Serum Levels Of Angiogenic Cytokines Research Articles

VEGF and IL-6 As Markers of Severity and Response to Treatment in Waldenstrom Macroglobulinemia and IgM-Monoclonal Gammopathy of Undetermined Significance: A Monocentric Experience

Background: Angiogenesis plays an important role in the development and the maintenance of hematolymphoid malignancies. Serum levels of angiogenic cytokines, such as vascular endothelial growth factor (VEGF) and interleukin 6 (IL-6), are known to be increased in patients with Waldenstrom Macroglobulinemia (WM) and IgM monoclonal gammopathy of undetermined significance (MGUS). Even if the role of IL-6 and VEGF as markers of disease status and severity has been recognized, only a limited number of studies have addressed the role of cytokines in WM and there is no proof about any correlation between their serum levels in WM and IgM-MGUS patients. Nevertheless, their dosage at various stages of the disease aims to understand if they have a comparable pattern, and therefore they could be used in clinical practice for disease monitoring. Aims: This study aims to find a correlation of serum levels of VEGF and IL-6 with the burden of disease in our patients with IgM-MGUS and WM and to evaluate a possible relationship between the two angiogenic cytokines, in an attempt to identify potential markers of severity. Methods: We performed a retrospective monocentric study of 18 patients (ten with WM and eight with IgM-MGUS) with a median age of 73 years (range: 52-90), whose 66% were male. Patients with WM were at different stages of disease (four were naive, four in remission, two relapsed/refractory). A total of 18 controls with a similar age and gender distribution were also tested. Controls were selected among patients with neuropathies in the absence of any hematological condition. We used the 1:1 ratio for the selection of controls: for every male or female WM patient, one control of the same gender and of similar age was included. In order to find a relationship of VEGF and IL-6 with a laboratory parameter, IgM levels dosage for each patient have been measured at the same time-point of the measurement of the cytokines. In all patients and controls, we evaluated serum levels of VEGF and IL-6 using Immunoassay panels (i.e., anti-IL-6 and anti-VEGF ELISA kit). Results: All patients showed elevated values of angiogenic cytokines when compared with controls (P value < 0.05). Both VEGF and IL-6 serum levels (median values: 623,5 pg/mL and 4,5 pg/mL, respectively; ranges: 113,4-1981 pg/mL and 0,3-21,9 pg/mL, respectively) showed a positive correlation with IgM serum levels (median value: 2055 mg/dL, range: 300-6123 pg/mL) (Fig. 1a and 1b). Furthermore, the relationship between IL-6 and VEGF was statistically significant and could be explained by a linear model (P value = 0,007) (Fig. 1c). No statistically significant differences were observed between the different phases of WM. Conclusions: Evidence from our sample shows that serum levels of angiogenic cytokines as VEGF and IL-6 have a comparable pattern with IgM levels. Therefore, it seems reasonable to suggest that they could be used in clinical practice as markers of severity of disease and response to treatment. Moreover, since the cytokines profiles are comparable, IL-6 can be used interchangeably with VEGF, allowing the monitoring of the disease with easily availability and unexpensive techniques. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Relevance of serum angiogenic cytokines in adult patients with dermatomyositis

BackgroundUntil now, there are few studies evaluating serum levels of angiogenic cytokines in dermatomyositis (DM). Therefore, the aims of the present study were: (a) to analyze systematically and simultaneously serum levels of angiogenin (ANG), angiopoietin (ANGPT)-1, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-1 and − 2, platelet derived growth factor (PDGF)-AA and -BB in DM; (b) to correlate the serum level of these cytokines with the DM clinical and laboratory features.MethodsThis is a cross sectional study, in which 48 patients with DM aged 18 to 45 years were gender-, age- and ethnicity-matched with 48 healthy individuals (control group). The serum levels of cytokines analyses were performed by multiplex immunoassay. The parameters of DM activity were based on the scores established by the International Myositis Assessment & Clinical Studies Group.ResultsThe mean ages, gender frequencies and ethnicities were comparable between the patients with DM and the control group. A significantly higher serum FGF-1 and FGF-2 levels (P < 0.001 and P < 0.001, respectively), lower VEGF and PDGF-AA levels (P = 0.009 and P = 0.022), and comparable ANG, ANGPT-1 and PDGF-BB levels were observed in DM patients compared to controls. There was a tendency for cytokines (with the exceptions of VEGF and PDGF-BB) to correlate positively with the DM activity parameters, whereas FGF-2 correlated inversely. Moreover, FGF-1 strongly correlated with DM cutaneous manifestations.ConclusionsThe present data provide the relevance of different serum angiogenic cytokines in patients with DM. Additional studies will be needed to validate the data obtained in this work.

