Anna Krawczynska

Circulating endothelial cells in patients with acute myeloid leukemia.

Abstract:  Objectives: The circulating endothelial cells (CEC) are proposed to be a non‐invasive marker of angiogenesis. The level of CEC in peripheral blood (PB) of acute myeloid leukemia (AML) patients has not been investigated prior to this study. We evaluated the count of resting (rCEC), activated (aCEC) and endothelial progenitor cells (CEPC) in the PB of AML and healthy subjects. In addition we correlated the levels of CEC with disease status, known prognostic factors and response to treatment. Methods: CEC were quantified by utilizing four‐color flow cytometry procedures in 48 AML patients at the time of diagnosis and 29 healthy controls. Additionally, measurements were again taken after the first course of induction treatment in 12 of the patients. Results: The numbers of aCEC, rCEC and CEPC were significantly higher in the AML patients than in the controls (P < 0.0001, P < 0.0001 and P < 0.001, respectively). The CEC count was significantly higher in the AML patients with white blood cell count (WBC) >15 G/L, elevated lactic dehydrogenase (LDH) levels and a higher (over median) absolute blasts count (ABC) in PB than in the group with WBC <15 G/L (P < 0.03), a normal LDH level (P < 0.03) and a lower (

Kinetics and apoptotic profile of circulating endothelial cells as prognostic factors for induction treatment failure in newly diagnosed acute myeloid leukemia patients

The circulating endothelial cells (CEC) are proposed to be a noninvasive marker of angiogenesis. Recent data suggest that endothelial cells may enhance the survival and proliferation of leukemic blasts and mediate chemotherapy resistance in acute myeloid leukemia (AML). We analyzed CEC count by the four-color flow cytometry in AML and healthy subjects. We evaluated the kinetics of mature CEC, both resting (rCEC) and activated (aCEC), as well as progenitor (CEPC) and apoptotic CEC (CECAnnV+) in AML patients treated with standard chemotherapy and their influence on response to treatment and overall survival. We found significantly higher numbers of aCEC, rCEC, CEPC, and CECAnnV+ in AML patients than in healthy controls. The elevated CEPC and absolute blood counts in peripheral blood as well as the low CECAnnV+ number were associated with higher probability of induction treatment failure. aCEC, rCEC, CEPC, and CECAnnV+ counts determined in complete remission (CR) were significantly lower than those found at diagnosis. In those CR patients, a significant decrease in the CEC count and increase in the number of CECAnnV+ were observed already 24h after the first dose of chemotherapy. In refractory AML, the aCEC, rCEC, CEPC, and CECAnnV+ counts assessed before and after induction chemotherapy did not differ significantly, and a significant decrease in CEC count and increase in CECAnnV+ number were noted only after the last dose of chemotherapy. The number of CEC is significantly higher in AML patients than in healthy subjects and correlates with response to treatment. The evaluation of CEC kinetics and apoptotic profile may be a promising tool to select AML patients with poor response to chemotherapy who may benefit from antiangiogenic therapies.

Circulating endothelial cells in essential thrombocythemia and polycythemia vera: correlation with JAK2-V617F mutational status, angiogenic factors and coagulation activation markers

Angiogenesis plays an important role in the biology of hematological malignancies, including essential thrombocythemia (ET) and polycythemia vera (PV). Some data suggests that it has a role in the pathogenesis of thrombosis, the major clinical problem in ET and PV. The number of different subpopulations of circulating endothelial cells (CECs), plasma levels of vascular endothelial growth factor (VEGF), soluble vascular endothelial growth factor receptor 1 and 2 (sVEGFR-1,2) and placenta growth factor (PlGF) were determined in 30 patients with ET and 16 patients with PV. Correlations between angiogenesis and JAK2-V617F mutational status, risk factors for thrombosis and coagulation activation markers were also assessed. The number of CEC subpopulations, were markedly higher in ET and PV patients, irrespective of JAK2-V617F status, when compared to the control group. The median values of activated CECs were markedly higher in PV patients with WBC >8.7 (×109/l). Significantly higher VEGF plasma levels were found in ET patients and a similar trend was seen in PV patients in relation to healthy volunteers. The plasma levels of sVEGFR-1 were significantly higher, and PlGF levels markedly lower, in the ET and PV group than in controls. Our study also demonstrated markedly increased levels of D-dimer and TAT complexes in the patient groups. In conclusion, we found that angiogenesis, as measured by CEC numbers, is increased in ET and PV patients regardless of JAK2-V617F mutational status. Our results demonstrated that angiogenic cytokines interact with known thrombotic risk factors. We confirmed the coagulation activation in ET and PV patients but found no differences in levels of coagulation activation markers in relation to JAK2-V617F mutational status.

Polymorphism of CD44 Influences the Efficacy of CD34+ Cells Mobilization in Patients with Hematological Malignancies

