Addition of cladribine to the standard induction treatment improves outcomes in a subset of elderly acute myeloid leukemia 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.

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.

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.

The kinetics and apoptotic profile of circulating endothelial cells in autologous hematopoietic stem cell transplantation in patients with lymphoproliferative disorders.

In neoplastic disorders, endothelial cells take part in tumor progression and also influence the recovery of hematopoiesis after high-dose chemotherapy. Measurements of circulating endothelial cells (CEC), their subsets and kinetics were taken in patients with lymphoid malignancies (37 multiple myeloma, ten lymphoma) during autologous hematopoietic stem cell transplantation (HSCT). CEC were evaluated by four-color flow cytometry at different time points. Additionally levels of angiopoietins 1 and 2 were evaluated by ELISA assay. The baseline number of CECs and their subsets in patients were higher than in the control group. The median CEC number dropped significantly after transplantation (from 9.5/µL to 6.2/µL, p < 0.001). Apoptosis of CECs 24 h after chemotherapy was enhanced in comparison to baseline values (median apoptotic CEC number 4.15/µL vs 3.1/µL; p < 0,001). The time for neutrophil engraftment was shorter for patients with a low apoptotic CEC count at baseline as compared to those with a high apoptotic CEC count (median time to engraftment 13 vs. 16 days respectively, p = 0.04). We observed an adverse correlation of progenitor CEC numbers measured 1 h after transplantation with the time to neutrophil engraftment (r = -0.49, p = 0.008). We also found a negative correlation between the number of CECs originating from microvessels measured 1 h after transplantation, and the time to neutrophil engraftment (r = -0.39, p = 0.04). Baseline angiopoietins 1 and 2 concentration did not influence the post-transplant regeneration time. CEC numbers significantly change during autologous HSCT. Our results suggest that progenitor CECs and CECs derived from microvessels both take part in successful engraftment.

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 × 10(6)/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.

The Association between Immune Checkpoint Proteins and Therapy Outcomes in Acute Myeloid Leukaemia Patients.

The development of novel drugs with different mechanisms of action has dramatically changed the treatment landscape of AML patients in recent years. Considering a significant dysregulation of the immune system, inhibitors of immune checkpoint (ICI) proteins provide a substantial therapeutic option for those subjects. However, use of ICI in haematological malignancies remains very limited, in contrast to their wide use in solid tumours. Here, we analysed expression patterns of the most promising selected checkpoint-based therapeutic targets in AML patients. Peripheral blood of 72 untreated AML patients was used for flow cytometric analysis. Expression of PD-1, PD-L1, CTLA-4, and B7-H3 was assessed within CD4+ (Th) lymphocytes and CD33+ blast cells. Patients were stratified based on therapy outcome and cytogenetic molecular risk. AML non-responders (NR) showed a higher frequency of PD-1 in Th cells compared to those with complete remission (CR). Reduced blast cell level of CTLA-4 was another factor differentiating CR from NR subjects. Elevated levels of PD-1 were associated with a trend for poorer patients' survival. Additionally, prognosis for AML patients was worse in case of a higher frequency of B7-H3 in Th lymphocytes. In summary, we showed the significance of selected ICI as outcome predictors in AML management. Further, multicentre studies are required for validation of those data.

Targeting Apoptosis in AML: Where Do We Stand?

More than 97% of patients with acute myeloid leukemia (AML) demonstrate genetic mutations leading to excessive proliferation combined with the evasion of regulated cell death (RCD). The most prominent and well-defined form of RCD is apoptosis, which serves as a defense mechanism against the emergence of cancer cells. Apoptosis is regulated in part by the BCL-2 family of pro- and anti-apoptotic proteins, whose balance can significantly determine cell survival. Apoptosis evasion plays a key role in tumorigenesis and drug resistance, and thus in the development and progression of AML. Research on the structural and biochemical aspects of apoptosis proteins and their regulators offers promise for new classes of targeted therapies and strategies for therapeutic intervention. This review provides a comprehensive overview of current AML treatment options related to the mechanism of apoptosis, particularly its mitochondrial pathway, and other promising concepts such as neddylation. It pays particular attention to clinically-relevant aspects of current and future AML treatment approaches, highlighting the molecular basis of individual therapies.