Beta Adrenergic Blockade Is Feasible During Primary Treatment for Ovarian Cancer [10J

INTRODUCTION: Adrenergic stimulation promotes EOC growth and metastasis. Nonselective beta-blocker (BB) women with EOC is associated with longer OS. The purpose of this study was to determine the feasibility of beta-adrenergic blockade in women with EOC during initial chemotherapy in a prospective clinical trial. METHODS: Women with suspected diagnosis of EOC, primary peritoneal or fallopian tube cancer with plan for tumor reductive surgery (TRS) or neoadjuvant chemotherapy were eligible. Patients (pts) began propranolol 10-20 mg BID 48 hrs prior to any treatment. QOL and serum levels of angiogenic cytokines were assessed. Random effect Tobit models were used to determine association between response and IL6, IL8 and VEGF. RESULTS: 32 pts enrolled, 6 ineligible leaving 26 for analysis. Of 26 pts, 8 did not complete 6 cycles on BB. Only 1 pt stopped due to fatigue after 4 days of BB. Eighteen pts (69%) completed all 6 cycles on BB; 69% were white. Mean age was 58; 80% were stage IIIC- IV, 30% were primarily debulked and 85% had optimal TRS. Serum IL 6, IL 8 and VEGF correlated with response (p &lt; 0.0001). For 18 pts completing 6 cycles on BB, overall QOL, anxiety and depression improved significantly (p=.03). CONCLUSION: The impact of adrenergic stimulation on cancer growth is well documented and deserves further evaluation in a randomized setting. Use of propranolol during primary treatment of EOC, PP/FT cancer is feasible and potentially results in improved QOL.

Role of Serum Levels of Angiogenic Cytokines in Assessment of Angiogenesis after Stem Cell Therapy of Diabetic Patients with Critical Limb Ischemia

The release of proangiogenic cytokines into the circulation after stem cell (SC) therapy and compensatory increase of angiogenesis inhibitors may reflect local vasculogenesis but also can increase the risk of side effects. The aim of our study was to evaluate serum levels of angiogenic cytokines with regard to the assessment of local and systemic vasculogenesis in diabetic patients with no-option critical limb ischemia (NO-CLI). Twenty-five diabetic patients with NO-CLI treated with SCs isolated from bone marrow or stimulated peripheral blood were included in the study. Serum levels of proangiogenic cytokines (VEGF, bFGF, Ang-1, PDGF-AA, and PDGF-BB) and an antiangiogenic cytokine (endostatin) were assessed 6 months after cell treatment, compared to baseline values, and correlated with the number of injected CD34(+) cells. The clinical effect of SC therapy (assessed by changes in TcPO2) and potential systemic vasculogenesis (assessed by eye fundus examination) were evaluated after 6 months. Serum levels of angiogenic inhibitor endostatin increased significantly after 1 and 3 months (p = 0.0003), but no significant increase in serum levels of proangiogenic cytokines was observed. A significant correlation between number of injected CD34(+) cells and serum levels of endostatin was observed (r = 0.41, p < 0.05); however, proangiogenic cytokines did not correlate with CD34(+) cells. No correlation between increase in TcPO2 after treatment and serum levels of any of the angiogenic cytokines were seen, and no signs of systemic vasculogenesis in the retina were observed after 6 months. Despite the significant increase in the levels of the angiogenic inhibitor endostatin following SC treatment, there was no risk of systemic vasculogenesis after SC therapy as documented by serum levels of proangiogenic cytokines or changes in the retina.

Both autologous bone marrow mononuclear cell and peripheral blood progenitor cell therapies similarly improve ischaemia in patients with diabetic foot in comparison with control treatment

The aim of our study was to compare the effect of bone marrow mononuclear cell and peripheral blood progenitor cell therapies in patients with diabetic foot disease and critical limb ischaemia unresponsive to revascularization with conservative therapy. Twenty-eight patients with diabetic foot disease (17 treated by bone marrow cells and 11 by peripheral blood cell) were included into an active group and 22 patients into a control group without cell treatment. Transcutaneous oxygen pressure and rate of major amputation, as the main outcome measures, were compared between bone marrow cells, peripheral blood cell and control groups over 6 months; both cell therapy methods were also compared by the characteristics of cell suspensions. Possible adverse events were evaluated by changes of serum levels of angiogenic cytokines and retinal fundoscopic examination. The transcutaneous oxygen pressure increased significantly (p < 0.05) compared with baseline in both active groups after 6 months, with no significant differences between bone marrow cells and peripheral blood cell groups; however, no change of transcutaneous oxygen pressure in the control group was observed. The rate of major amputation by 6 months was significantly lower in the active cell therapy group compared with that in the control group (11.1% vs. 50%, p = 0.0032), with no difference between bone marrow cells and peripheral blood cell. A number of injected CD34+ cells and serum levels of angiogenic cytokines after treatment did not significantly differ between bone marrow cells and peripheral blood cell. Our study showed a superior benefit of bone marrow cells and peripheral blood cell treatments of critical limb ischaemia in patients with diabetic foot disease when compared with conservative therapy. There was no difference between both cell therapy groups, and no patient demonstrated signs of systemic vasculogenesis.

Serum levels of angiogenic cytokines in psoriatic arthritis and SAPHO syndrome

Angiogenesis is involved in the pathogenesis of arthritis. The aim of the study was to assess the serum levels of selected angiogenic cytokines and their association with clinical presentation in patients with psoriatic arthritis (PsA) and SAPHO syndrome. We studied 98 patients: 80 with PsA and 18 with SAPHO syndrome. The following data were recorded: age, sex, disease duration, joint involvement, type of psoriasis, nail involvement, and treatment. The following indices used to assess the activity of PsA and SAPHO were measured: PASI, BASDAI, BASFI, BASMI, BASG, and VAS pain. We determined erythrocyte sedimentation rate, C‑reactive protein (CRP), and platelet count. The serum levels of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic and acidic fibroblast growth factors (FGFb and FGFa) were determined using an enzyme‑linked immunosorbent assay. In patients with PsA, VEGF levels were positively correlated with CRP (P = 0.04), BASFI (P = 0.03), and disease duration (P = 0.007). No differences were found between patients with and without nail psoriasis in the VEGF or EGF levels (P = 0.32 and P = 0.85, respectively). There were no differences between patients with the peripheral and axial forms of arthritis in VEGF or EGF levels (P = 0.56 and P = 0.28, respectively). No significant correlations were observed between EGF and FGF levels and clinical presentation in patients with PsA. In patients with SAPHO, no significant correlations were found between angiogenic cytokine levels and clinical presentation. Our data suggest a role of VEGF in the pathogenesis of PsA. Further studies are required to better understand the role of angiogenic cytokines in PsA.

