Anna Szmigielska-Kaplon

Rituximab combined with cladribine or with cladribine and cyclophosphamide in heavily pretreated patients with indolent lymphoproliferative disorders and mantle cell lymphoma

In vitro studies have shown synergistic or additive interactions between rituximab and purine nucleoside analogues. The results of recent clinical trials seem to confirm these preclinical observations.

Proapoptotic activity of alemtuzumab alone and in combination with rituximab or purine nucleoside analogues in chronic lymphocytic leukemia cells

Proapoptotic activity of anti-CD52 monoclonal antibody, alemtuzumab (ALT) as well as ALT-affected apoptosis-regulatory mechanisms were assessed in tumor cells from 36 patients with chronic lymphocytic leukemia (CLL). Cells were treated in vitro for 24 - 48 h with ALT alone or in combination with rituximab (RTX), or purine nucleoside analogues (PNA), fludarabine and cladribine. Moreover, eight ALT-treated patients were examined in vivo. In 22/36 patients with the pre-treatment overexpression of Bax, Bak and Bid proteins, ALT induced a distinct (more than 50% from the baseline) increase in the incidence of apoptosis after 24 h of in vitro treatment. ALT-attributed CLL cell apoptosis was also detected after 24 h from in vivo ALT administration, with significantly downregulated Bcl-2 (P = 0.012) and Mcl-1 (P = 0.031). ALT combined with PNA or RTX exerted significantly higher proapoptotic effect in vitro than single agents, downregulating FLIP and Bcl-2 (ALT + PNA) or significantly increasing Bax expression (ALT + RTX; P = 0.007). In conclusion, the evidence of apoptotic CLL cells death in response to ALT, with deregulation of intrinsic apoptotic pathway, is presented. ALT and PNA or RTX trigger complementary changes in expression of proteins regulating cell propensity to undergo apoptosis, what provides molecular rationale for combining ALT with those agents.

Hodgkin's type of Richter's syndrome in familial chronic lymphocytic leukemia treated with cladribine and cyclophosphamide.

Second malignancies are frequent complications in patients with chronic lymphocytic leukemia (CLL). Hodgkins disease (HD) has been observed in approximately 0.5% of the patients with CLL and is known as Hodgkins type Richters syndrome (H-RS). We present a 64-year-old male patient with a familial history of CLL who developed H-RS in abdominal lymph nodes 6 years after CLL diagnosis and 18 months after treatment with cladribine (2-CdA) and cyclophosphamide. HD was diagnosed by fine needle aspiration. The disease was refractory to treatment with two courses of CHOP and three courses of ABVD chemotherapy. In the current literature we found case reports of only 6 patients with H-RS who were treated with fludarabine (FA) before transformation, and, to our knowledge the presented patient is the first to develop H-RS after treatment with 2-CdA combined with cyclophosphamide. He is also the first published patient with familial CLL in whom this complication developed.

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.

Anthracyclines potentiate activity against murine leukemias L1210 and P388 in vivo and in vitro

Abstract:  The interactions of 2‐chlorodeoxyadenosine (2‐CdA, cladribine) and three anthracyclines: doxorubicin (DOX), idarubicin (IDA) and mitoxantrone (MIT) were evaluated on murine leukemias P388 and L1210. Prolongation of survival time of animals receiving drugs in combination compared to mice treated with drugs in monotherapy was tested. We have also evaluated interactions of the cytostatics on murine leukemias in vitro by measuring their inhibitory effects on P388 and L1210 cell proliferation. We have observed a synergistic effect of MIT and IDA in combination with 2‐CdA on P388 leukemia resulting in an increase of life span (ILS)=226% in case of MIT+2‐CdA and ILS=126% in the case of IDA+2‐CdA, whereas 2‐CdA used as a sole drug resulted in an ILS=47%. The survival time of animals inoculated with P388 leukemic cells and treated with DOX+ 2‐CdA was similar to ILS gained by DOX monotherapy (178% and 200% respectively). The mice bearing L1210 leukemia receiving combined chemotherapy lived significantly longer than the animals on single agent regimens. The animals treated with schedule 2‐CdA+MIT lived significantly longer (P=0.004) as compared to the groups receiving drugs in monotherapy (ILS of 2‐CdA+MIT group=60%, ILS of MIT group 33%, and 2‐CdA group 33%). Finally, combination of DOX or IDA with 2‐CdA resulted in ILS =73% (2‐CdA+DOX regimen), and ILS=60% in case of 2‐CdA+IDA regimen, which is significantly higher than ILS gained on monotherapy schedules. In vitro tests revealed that all tested anthracyclines enhance the antiproliferative activity of 2‐CdA against L1210 and P388 leukemic cells (P<0.05).

