Agnieszka Pluta

Cladribine combined with high doses of arabinoside cytosine, mitoxantrone, and G-CSF (CLAG-M) is a highly effective salvage regimen in patients with refractory and relapsed acute myeloid leukemia of the poor risk: a final report of the Polish Adult Leukemia Group.

Objectives: Patients with primary refractory AML and with early relapses have unfavorable prognoses and require innovative therapeutic approaches. Purine analogs fludarabine (FA) and cladribine (2‐CdA) increase cytotoxic effect of Ara‐C in leukemic blasts and inhibit DNA repair mechanisms; therefore its association with Ara‐C and mitoxantrone (MIT) results in a synergistic effect. In the current report, we present the final results of multi‐center phase II study evaluating the efficacy and toxicity of CLAG‐M salvage regimen in poor risk refractory/relapsed AML patients.

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.

Influence of high expression of Smac/DIABLO protein on the clinical outcome in acute myeloid leukemia patients

The role of the Smac/DIABLO protein, a novel apoptosis agonist, in acute myeloid leukemia (AML) is not clearly determined. The expression of Smac/DIABLO protein in AML leukemic cells and its relationship with clinical outcome was evaluated in this study. The intracellular expression of Smac/DIABLO protein was assessed using multi-color flow cytometry in 71 newly diagnosed AML patients treated with conventional chemotherapy. It was found that the high expression of Smac/DIABLO protein was an independent prognostic factor in terms of higher complete remission rate (p<0.001) and longer overall survival (p=0.003). Moreover the low expression of Smac/DIABLO protein was associated with poor karyotype.

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.

Addition of cladribine to the standard induction treatment improves outcomes in a subset of elderly AML patients. Results of a randomized Polish Adult Leukemia Group (PALG) phase II trial

Intensive induction chemotherapy using anthracycline and cytarabine backbone is considered the most effective upfront therapy in physically fit older patients with acute myeloid leukemia (AML). However, outcomes of the standard induction in elderly AML are inferior to those observed in younger patients, and they are still unsatisfactory. As addition of cladribine to the standard induction therapy is known to improve outcome in younger AML patients. The present randomized phase II study compares efficacy and toxicity of the DAC (daunorubicin plus cytarabine plus cladribine) regimen with the standard DA (daunorubicin plus cytarabine) regimen in the newly diagnosed AML patients over 60 years of age. A total of 171 patients were enrolled in the study (DA, 86; DAC, 85). A trend toward higher complete remission (CR) was observed in the DAC arm compared to the DA arm (44% vs. 34%; P = .19), which did not lead to improved median overall survival, which in the case of the DAC group was 8.6 months compared to in 9.1 months in the DA group (P = .64). However, DAC appeared to be superior in the group of patients aged 60‐65 (CR rate: DAC 51% vs. DA 29%; P = .02). What is more, a subgroup of patients, with good and intermediate karyotypes, benefited from addition of cladribine also in terms of overall survival (P = .02). No differences in hematological and nonhematological toxicity between the DA and DAC regimens were observed.

Different prognosis of acute myeloid leukemia harboring monosomal karyotype with total or partial monosomies determined by FISH: Retrospective PALG study

A monosomal karyotype (MK) was identified by banding techniques (BT) in acute myeloid leukemia (AML). However, BT may be insufficient to determine the actual loss of a complete chromosome, especially in complex karyotypes. We have investigated the effect of monosomy type, total (MK-t) and partial (MK-p), reevaluated by FISH, on prognosis. We have found that complete remission rate and probability of overall survival at 1 year was higher in MK-p (n=27) than MK-t (n=15) group (40% vs. 15.4%, P=0.19 and 30% vs. 9%, P=0.046, respectively). Our results indicate for the first time that monosomy type influences the prognosis of MK-AML.

Concomitance of monosomal karyotype with at least 5 chromosomal abnormalities is associated with dismal treatment outcome of AML patients with complex karyotype – retrospective analysis of Polish Adult Leukemia Group (PALG)

Abstract Monosomal karyotype (MK) and complex karyotype (CK) are poor prognostic factors in acute myeloid leukemia (AML). A comprehensive analysis of cytogenetic and clinical factors influencing an outcome of AML-CK+  was performed. The impact of cladribine containing induction on treatment results was also evaluated. We analyzed 125 patients with AML-CK+  treated within PALG protocols. MK was found in 75 (60%) individuals. The overall complete remission (CR) rate of 66 intensively treated patients was 62% vs. 28% in CK+ MK− and CK+ MK+  group (p = .01). No difference in CR rate was observed between DA and DAC arms. The overall survival (OS) in intensively treated patients was negatively influenced by MK, karyotype complexity (≥5 abnormalities), and WBC >20 G/L in multivariate analysis. The addition of cladribine to DA regimen improved OS only in MK− but not in MK+  group. In conclusion, concomitance of MK with ≥5 chromosomal abnormalities is associated with dismal treatment outcome in AMK-CK+.

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.

Significance of Bax expression in breast cancer patients.

UNLABELLED Bax protein, the proapoptotic member of Bcl-2 protein family, plays the key role in apoptosis pathway. THE AIM OF THE STUDY was to assess the expression of Bax protein in breast cancer cells. MATERIAL AND METHODS Sixty-two breast cancer patients were included in the study. The control group encompassed 11 fibroadenoma patients. Single cells were isolated from defrosted samples and prepared for flow cytometry measurement. RESULTS Median expression of Bax protein in study group was 7.9% (range: 0-49.4%) and was significantly lower than in control (median expression 15.8%; range 4.9-30.9%; p=0.034). Expression of Bax correlated with expression of p53 and caspase-3 proteins (p<0.01, rank Spearman test). In patients under 70 years old and with positive estrogen receptors status the expression of Bax protein was significantly higher (p=0.03 and p=0.01 respectively). CONCLUSIONS Lower expression of Bax protein in breast cancer cells may suggest the potential way of apoptosis avoidance of tumor cells. Correlations among Bax protein, p53 and caspase-3 are likely associated with active apoptotic mechanism in breast cancer cells expressing Bax protein. Further investigation with long time follow-up should be performed to establish the prognostic role of Bax protein expression in breast cancer patients.