levels and correlated them with parameters of disease activity, as serum levels of tumor necrosis factor-α and lactate dehydrogenase, bone marrow microvascular density and proliferating cell nuclear antigen expression, in 50 myeloma patients, in 22 of them in plateau phase and in 20 controls. All of them were higher in patients and in advanced disease while reduced in plateau phase. BAFF correlated with all the above markers. Higher BAFF levels predicted a shorter survival, suggesting an important prognostic marker and a possible therapeutic target in myeloma.

Serum levels of leptin in multiple myeloma patients and its relation to angiogenic and inflammatory cytokines.

BACKGROUND Leptin, apart from the regulation of food intake, has been implicated in hematopoiesis, the immune response and angiogenesis. Leptin has been found to be decreased in various hematological malignancies. In the present study leptin was measured in multiple myeloma (MM) patients before and after treatment and correlated with other angiogenic molecules and markers of disease activity. METHODS Serum leptin, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FGF), interleukin-1 beta (IL-1beta), beta 2 microglobulin (beta2M) and C-reactive protein (CRP) were measured in 62 newly diagnosed MM patients, 22 of whom obtaining disease stabilization after treatment. The same parameters were measured in 20 healthy controls. Disease stage was defined according to the Durie-Salmon criteria. RESULTS Leptin, VEGF, b-FGF, IL-1beta, and beta2M were significantly higher in newly diagnosed MM patients than in controls (p<0.05). VEGF, b-FGF, IL-1beta, beta2M, CRP but not leptin increased with advancing stage of disease (p<0.01). All parameters decreased significantly following treatment (p<0.001). Although IL-1beta correlated positively with VEGF, beta2M, b-FGF and CRP, leptin did not correlate with any of the measured parameters. CONCLUSION Leptin serum levels do not reflect disease severity in MM. However, there seems to be a decrease in leptin following treatment, which may be associated with an alteration in the metabolic state or the chemokine milieu.

Interleukin-10 Induces Both Plasma Cell Proliferation and Angiogenesis in Multiple Myeloma.

In multiple myeloma the angiogenic process is enhanced by various mediators. Among them interleukin-10 (IL-10), secreted mainly by myeloma-associated macrophages seems to participate in myeloma progression with variable manners. The aim of the study was to measure serum levels of IL-10 in various stages of MM patients and to correlate them with various angiogenic cytokines, such as vascular endothelial growth factor and angiopoietin-2 and with known proliferation parameters, such as serum levels of B-cell activating factor and bone marrow infiltration by myeloma plasma cells, in order to explore their clinical significance. We measured serum levels of the above parameters by ELISA in 54 newly diagnosed MM patients. All of them were higher in MM patients and were increasing in parallel with disease progression. Furthermore, IL-10 correlated positively with both angiogenic cytokines and proliferation markers. This correlation of IL-10 with both angiogenic cytokines and markers of disease activity implicates that they all have an important role in MM pathogenesis and progression.

Monitoring serum levels ELR+ CXC chemokines and the relationship between microvessel density and angiogenic growth factors in multiple myeloma