Hypomethylating agents in the treatment of myelodysplastic syndromes and myeloid leukemia.

Epigenetic changes play an important role in cancer pathogenesis. Hypermethylation of DNA generally results in decreased expression of tumor suppressor genes and defective cell cycle control. This is a hallmark of myelodysplastic syndromes (MDS) and acute myeloid leukemia. Fortunately, epigenetic changes are potentially reversible and thus remain an attractive target for anticancer therapy. Inhibitors of DNA methyltransferase cause demethylation of DNA and exert their activity in myelodysplastic syndromes and acute myeloid leukemia with good safety profile. Decitabine and azacytidine are approved for treatment of patients with high-risk MDS. Demethylating agents seem to be the best choice for elderly patients with myelodysplastic syndromes and acute myeloid leukemia, even in case of high risk cytogenetic changes in the karyotype. The mechanisms of action, pharmacokinetics and antileukemic activity of azacytidine and decitabine are the subjects of this review.

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.

Activity of Cladribine Combined with Etoposide in Heavily Pretreated Patients with Indolent Lymphoid Malignancies

We determined the effectiveness and toxicity of combined chemotherapy consisting of etoposide 100 mg/m2/day i.v. and cladribine (2-CdA) 0.12 mg/kg/day i.v. each for 5 days (EC regimen) in the treatment of refractory or relapsed low-grade non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. The cycles were repeated every 28 days, reaching a maximum of six courses. Twenty patients entered the study. All patients had received three or more cycles of chemotherapy before the EC regimen (median 8, range 3–19). Thirteen patients received 2-CdA before the EC regimen. Seven out of 20 patients (35%) responded, including one complete response. Median overall survival time of responding patients was 22 months (range 3–30). Myelosuppression and infections were the major toxicity of the EC regimen.

Prognostic value of the bone marrow microvessel density in progressive B-cell chronic lymphocytic leukemia

Angiogenesis is a complex, multifactorial phenomenon leading to new vessel formation. Numerous clinical studies indicate that angiogenesis plays a key role in the development of multiple myeloma, leukemia, and lymphoma, including chronic lymphocytic leukemia (CLL) [1–8]. Evaluation of angiogenic status as a prognostic factor in CLL seems to be of great interest. The clinical course of CLL is heterogeneous. Although numerous new prognostic factors are evaluated to predict the disease course, among which cytogenetics, ZAP70, CD38, and immunoglobulin variable heavy chain (IgVH) region somatic hypermutation status are the most important, there is still a need for new ones. Several clinical studies have evaluated the role of angiogenesis in CLL, but most of these were based on indirect methods—measuring the level of proand antiangiogenic cytokines in the serum [4,6,7]. The imbalance between proand antiangiogenic cytokines produced by CLL cells was reported [4,6]. However, the assessment of cytokine levels in serum does not precisely reflect the bone marrow vasculature [8]. Microvessels can be identified by staining for CD34 in endothelial cells or by more detailed transmission electron microscopy (TEM) [9]. Only a few authors have assessed angiogenesis by the direct method, i.e. the evaluation of bone marrow microvessels, and the results were inconsistent [6,8,10]. Therefore, the aim of our study was to evaluate the influence of bone marrow angiogenesis on treatment results and prognosis in patients with CLL. The study involved 75 patients with previously untreated CLL at the time of disease progression. All patients fulfilled the National Cancer Institute (NCI) Working Group diagnostic criteria for CLL. Bone marrow biopsy was taken at the time of CLL progression, directly before the beginning of treatment. Paraffin-embedded trephine biopsies were stained with anti-CD34 antibody specific for endothelial cells. Microvessels were defined as any group of endothelial cells distinct from other cells. The number of vessels was calculated in hot-spot locations, the areas with the highest vessel density, under the microscope at6200 magnification. Clinical characteristics of the patients enrolled in the study are presented in Table I. All the participants were observed for CLL in our Department of Hematology. Patients who progressed between October 2000 and April 2005 and were eligible for treatment with purine analogs were enrolled in the study. The median time from diagnosis to CLL progression was 8 months (range 3–180). Among 75 patients, first-line chemotherapy based on purine analogs was given to 72 subjects. Three patients were not treated during the study period. Patients received purine analogs alone or in combinations (Table I). The treatment was planned to complete 3–6 cycles of chemotherapy. Seven patients received only one cycle, and for these patients the response to chemotherapy was not

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.