Increased Angiogenesis and Enhanced Bone Formation in Patients with IgM Monoclonal Gammopathy and Urticarial Skin Rash: New Insight Into the Biology of the Schnitzler Syndrome

Abstract 1802Schnitzler syndrome is a rare plasma cell disorder which is characterized by the presence of a monoclonal IgM component in association with a chronic urticarial skin rash and at least 2 of the following criteria: fever, joint and/or bone pain, organomegaly (enlarged lymph nodes, spleen and/or liver), increased neutrophil counts and increased ESR. Abnormal bone findings with imaging evidence of osteosclerosis are also frequent. The pathogenesis of this syndrome is unclear and there is no information on bone remodeling and angiogenesis in these patients. To address this issue we studied 13 patients (12M/1F, median age 55 years, range: 39–79 years) with a well characterized Schnitzler syndrome. Patients were diagnosed between 1989 and 2009 and were treated and followed in Hôpital Saint-Louis, Paris (France) and in the University of Athens (Greece). We evaluated the following serum indices of bone remodeling and angiogenesis at diagnosis and after treatment: i) osteoclast regulators [soluble receptor activator of nuclear factor kappa-B ligand (sRANKL) and osteoprotegerin (OPG)]; ii) osteoblast inhibitor dickkopf-1 (Dkk-1); iii) bone resorption marker C-telopeptide of collagen type-1 (CTX); iv) bone formation markers [bone-specific alkaline phosphatase (bALP) and osteocalcin (OC)]; and v) angiogenic cytokines [vascular endothelial growth factor (VEGF), angiogenin (ang), angiopoietin (angp)-1 and -2]. The above molecules were also measured in 24 gender- and age-matched controls.All patients presented with urticaria and monoclonal IgM(kappa) component (median M-peak 0.79 g/dl; range 0.1–2.36 g/dl), while 10 (76%) patients had fever, 9 (69%) had joint and/or bone pain, 5 (38%) had lymphadenopathy and 2 (15%) had splenomegaly. Four patients had documented sclerotic bone lesions in plain X-rays. A technetium bone scintigraphy was performed in 11 patients; all of them had high uptake in at least one site. At diagnosis, 11/13 patients received various symptomatic therapies, including low dose corticosteroids that were ineffective. Two patients at diagnosis and 6 after initial symptomatic therapy were treated with the quinolone antibiotic pefloxacin, while 7 patients were treated with the interleukin-1 inhibitor anakinra (including 3 previously treated by pefloxacine) usually with a complete and sustained efficacy on clinical manifestations of the disease. In a median follow up time of 10 years (range 1–20 years), 3 patients developed overt Waldenström macroglobulinemia (WM) at 5, 7 and 20 years post diagnosis.At diagnosis patients had increased serum levels of bALP (mean±SD: 36.5±15.0 IU/L vs. 26.8±7.1 IU/L; p=0.049), osteocalcin (20.5±18.6 ng/ml vs. 7.4±3.5 ng/ml; p<0.001), Dkk-1 (49.9±13.0 pmol/L vs. 30.9±11.1 pmol/L; p<0.001) and OPG (6.7±1.3 pmol/L vs. 3.5±1.8; p<0.001) compared to controls, while there were no differences between patients and controls for sRANKL and CTX. The increase of Dkk-1 may suggest a balance effect on osteoblast increased activity, while the lack of increase in osteoclast function is in accordance with data on idiopathic osteosclerosis.At diagnosis, patients had also elevated VEGF (809±598 pg/ml vs. 263±257 pg/ml; p=0.001) and ang (221±96 ng/ml vs. 169±33; p=0.013) and decreased angp-1 (18.7±9.1 ng/ml vs. 25.4±10.9 ng/ml; p=0.048) and angp-1/angp-2 ratio (13.9±8.7 vs. 63.4±102.1; p=0.005). Patients presenting with fever had reduced angp-1 levels (14.8±5.2 ng/ml) compared to others (32.7±10.3 ng/ml; p=0.03). Interestingly, patients who progressed to WM had also decreased levels of angp-1 (10.2±7.5 ng/ml) compared to others (21.2±8.2 ng/ml; p=0.04) and angp-1/angp-2 ratio (5.3±4.6 vs. 16.5±8.0; p=0.04), suggesting that low angp-1/angp-2 ratio at diagnosis, which reflects increased angiogenesis, may indicate a predisposition for evolution of the gammopathy towards an overt WM. After successful treatment, VEGF levels decreased (387±207 pg/ml) compared to baseline (770±456 pg/ml; p=0.04).In conclusion our analysis shows increased serum levels of angiogenic cytokines in Schnitzler syndrome and documents enhanced bone formation which appears not to be balanced by a comparable increase in bone resorption; this result may explain the presence of sclerotic bone lesions in this entity. Successful therapy with either anakinra or pefloxacin is associated with reduction of the major angiogenic cytokine VEGF. Disclosures:No relevant conflicts of interest to declare.