BACKGROUND The ELR+ CXC chemokines are important mediators of tumorigenesis, related to their angiogenic properties. Angiogenesis appears to be a prominent feature in the progression of multiple myeloma (MM). CXC chemokines have four highly conserved cysteine amino acid residues, with the first two cysteine molecules separated by a single amino acid. The angiogenic potential of this group is determined by the presence of three amino acid residues (Glu-Leu-Arg: the ELR motif) preceding the first cysteine amino acid, in the NH2 terminus. AIMS The purpose of this study was to determine serum concentrations of angiogenesis-related chemokines ELR+ motif, such as interleukin-8 (IL-8), epithelial neutrophil activating protein-78 (ENA-78) and growth-related gene alpha (GRO-α), as well the bone marrow microvascular density (MVD) in patients with MM at diagnosis and after treatment, in plateau phase. We also evaluated the relationship among them with other known growth factors involved in angiogenesis. METHODS Serum levels of the ELR+ CXC chemokines: IL-8, ENA-78 and GRO-α as well as of the angiogenic factors: hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNF-α) were determined in 63 newly diagnosed MM patients, in 30 in plateau phase and in 20 healthy controls. Serum measurements of them were performed with commercially available kits for ELISA. Bone marrow biopsies were performed before and after treatment, in plateau phase, in order to determine MVD by staining vessels with anti-CD31. RESULTS Serum concentrations of IL-8, ENA-78, GRO-α and TNF-α were significantly higher in the group of MM patients (44.5±25.3, 765±572.1, 186.5±129.1 and 4.2±2.8 pg/ml, respectively) in comparison to control group (27.3±6.4, 335.1±268.6, 112.5±76.1 and 1.3±0.8 pg/ml) (p<0.02 for GRO-α, p<0.001 for other cases). We also found that untreated patients had higher levels of IL-8, ENA-78, GRO-α than post treatment patients, but statistical significant difference was found only for IL-8 (48.36±30.93 pg/ml vs. 35.05±19.77 pg/ml, p<0.001). Furthermore IL-8, GRO-α, TNF-α, HGF and VEGF were significantly higher with increasing disease stage (p<0.001 in all cases). ENA-78 serum levels were higher in stage III than in stage I and II, but without statistical significance. Additionally we correlated each proinflammatory cytokine with well known angiogenic factors such as HGF, VEGF and TNF-α. A positive correlation was found between serum HGF and IL-8 and GRO-α (r=0.316 p<0.01, r=0.297 p<0.02, respectively). Similarly serum VEGF correlated with ENA-78 and GRO-α (r=0.323 p<0.01, r=0.469 p<0.001, respectively). In the pretreatment group of patients a positive correlation between bone marrow MVD and serum levels of GRO-α was found (r=0.304 p<0.01). There was a difference in survival times between patients with higher than median versus low IL-8, ENA-78 and GRO-α levels, but the differences could not reach statistical significance in either case. CONCLUSIONS These findings support the hypothesis that ELR+ motif CXC chemokines, such as IL-8, ENA-78 and GRO-α correlate with angiogenic growth factors and may play a role in the progression of MM. Further studies are needed to determine their prognostic and predictive significance.

Circulating osteopontin: a dual marker of bone destruction and angiogenesis in patients with multiple myeloma

The matrix protein osteopontin has been shown to be a marker of osteoclastic activity in multiple myeloma patients, as well as a regulator of angiogenesis. We measured serum levels of osteopontin in 50 untreated multiple myeloma patients (in 25, also after treatment) and examined the relation to markers of osteolytic and angiogenic activity. The median (range) of serum osteopontin was 85 (5-232) in the patient group vs. 36 (2-190) ng/ml in the control group. Serum osteopontin levels were significantly higher in patients with advanced stage or grade of myeloma disease. All patients with serum osteopontin levels >100 ng/ml had advanced stage (II or III) or high grade bone disease, whereas stage I or low grade patients had serum osteopontin levels <100ng/ml. Serum osteopontin levels significantly decreased after treatment. There was a positive correlation of osteopontin with the bone turnover marker N-terminal propeptide of procollagen type I (NTx) and the angiogenic markers vascular endothelial growth factor (VEGF) and bone marrow microvessel density (r: 0.35, 0.47 and 0.30 respectively, p < 0.05). These results support osteopontin as a dual marker of bone destruction and angiogenic activity in myeloma patients. Osteopontin represents a useful biomarker for monitoring myeloma disease activity.

Emerging roles of endoglin/CD105 and angiogenic cytokines for disease development and progression in multiple myeloma patients

Angiogenesis is an essential process for the expansion of multiple myeloma (MM), in which many angiogenic factors participate. Endoglin (CD105) is a transforming growth factor‐β co‐receptor, being mainly expressed in angiogenic endothelial cells and has been used as a marker of tumor angiogenesis, having prognostic potential. The aim of the study was to evaluate serum levels of soluble CD105 (sCD105) in MM patients, both during diagnosis and after effective conventional chemotherapy, in the plateau phase, and to correlate them with the clinical stage of the disease, as well as with the known angiogenic factors vascular endothelial growth factor, angiogenin and interleukin‐18 (IL‐18). Serum levels of the aforementioned factors were measured, by enzyme‐linked immunosorbent assay, in 56 newly diagnosed MM patients, in 35 of them who entered plateau phase and in 24 healthy controls. Bone marrow aspirations were also performed in all patients to determine plasma cell infiltration. All measured cytokines were higher in MM patients compared with controls and with advancing disease stage (p < 0.001 for all cases). Furthermore, the values of all factors decreased significantly in the plateau phase (p < 0.001 for all cases). Serum levels of sCD105 correlated with the other angiogenic cytokines, whereas only serum levels of angiogenin had prognostic value for the survival. In conclusion, CD105 and the angiogenic cytokines vascular endothelial growth factor, angiogenin and IL‐18, seem to have emerging roles both in angiogenesis and tumor growth in MM. Copyright