In the last decade, peripheral blood was the main source of hematopoietic stem cells (HSC) for autologous and allogeneic transplantation. The exact mechanisms of HSC mobilization are still not clear and the efficacy of the procedure is hardly predictable. Ligand-receptor interactions of adhesion molecules, such as SDF1/CXCR4, VLA4/VCAM-1, or CD44/osteopontin, play an important role in homing of HSC in the hematopoietic niche. There is some evidence that disruption of the ligand-receptor complex leads to the egress of HSCs to the peripheral blood. The aim of the present study was the evaluation of constitutive polymorphism of genes encoding cytokines and receptors present in the HSC niche and their impact on the efficacy of mobilization of HSCs in patients with hematological malignancies. We enrolled 110 patients (60 females and 50 males) in the study. The median age of the patients was 55 (range, 22 to 69) years. The group consisted of patients with multiple myeloma (n = 74), non-Hodgkin lymphoma (n = 19), Hodgkin lymphoma (n = 15), or acute myeloid leukemia (n = 2). The mobilization procedures comprised chemotherapy and subsequent granulocyte-colony stimulating factor (G-CSF) at a dose of 10 μg/kg daily. The poor mobilizers group was defined according to Italian National Bone Marrow Transplant Registry criteria: patients with peak CD34(+) in the peripheral blood < 20/μL or total yield < 2 × 10(6) CD34(+) cells/kg body weight in maximum 3 aphereses. Genotyping was performed using standard PCR-based assays. The group of patients (N = 108) who achieved minimal threshold for collections (CD34(+) at least 10/μL) proceeded to apheresis. The median total yield of CD34(+) in this group was 5.6 × 10(6) cells/kg body weight, whereas the median number of cells collected during the first apheresis was 3.3 × 10(6) cells/kg body weight. Median number of days of G-CSF treatment before first apheresis was 10. Fifteen patients fulfilled the criteria for poor mobilizer. The group of poor mobilizers had higher frequency of TT genotype in rs13347 (CD44) gene (CC+ CT versus TT P = .047). Patients homozygous for T allele had a lower total yield of CD34(+) cells/kg body weight than the group with allele C (median, 3.7 × 10(6)/kg versus 5.8 × 10(6)/kg; P = .019) and a lower number of CD34(+) cells gathered during first apheresis (.95 × 10(6)/kg versus 3.3 × 10(6)/kg, P = .04). Multivariate logistic regression analysis revealed that the CD44 TT genotype was the only factor associated with 5-fold higher risk of poor mobilization (P = .037). Polymorphic variants of CXCR4 and VCAM-1 did not significantly influence the efficacy of HSCs mobilization in our group of patients. In conclusion, our results indicate that among investigated single nucleotide polymorphisms (SNPs), only CD44 rs13347 has an impact on the efficacy of HSCs mobilization in patients with hematologic malignancies. CD44 SNPs analysis may be helpful for predicting the poor mobilizers population who may benefit from newer modalities using adhesion molecules inhibitors.

Circulating endothelial cell kinetics and their potential predictive value during mobilization procedure.

Objective: Circulating endothelial cells (CECs) in patients with hematological malignancies are assessed as a noninvasive marker of angiogenesis. The aim of this study was to evaluate the numbers of CECs and their subsets during mobilization of hematopoietic stem cells. Patients and methods: Thirty‐eight patients were enrolled to the study (19 females and 19 males) at median age of 56.5 years. The group consisted of patients with multiple myeloma (26), lymphoma (10), and acute myeloid leukemia (2). Blood samples were collected before chemotherapy (0), 1 day after chemotherapy (Cht+1), on the day G‐CSF commenced (G0), after 1 day of G‐CSF (G+1), and on the day of the first apheresis. CECs were evaluated by four‐color flow cytometry. Circulating progenitor cells were defined as CD45−/CD34+/CD31+/CD133+. Apoptotic CECs (ApoCECs) were defined as CD146+/AnnexinV+. Results: Median (Me) CECs number was 10.5/µl and it decreased after chemotherapy (Me = 8.3/µl, P < 0.001 when compared with baseline). Based on the number of aphereses needed to obtain 2 × 106/kg CD34+ cells, patients were divided into “highly efficient” (one apheresis) and “poorly efficient” mobilizers (two or more aphereses). Median ApoCEC at Day G+1 was lower in highly efficient than in poorly efficient mobilizers (Me = 3.1/µl vs. Me = 5.1/µl, P = 0.02). ApoCEC at Day G+1 correlated with the number of aphereses (r = 0.48, P = 0.03). In multivariate analysis, ApoCEC at Day G+1 was an independent factor for successful mobilization during one apheresis. Conclusions: CECs and their subsets change significantly during mobilization of HSCs. ApoCECs measured at the time of G‐CSF commencement can predict the efficacy of HSC collection. J. Clin. Apheresis 28:341–348, 2013.

The kinetics of hematopoietic niche cytokines and their influence on mobilization efficacy and timing in patients with hematological malignancies.

The bone marrow niche functions are modulated by complicated cytokines network. The aim of our study was to evaluate the levels of VCAM‐1, VEGF, MMP‐9 and SDF during mobilization of CD34+ cells in patients with hematological malignancies. Thirty four patients were enrolled to the study (19F, 15 M) at median age of 57 years. The group consisted of patients with multiple myeloma (26) and lymphoma (8). The mobilization procedures comprised chemotherapy and then G‐CSF. Blood samples were collected before chemotherapy (N = 34) and on the day of the first apheresis (N = 26). Cytokines were evaluated with ELISA assay. We observed significant increase in VCAM‐1 levels during mobilization. On contrary, VEGF and SDF levels decreased during mobilization procedure. The levels of MMP‐9 were stable during mobilization. We divided patients according to baseline cytokines levels below and above median into “low” and “high” expressors. The group of VEGF “low” expressors had longer median time of G‐CSF treatment before first apheresis than ‘high’ expressors. Baseline VEGF levels correlated adversely with duration of G‐CSF treatment before first apheresis. Patients were also divided according to median cytokines levels at apheresis into “low” and “high” expressors. “High” VCAM‐1 expressors had higher CD34+in peripheral blood as well as higher CD34+numbers collected during first apheresis than “low” expressors. In conclusion, the levels of niche cytokines change significantly during mobilization in patients with hematopoietic malignancies. Baseline VEGF can influence timing of mobilization. Higher VCAM‐1 corresponds with higher mobilization efficacy. J. Clin. Apheresis 30:247–251, 2015.