Circulating levels of angiogenic cytokines in advanced breast cancer patients with system chemotherapy and their potential value in monitoring disease course

The low-dose metronomic chemotherapy was reported to inhibit directly tumor angiogenesis or VEGF secretion. The study aimed to seek for this effect of system chemotherapy by observing the changes in serum levels of angiogenic cytokines during treatment and assessing their value in monitoring the advanced breast cancer. In sixty-one patients with advanced breast cancer, serum levels of vascular endothelial growth factor (VEGF) and endostatin (ES) were compared at baseline (B0), after one cycle (B1), after 3 cycles (B3), and after 5-6 cycles (B5-6) of system chemotherapy using a quantitative ELISA. Data were correlated with treatment response and total survival. The response to chemotherapy did not correlate with serum VEGF level before therapy or after one cycle, but the changes in VEGF levels after 3 cycles and 5-6 cycles showed good association with clinical responses, i.e., the patients with disease control had a decreased VEGF value, whereas the progressive patients had an increased value. The Cox proportional hazard model revealed that a normalized VEGF level after therapy and an increase in VEGF level after 5-6 cycles were independent predictors for survival. System chemotherapy for advanced breast cancer lead to a significant decrease in serum VEGF level in patients with disease control, and this anti-VEGF efficacy may be mainly due to the reduction in tumor burden. Sequential measurement of serum VEGF could be useful for evaluating treatment efficacy and prognosis.

Angiogenic Molecules and Inflammatory Cytokines in Patients with Thalassemia Major and Double Heterozygous HbS/Beta-Thalassemia; the impact of Deferasirox.

Abstract 2018Poster Board I-1040Angiogenesis is a crucial process in the pathogenesis of several inflammatory, autoimmune and malignant diseases. Endothelial damage and inflammation make a significant contribution to the pathophysiology of sickle cell disease (SCD) and the beta-thalassemia syndromes. However, there are very limited data in the literature for the role of angiogenic cytokines in the pathogenesis of thalassemia major (TM) and of double heterozygocity of SCD and beta-thalassemia (HbS/beta-thal). Deferasirox (Exjade®) is a once-daily orally administered iron chelator approved for the treatment of transfusional iron overload in patients with transfusion-dependent anemia which seems to reduce markers of inflammation in TM. The aim of this prospective study was to evaluate the levels of angiogenic and inflammatory cytokines in patients with TM and HbS/beta-thal with iron overload who received chelation therapy with deferasirox. Forty-five patients (16M/29F) with TM and 20 patients (7M/13F) with HbS/beta-thal were evaluated. Deferasirox was administered at a dose between 10-30 mg/kg/day based on the number of blood transfusions received before the initiation of treatment. After 3 months, dose adjustments (increases) were allowed in increments of 5 mg/kg/day every 3 months as required to reduce markers of iron overload. Deferasirox was given for a period of 12 months. Serum levels of angiogenic cytokines, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiogenin (Ang), angiopoietin (Angp)-1 and -2 and of inflammatory cytokines including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-á), IL-1á, IL-1β, IL-4, IL-10 and transforming growth factor (TGF)-β1 and β2, were measured at baseline (day 1 of deferasirox administration) and then after 12 months post-deferasirox therapy, using ELISA methodology (R&D Systems, Minneapolis, MN, USA, for angiogenin cytokines and ILs and Diaclone, Bensancon, France for TNF-á, TGF-β1 and TGF-β2). Twenty healthy blood donors of similar age and gender were also evaluated as control group. Standard hematology and biochemistry was evaluated monthly in all patients. Patients with both TM and HbS/beta-thal had increased levels of all studied angiogenic cytokines compared with healthy controls but reduced levels of Angp-1/Angp-2 ratio (p<0.01 for all comparisons). Patients with HbS/beta-thal had increased levels of Angp-2 (p=0.002) and reduced levels of Angp-1/Angp-2 ratio (p<0.001) compared to TM patients. Both TM and HbS/beta-thal patients had also increased levels of IL-6 (p=0.008), IL-1á (p=0.01), TGF-β2 (p=0.01), IL-10 (p=0.02), IL-4 (p=0.03), and TNF-á (p=0.01) compared to controls. There were no differences between TM and HbS/beta-thal patients in terms of inflammatory cytokines. In TM patients, VEGF strongly correlated with TGF-β1 (r=0.652, p<0.001), platelet count (r=0.503, p=0.001) and white blood cell (WBC) count (r=0.484, p=0.002), while Angp-2 strongly correlated with TNF-á (r=0.536, p=0.001), WBC count (r=0.430, p=0.008) and platelet count (r=0.330, p=0.01). In HbS/beta-thal patients Angp-1/Angp-2 ratio strongly correlated with ALT (r=-0.611, p=0.007) and Hb (r=0.498, p=0.036). Twelve months post-deferasirox administration, there was a dramatic reduction of ferritin, SGOT and SGPT compared with baseline values in both patient groups (p<0.0001). TM patients showed a continuous increase of both VEGF (mean±SD: from 680±489 ng/ml prior to deferasirox to 801±547 ng/ml post-deferasirox; p=0.002) and bFGF (from 16.2±17.1 ng/ml prior to deferasirox to 33.1±28.8 ng/ml post-deferasirox; p=0.001) after 12 months of deferasirox therapy. No other changes were observed in TM group regarding angiogenic molecules. Patients of HbS/beta-thal showed no alterations in terms of angiogenic cytokines that continued to be elevated compared to controls. In terms of inflammatory cytokines deferasirox produced a reduction of TNF-á in both studied groups (p<0.01 for all comparisons). Our study suggests that angiogenic and inflammatory cytokines are increased in patients with TM and HbS/beta-thal and thus they have a role in the pathogenesis of these disorders. Angiopoietins seem to be strongly implicated in the biology mainly of HbS/beta-thal. However, further studies are needed to fully elucidate this role as well as the effect of deferasirox, if any, on inflammation. Deferasirox seems to have no impact on angiogenic cytokines. Disclosures:No relevant conflicts of interest to declare.