Alterations in microRNA Expression during Hematopoietic Stem Cell Mobilization.

microRNAs play an important role in the regulation of gene expression, cell fate, hematopoiesis, and may influence the efficacy of CD34+ cell mobilization. The present study examines the role of hsa-miR-15a-5p, hsa-miR-16-5p, hsa-miR-34a-5p, hsa-miR-126-3p, hsa-miR-146a-5p, hsa-miR-155-5p, and hsa-miR-223-3p in the course of hematopoietic stem cell mobilization. The numbers of CD34+ cells collected in patients with hematological malignancies (39 multiple myelomas, 11 lymphomas) were determined during mobilization for an autologous hematopoietic stem cell transplantation. The miRNA level was evaluated by RT-PCR. Compared to baseline, a significant decline in hsa-miR-15a-5p, hsa-miR-16-5p, hsa-miR-126-3p, hsa-miR-146a-5p, and hsa-miR-155-5p was observed on the day of the first apheresis (day A). An increase was observed only in the expression of hsa-miR-34a-5p. On day A, a negative correlation was found between hsa-miR-15a-5p and hsa-miR-146a-5p levels and the number of CD34+ cells in peripheral blood. A negative correlation was observed between hsa-miR-146a-5p and the number of collected CD34+ cells after the first apheresis. Good mobilizers, defined according to GITMO criteria, demonstrated a lower hsa-miR-146a-5p level on day A than poor mobilizers. Patients from the hsa-miR-146a-5p "low expressors" collected more CD34+ cells than "high expressors". Our results suggest that the investigated miRNAs, especially hsa-miR-146a-5p, may influence the efficacy of HSC mobilization.

The role of NF-κB and Smac/DIABLO proteins in the treatment response and survival of acute myeloid leukemia patients.

INTRODUCTION: The misbalance between a family of inhibitor of apoptosis proteins (IAP), regulated by the nuclear factor kappa B (NF-κB) and their natural antagonist second mitochondrial-derived activator of caspases/direct IAP binding protein with low pI (Smac/DIABLO) are important to biology of acute myeloid leukemia (AML). MATERIAL AND METHODS: The aim of the study was to assess NF-κB and Smac/DIABLO proteins expression in blasts of 109 newly diagnosed AML patients using the multicolor flow cytometry and evaluate their influence on AML patients outcome. RESULTS: Expression of NF-κB and of Smac/DIABLO proteins were found in 95% and 98% of the patients, respectively. A negative correlation between Smac/DIABLO and NF-κB was observed. Age < 60 years old as well as higher Smac/DIABLO expression were associated with a higher probability of complete response achievement in the multivariate analysis. Longer overall survival (OS) in the univariate and multivariate analyses was influenced by age < 60 years old, a favorable or intermediate-risk karyotype and high Smac/DIABLO expression. Additionally, in the survival analysis of the subgroups, the patients aged < 60 years old, with high Smac/DIABLO expression, lower NF-κB expression and < 50% of bone marrow blasts who were treated with standard treatment had better OS. CONCLUSIONS: Lower NF-κB and higher Smac/DIABLO expression may influence AML patients outcome.

Low serum albumin level deteriorates prognosis in azacitidine-treated myelodysplastic syndromes patients - results of the PALG study 'PolAZA'.

BACKGROUND: Azacitidine (AZA) is the standard of care for higher-risk myelodysplastic syndrome (HR-MDS) patients ineligible for intensive therapy. Clinical outcome discrepancies reported in clinical trials and real-life settings stimulate the search for new prognostic factors. METHODS: We retrospectively evaluated 315 MDS, 20-30% blast acute myeloid leukemia (AML) and chronic myelomonocytic leukemia (CMML) patients treated with azacitidine in 12 centers cooperating within the Polish Adult Leukemia Group (PALG). RESULTS: The median number of AZA cycles was 7 (1-69) and 24% patients received fewer than 4 cycles (early failure, EF). Serum albumin level was an independent predictor of EF occurrence. Complete remission (CR) was obtained in 20% and partial remission (PR) in 12% of patients. Hematologic improvement - erythroid (HI-E), neutrophil (HI-N), or platelet (HI-P) was achieved in 51%, 36%, and 48% of patients, respectively. No factors significantly predicted CR or PR in the multivariate analysis. For HI-E and HI-P, lower LDH level predicted response. Median survival was 15 (13-19) months. Lower serum albumin level, serious infection and receiving <4 AZA cycles independently predicted a worse overall survival (OS) (p < 0.05). CONCLUSION: Serum albumin assessment before azacitidine treatment can help to identify patients with higher risk of early failure and worse clinical outcome.

Early induction intensification with cladribine, cytarabine, and mitoxantrone (CLAM) in AML patients treated with the DAC induction regimen: a prospective, non-randomized, phase II study of the Polish Adult Leukemia Group (PALG).

We present the results of a prospective, non-randomized phase 2 trial in which 253 AML patients (pts) under 60 years old received DAC (Daunorubicin + AraC + Cladribine) as first induction followed by CLAM (Cladribine + AraC + Mitoxantrone) as early second induction on day 16 based on bone marrow (BM) blasts on day 14 (D14). The CR/CRi rate after a single course of DAC was 83% for pts with D14 BM blasts less than 10%. Forty-six pts had >10% BM blasts on D14, of whom 35 received CLAM with rates of CR/CRi 60% and early death (ED) 23%. The remaining 11 pts were not fit to receive CLAM, with rates of CR/CRi 28%, PR 18%, and ED 18%. Median OS was 7.2 versus 7.5 months, respectively. The overall CR/CRi rate was 77% after the first induction, with final CR/CRi rate 80% after DAC reinduction for pts who achieved PR with initial DAC course. CLAM used as early second induction might improve CR/CRi rates for younger AML pts with poor early response to DAC induction, but may be associated with higher mortality.