The Administration of Bortezomib, Dexamethasone and Thalidomide (VTD) after ASCT in Myeloma Patients Who Do Not Receive Bisphosphonates Normalizes sRANKL, Dickkopf-1 and Improves Abnormal Osteoclast Function and Impaired Angiogenesis.

Bortezomib (V) monotherapy has been associated with increased osteoblastic activity, reduced osteoclast function and decreased angiogenesis in relapsed/refractory myeloma. The effects of V on bones may be direct or may reflect the reduction of myeloma burden. The co-administration of zoledronic acid, in all reported studies to-date, may also suggest a synergistic stimulation on osteoclast/osteoblast interactions by the two agents but has not allowed the independent evaluation of V on bone metabolism. Previous studies have shown that the combination of V with other agents, such as thalidomide (T) and melphalan, although reduced osteoclast activity, did not enhance osteoblast function. We evaluated the effect of VDT consolidation therapy on bone metabolism and angiogenesis in myeloma patients who underwent high dose melphalan (200 mg/m2) followed by autologous stem cell transplantation (ASCT). In this prospective study, only patients in first remission or with primary refractory disease to one frontline treatment were included. Patients did not receive any bisphosphonate during or post-ASCT as throughout the period of VDT consolidation. VDT was given on day 100 after ASCT: V was administered at a dose of 1.0 mg/m2 on days 1,4,8,11, while T was given at a dose of 100 mg/day, po, on days 1–21 and D at a dose of 40mg/day on days 1–4 of a 21-day cycle. Patients received 4 VDT cycles (first course) and 4 more VDT cycles (second course) 100 days post-day 21 of the 4th cycle. Bone remodeling was studied by the measurement of a series of serum indices on day 1 of cycles 1 & 5 and on day 21 of cycle 8: i) osteoclast regulators [soluble receptor activator of nuclear factor-κB ligand (sRANKL) and osteoprotegerin], ii) osteoblast inhibitor dickkopf-1 (Dkk-1), iii) bone resorption markers [C-telopeptide of collagen type-I (CTX) and tartrate resistant acid phosphatase isoform 5b (TRACP-5b)], and iv) bone formation markers [bone-specific alkaline phosphatase (bALP) and osteocalcin (OC)]. Effect of VDT on angiogenesis was evaluated by measuring the serum levels of angiogenic cytokines, such as vascular endothelial growth factor (VEGF), angiogenin, angiopoietin-1, angiopoietin-2, and basic fibroblast growth factor (bFGF) on the same dates. The above biochemical markers and cytokines were also measured in 18, gender and age-matched, controls. To-date, 18 patients (7M/11F, median age 60 years) have been entered into this study. On day 100 after ASCT, just before VDT, 5 patients were in CR (2 in sCR), 11 in vgPR and 2 in PR, according to IMWG response criteria. At baseline, although the vast majority of patients were in ≥vgPR, they had increased serum levels of sRANKL (p=0.015), Dkk-1 (p=0.025), CTX (p=0.001), TRACP-5b (p=0.032), VEGF (p<0.0001), bFGF (p<0.0001), angiogenin (p=0.01) and reduced levels of angiopoietin-1/angiopoietin-2 ratio (p<0.0001) compared to controls. Serum levels of sRANKL strongly correlated with CTX (r=0.701, p=0.002) and OPG (r=0.733, p=0.0001), while levels of CTX correlated with TRACP-5b (r=0.619, p=0.0008). The administration of 4 VDT cycles resulted in a significant reduction of sRANKL (p=0.02), TRACP-5b (p=0.016), angiogenin (p=0.03), Dkk-1 (p=0.047), but also of bALP (p=0.003) and OC (p=0.016). A borderline reduction of CTX (p=0.071), and VEGF (p=0.056) was also observed. The levels of sRANKL, Dkk-1, TRACP and angiogenin did not differ from control values, while the reduction of bALP and OC did not produce significant differences compared to controls, after 4 VDT cycles. However, levels of VEGF, angiopoietin-1/angiopoietin-2 ratio and bFGF values continued to be elevated compared to controls' values, while CTX levels were slightly increased compared to controls (p=0.049). The results of this on going study suggests that VDT consolidation post-ASCT normalizes sRANKL and Dkk-1 serum levels and improves impaired angiogenesis. However, as in other combination regimens (i.e. VMDT in relapsed/refractory patients), bortezomib could not produce an anabolic effect on bones when combined with TD even in these patients with low myeloma burden.