The efficacy of sapacitabine in treating patients with acute myeloid leukemia.

INTRODUCTION: Acute myeloid leukemia (AML) remains a poor prognosis hematological malignancy. The introduction of aggressive chemotherapy with allogeneic stem cell transplantation has resulted in improved clinical outcomes in younger patients. However, the treatment results in unfit elderly AML population remain disappointing. New strategies should be introduced to improve the prognosis in this group of patients. Areas covered: This review presents and discusses the mechanism of action, safety and efficacy of sapacitabine in AML patients. Expert opinion: Sapacitabine, a novel nucleoside analog, seemed to be a promising new agent for AML treatment. Its oral bioavailability and tolerable toxicity profile allow the drug to be used in an outpatient setting, especially in elderly unfit patients. Sapacitabine is known to have antileukemic activity in randomized clinical trials. In AML patients, sapacitabine monotherapy offered no advantage over low-intensity cytarabine treatment, and the combination of sapacitabine with decitabine was not significantly more effective than decitabine alone. However, the oral administration of sapacitabine allows it to be used in AML maintenance therapy.

Angiopoietins in haematopoietic stem cell mobilisation in patients with haematological malignancies.

BACKGROUND: The bone marrow niche contains different types of cells including osteoblasts and endothelial progenitors, all of which interact and take part in the process of mobilisation. The aim of our study was to evaluate the levels of cytokines (osteopontin and angiopoietins 1 and 2) active in the bone marrow niche during the mobilisation of haematopoietic stem cells for autologous transplantation. MATERIALS AND METHODS: Forty-eight patients (24 females, 24 males), median age 56.5 years, entered the study. The group consisted of patients with multiple myeloma (n=34), lymphoma (n=13) and acute myeloid leukaemia (n=1). Blood samples were collected before chemotherapy and on the day of the first apheresis. Cytokines were evaluated by enzyme-linked immunosorbent assays. Additionally, circulating endothelial cells were assessed by flow cytometry. RESULTS: The median concentration of angiopoietin 1 at the time of apheresis was lower than that at baseline (2.7 vs 7.8 ng/mL, p<0.001). In contrast, the median level of angiopoietin 2 increased during the mobilisation procedure (3.6 vs 2.8 ng/mL, p=0.001). The patients were divided according to the number of days of granulocyte colony-stimulating factor treatment before the first apheresis into "early" (median) mobilisers. The group of "early mobilisers" had higher baseline angiopoietin 1 levels (median=11.6 ng/mL) than those of the "late mobilisers" (median=6.0 ng/mL, p=0.05). An adverse correlation was observed between duration of granulocyte colony-stimulating factor treatment and baseline angiopoietin 1 level. Baseline angiopoietin 1 levels correlated with numbers of circulating endothelial cells. Low angiopoietin 2 level increased the chance of poor mobilisation. CONCLUSIONS: The angiogenic processes can influence the timing of mobilisation. Angiopoietins 1 and 2 need further evaluation in the context of mobilisation.

Prognostic value of inhibitor of apoptosis protein family expression in patients with acute myeloid leukemia.

The inhibitor of apoptosis protein (IAP) family acts as an inhibitor of apoptosis pathways. The potential prognostic value of the expression of selected IAP family members, XIAP, cIAP-1, cIAP-2 and survivin protein, was evaluated with regard to treatment response and survival of 56 newly diagnosed adult patients with acute myeloid leukemia (AML). The presence of these IAP members influenced the achievement of a complete response (CR). In addition, overall survival (OS) was influenced by low survivin expression in univariate and multivariate analysis (p = 0.014 and p = 0.013, respectively). A strong correlation was observed between members of the IAP family (XIAP and cIAP-1, XIAP and cIAP-2, cIAP-1 and cIAP-2, p < 0.001 for all comparisons), while Smac/DIABLO demonstrated an inverse correlation with XIAP, cIAP-1 and cIAP-2 (p < 0.001 for all comparisons). Further studies should be undertaken to better demonstrate the mode of action of IAP members, as well as their prognostic and therapeutic potentials.

Jagged-1: a new promising factor associated with favorable prognosis in patients with acute myeloid leukemia.

In this study Jagged-1 and Dll-1 surface expression as well as Notch-1 receptor intracellular domain (Notch-1-IC) expression were assessed by multi-color flow cytometry in leukemic blasts obtained from 88 patients with acute myeloid leukemia (AML). CD34+peripheral blood stem cells (PBSCs) were used as a control. The median expression of Jagged-1 and Dll-1 was significantly higher in AML blasts than in PBSCs (p=0.001 and p=0.002, respectively). Higher expression of Notch-1-IC was detected in patients with poor-risk karyotype as compared to good- and intermediate-risk groups (p=0.035). In our study, poor-risk cytogenetics and low (

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.