Serum levels of angiogenic cytokines decrease after radiotherapy in non-Hodgkin lymphomas

Serum levels of angiogenic cytokines decrease after radiotherapy in patients with cancer, and this may be relevant for treatment response and progression-free survival. Herein, we set out to determine whether circulating fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and platelet-derived growth factor-beta (PDGF-beta) decrease after radiotherapy in patients with non-Hodgkin lymphomas (NHLs) and if so, whether their decrease correlates with age, tumour histotype and stage, and radiation dose. The serum levels of FGF-2, VEGF, HGF and PDGF-beta were evaluated before and after radiotherapy by an enzyme-linked immunosorbent assay (ELISA). These levels were correlated both reciprocally and with age, histotype, stage and radiation dose. After radiotherapy, FGF-2, VEGF and PDGF-beta, but not HGF, significantly decreased in relation to the radiation dose and response. No correlation was established between cytokine levels, except for VEGF and PDGF-beta, which decreased in parallel. Haemoglobin levels did not decrease after radiotherapy, while FGF-2, VEGF, HGF and PDGF-beta levels did not correlate with age, NHL stage and histotype. Soluble FGF-2, VEGF and PDGF-beta levels decline after radiotherapy in NHLs, and may have predictive significance for response to treatment and recurrence.

Bone marrow angiogenesis and angiogenic factors in multiple myeloma treated with novel agents

Introduction. An increased bone marrow (BM) angiogenesis is associated with poor outcome in multiple myeloma (MM). Objective. Angiogenesis study in MM treated with novel antimyeloma agents: thalidomide, lenalidomide, bortezomib, and with dexamethasone. Patients and methods. Forty-four patients with MM (14 newly diagnosed, 30 refractory/relapsed) were treated with novel agents at our institution. A BM biopsy was obtained before the initiation of therapy in 19. Angiogenesis was assessed by microvessel density (MVD) estimation in BM biopsies stained with the monoclonal anti-CD34 antibody, and by serum levels of angiogenic factors (VEGF, bFGF, and HGF) and cytokines (IL-6 and TNF-α). Results. A positive correlation was found between BM plasma cell involvement and MVD estimation ( p = 0.01). However, MVD was not significantly correlated with either disease phase ( p = 0.065) or response to therapy ( p = 0.79). Neither baseline serum levels of angiogenic cytokines correlated to response to treatment. No significant correlation was found between BM MVD and serum levels of angiogenic cytokines. Serum levels of angiogenic cytokines before and after therapy showed a significant increase of bFGF ( p = 0.008). Conclusion. There is no relationship between MVD estimation and baseline serum levels of angiogenic cytokines, neither between each of them and response to therapy.

High Serum Levels of Angiogenic Cytokines in AL Amyloidosis Correlates with Disease Features.

Angiogenesis is a crucial step for disease progression in several hematological malignancies, including plasma cell dyscrasias like multiple myeloma (MM). Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are antagonistic ligands for the receptor tyrosine kinase Tie-2. These cytokines are crucial for maturation and stabilization of the vascular wall: Ang-1 binds to Tie-2 and stabilizes the vascular wall, while Ang2- antagonizes Tie-2 binding and induces vessel destabilization, which leads to the angiogenic sprouting in conjunction with onset of angiogenesis. Vascular endothelial growth factor (VEGF), angiogenin and basic fibroblast growth factor (bFGF) are also potent stimulators of both physiological and pathological angiogenesis. However little is known about the role of these molecules in AL amyloidosis and their possible correlations with the features of the disease. Serum levels of Ang-1, Ang-2, VEGF, and VEGF-A (the major angiogenesis component of VEGF), angiogenin, and bFGF were evaluated using ELISA methodology (R&D Systems, Minneapolis, MN, USA, for all, except VEGF-A: Diaclone, Bensancon, France). Serum samples were collected from 62 previously untreated AL amyloidosis patients as well as from 35 age-matched healthy controls and 35 newly diagnosed, untreated, myeloma patients. Microvascular Density (MVD) of the bone marrow (BM) was also measured in 15 AL patients who had both serum and trephine biopsies available. Definition of organ involvement was based on established criteria (Gerrtz et al Am J Hematol 2005). Serum levels of VEGF (p<0.001), angiogenin (p<0.001), Ang-1(p=0.002) and Ang-2 (p<0.001) were significantly higher in AL patients than in controls; however the ang1/ang2 ratio was not different. The levels of Ang-2 (r=0.776, p=0.005), angiogenin (r=0.746, p=0.008) and bFGF ((r=0.743, p=0.009) correlated with MVD in BM biopsies of AL patients while VEGF, VEGF-A and Ang-1 were not. When compared to MM patients, AL patients had significantly higher VEGF levels (p=0.007), angiogenin (p=0.003) and Ang-1(p<0.001) levels but lower angiopoietin-2 levels (p=0.09); thus the Ang-1/Ang-2 ratio was higher in AL compared to MM (p=0.08). Both VEGF and VEGF-A were increased in patients with symptoms of peripheral neuropathy (p=0.016 and 0.029 respectively), while Ang-2 was increased in patients with heart involvement (p=0.03). There were no differences between AL patients with a creatinine ≥2 mg/dl and those with creatinine<2 mg/dl. In patients with BM plasma cells (PCs)>10%, Ang-1 was lower than in those with PCs<10% (p=0.013), while Ang-2 was not different (p=0.170); thus the Ang-1/Ang-2 ratio was lower in patients with BMPCs >10% (p=0.021). There was no correlation among the number of involved organs, a significant predictor of survival in AL amyloidosis, and levels of angiogenic cytokines. In conclusion, serum markers of angiogenesis are significantly higher in AL patients compared to both healthy individuals and MM patients indicating a possible pathogenetic role in some features of the disease and also a possible therapeutic target.

Angiogenetic Factors and Biochemical Markers of Bone Metabolism in POEMS Syndrome Treated with High-Dose Therapy and Autologous Stem Cell Support

The cause of the polyneuropathy, organomegaly, endocrinopathy, monoclonal plasma cell disorder and skin changes (POEMS) syndrome is currently unknown, but increased levels of vascular endothelial growth factor (VEGF) appear to play a pathogenetic role. Osteosclerotic bone lesions are a characteristic finding in POEMS, but there are no data about the specific role of various molecules that control bone remodeling in patients with POEMS. Serum levels of angiogenic cytokines (VEGF, angiogenin, angiopoietin-2, and basic fibroblast growth factor) along with a series of bone remodeling indices (C-telopeptide of type I collagen, bone-alkaline phosphatase [bALP], osteocalcin [OC], soluble receptor activator of nuclear factor–κB ligand [RANKL], osteoprotegerin [OPG], and macrophage inflammatory protein-1α) were measured in 2 patients with POEMS before and after high-dose therapy (HDT) with autologous stem cell transplantation and in age- and sex-matched controls. Increased VEGF levels before HDT were reduced significantly after treatment, although levels of the other angiogenetic factors did not differ from that of controls and were less influenced by HDT. Serum RANKL levels were increased before HDT, whereas OPG levels were within the levels of healthy controls, resulting in an abnormal soluble RANKL to OPG ratio. Levels of bone resorption markers (C-telopeptide of type I collagen) were very low or undetectable before HDT, although bALP and OC levels were similar to that of controls. After HDT, soluble RANKL levels decreased, OPG remained rather stable, bone resorption markers increased to levels of normal individuals, bALP levels were rather unchanged, and OC levels increased. Decreasing VEGF levels parallel clinical improvement, and the restoration of normal bone metabolism follows HDT.

The Combination of Bortezomib, Melphalan, Dexamethasone and Intermittent Thalidomide (VMDT) Is an Effective Treatment for Relapsed/Refractory Myeloma: Results of a Phase II Clinical Trial.

Interactions between myeloma (MM) cells and marrow microenvironment are crucial for myeloma growth and resistance to anti-myeloma therapy. Bortezomib (VELCADE®; V) and thalidomide (T) have proven anti-MM effect and exert their action partly through perturbation of the MM microenvironment. Furthermore, bortezomib enhances the cytotoxic potential of other agents, such as melphalan (M), and dexamethasone (D) in resistant cell lines. We hypothesize that combining VT (to target both MM cells and microenvironment) with M and D may help overcome resistance and increase clinical efficacy of these agents in relapsed/refractory disease. The aim of this phase II study was to determine the efficacy and safety of the VMDT regimen and its effect on angiogenesis and bone remodeling in relapsed/refractory MM. Bortezomib (1.0 mg/m2) was given iv, on days 1, 4, 8, and 11; oral melphalan (0.15 mg/kg) was administered on days 1–4, while thalidomide (100 mg/day) and dexamethasone (12 mg/m2) were given on days 1–4 and 17–20 of a 28-day cycle, for 4 cycles. Responders and patients with SD continued for up to 8 cycles. Effect of VMDT on angiogenesis was evaluated by measuring the serum levels of angiogenic cytokines, such as VEGF, angiogenin, angiopoietin-2, and bFGF at baseline and after cycles 4 and 8. Bone remodeling was studied by the measurement of a series of serum indices: i) osteoclast stimulating factors [sRANKL, osteoprotegerin (OPG), osteopontin, MIP-1α ], ii) bone resorption markers (CTX, TRACP-5b), and iii) bone formation markers [bALP, osteocalcin (OC), and CICP]. Thirty-one pretreated pts (median age: 66 y; range: 45–83 y) have been enrolled in this study, including 20 pts treated during refractory relapse. Median time from 1st treatment to VMDT was 40 months. Many pts had features of advanced disease including ISS stage 3 (32%), high LDH (23%), and creatinine >2mg% (10%). The median number of previous treatments was 2 (range: 1–6), including M (48% of pts), T (65%), D (100%), V (3%) and ASCT (39%). Among 25 pts evaluable for response so far, 14 (56%) achieved an objective response (CR 8% and PR 48%). Furthermore, 2 pts (8%) achieved a MR and 5 (20%) SD. Median time to response was 30 days. Adverse events included fatigue (56%), thrombocytopenia (12% grade 3/4), neutropenia (8% grade 3/4), anemia (8% grade 3), neuropathy (48% grade 1/2, no grade 3/4 observed), infections (36%, including 2 HZ cases), and hyponatremia (12%). No pt experienced DVT, while 2 pts died due to sepsis. At baseline, MM pts had increased serum levels of sRANKL, sRANKL/OPG ratio, MIP-1α , VEGF, angiogenin, angiopoietin-2, and bFGF (p<0.01) compared with controls, while serum levels of CTX, TRACP-5b, bALP, OC were decreased (p<0.03) since all pts have been on zoledronic acid treatment. Our preliminary analysis showed that sRANKL and MIP-1α levels reduced after 4 cycles of VMDT. In conclusion the VMDT combination with intermittent T is associated with significant activity in pts with heavily pretreated MM. The toxicities are manageable; due to intermittent T administration there was no severe neurotoxicity and no DVT. Our study will also provide further data regarding post-treatment changes of cytokines involved in interactions between MM and stromal cells.

The combination of intermediate doses of thalidomide and dexamethasone reduces bone marrow micro‐vessel density but not serum levels of angiogenic cytokines in patients with refractory/relapsed multiple myeloma

The aim of the study was the evaluation of anti-angiogenic activity of the combination of intermediate doses of thalidomide and dexamethasone in patients with refractory/relapsed myeloma. Twenty-five patients were included in the study. Microvessel density (MVD) was evaluated in marrow biopsies before and after treatment. Serum levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FGF), tumor necrosis factor-alpha (TNF-alpha), which have angiogenic potential and interleukin-6 (IL-6), IL-1beta, soluble IL-6 receptor (sIL-6R), and transforming growth factor-beta (TGF-beta) which are involved in the disease biology, were measured before treatment and then every 2 weeks for 8 weeks. Pretreatment levels of MVD, VEGF, b-FGF, IL-6, sIL-6R were increased in the patients compared to controls. The overall response rate to therapy was 72%. The administration of the combined regimen produced a significant reduction in MVD in responders. However, an increase in serum levels of VEGF, b-FGF, IL-6, sIL-6R was observed post-treatment in responders. In contrast, serum levels of TNF-alpha, TGF-beta, IL-1beta did not differ between patients and controls and remained unchanged during the study. These results suggest that the combination of thalidomide plus dexamethasone is an effective treatment for myeloma reducing MVD marrow levels but not serum levels of angiogenic cytokines or cytokines implicated in myeloma biology.

Serum levels of angiogenic cytokines decrease after antineoplastic radiotherapy

Serum levels of angiogenic cytokines decrease after radiotherapy in patients with cancer and their may have an impact on response to treatment and progression-free survival. Here, we have evaluated sera of patients before and after radiotherapy for various tumour types for levels of soluble fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) to assess whether these factors decrease after radiotherapy, and whether their diminution is related to the radiation dose, tumour type, age and haemoglobin level. We demonstrate that levels of FGF-2 and VEGF, but not HGF, decrease significantly, and that the extent of their diminution is related to the radiation dose and response.

Elevated preoperative serum levels of angiogenic cytokines correlate to larger primary tumours and poorer survival in non-small cell lung cancer patients

We have analysed the predictive and prognostic information in preoperatively collected serum levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in patients clinically evaluated as operable non-small cell lung cancer (NSCLC). Fifty-eight patients with operable NSCLC were included. VEGF and bFGF levels in serum were analysed using enzyme linked immunosorbent assays (Quantikine™ human VEGF and Quantikine™ HS human FGF basic, R&D Systems). Univariate analysis demonstrated that tumour volume, platelet counts, VEGF and bFGF were significant prognostic factors. However, only bFGF remained significant in the multivariate analysis ( P=0.014). Significant correlation's were demonstrated between VEGF levels and tumour volume ( r=0.33; P=0.012) and platelet count ( r=0.43; P=0.001). bFGF levels correlated significant with recurrent disease ( r=0.34; P=0.01), platelet count ( r=0.53, P<0.001) and performance status ( r=0.29; P=0.029). Furthermore, bFGF levels and VEGF levels correlated significantly ( r=0.44; P<0.001). We conclude that elevated circulating angiogenic cytokines correlate with tumour volume, higher relapse risk and poorer survival in patients with operable non-small cell lung cancer.

Plasma fibrin D-dimer levels correlate with tumour volume, progression rate and survival in patients with metastatic breast cancer.

Plasma levels of D-dimer are elevated in cancer patients. Activation of the extrinsic coagulation system and the fibrinolytic cascade within a tumour is thought to be related with growth, invasion and metastasis. We have investigated the relationship between these markers of fibrin metabolism, standard clinicopathological variables and serum levels of angiogenic cytokines in three cohorts: group A (n=30) consisted of 30 healthy female volunteers, group B (n=23) of consecutive patients with operable breast cancer and group C (n=84) of patients with untreated or progressive metastatic breast cancer. Plasma D-dimers, fibrinogen, IL-6, vascular endothelial growth factor and calculated vascular endothelial growth factor load in platelets are clearly increased in patients with breast cancer. D-dimers were increased in nearly 89% of patients with progressive metastatic disease. The level of D-dimers was positively correlated with tumour load (P<0.0001), number of metastatic sites (P=0.002), progression kinetics (P<0.0001) and the cytokines related to angiogenesis: serum vascular endothelial growth factor (P=0.0016, Spearman correlation=0.285), calculated vascular endothelial growth factor load in platelets (P<0.0001, Spearman correlation=0.37) and serum interleukin-6 (P<0.0001, Spearman correlation=0.59). Similarly increased D-dimer levels were positively correlated with increased fibrinogen levels (P<0.0001, Spearman correlation=0.38). The association between markers of fibrin degradation in patients with progressive breast cancer suggests that the D-dimer level is a clinically important marker for progression and points towards a relation between haemostasis and tumour progression. A role of interleukin-6, by influencing both angiogenesis and haemostasis, is suggested by these observations.British Journal of Cancer (2002) 86, 389–395. DOI: 10.1038/sj/bjc/6600069 www.bjcancer.com© 2002 The Cancer Research Campaign