Barbara Cebula

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.

Expression and prognostic significance of the inhibitor of apoptosis protein (IAP) family and its antagonists in chronic lymphocytic leukaemia.

Impaired apoptosis is still considered to be an important event in the development and progression of chronic lymphocytic leukaemia (CLL). However, mechanisms of this defect have not been fully elucidated. In this study, expression of inhibitor of apoptosis proteins, IAPs (cIAP1, cIAP2, XIAP and survivin), and their antagonists (Smac/DIABLO and HtrA2/Omi) was comprehensively analysed in 100 untreated CLL patients, using flow cytometry and Western blot techniques. Expression of anti-apoptotic cIAP1 and cIAP2 in leukaemic cells was significantly higher than in non-tumour lymphocytes (p=0.000001 and p=0.014, respectively), whereas the IAP-antagonist, Smac/DIABLO, was decreased in CLL (p=0.010). Higher expression of all analysed IAPs (cIAP1, p=0.002; cIAP2, p=0.026; XIAP, p=0.002; survivin, p=0.00006) and lower levels of Smac/DIABLO (p=0.006) were found in patients with progressive disease, compared to those with stable CLL. High baseline expression of cIAP1 and survivin correlated with worse response to treatment. Co-expression of these proteins was associated with shorter overall survival of CLL patients (p=0.005). In conclusion, CLL cells show the apoptosis-resistant profile of IAPs/IAP-antagonist expression. Upregulation of IAPs is associated with a progressive course of the disease. Co-expression of cIAP1 and survivin seems to be an unfavourable prognostic factor in CLL patients. Further studies with longer follow up are warranted to confirm and expand these findings.

Rituximab plus cladribine with or without cyclophosphamide in patients with relapsed or refractory chronic lymphocytic leukemia.

Objectives: The aim of our study was to determine the feasibility, effectiveness and toxicity of combined regimens consisting of rituximab and cladribine (2‐CdA) (RC) and RC plus cyclophosphamide (RCC) in the treatment of patients with recurrent or refractory chronic lymphocytic leukemia (CLL).

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.

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 (

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.

In vitro cytotoxic effect of proteasome inhibitor bortezomib in combination with purine nucleoside analogues on chronic lymphocytic leukaemia cells

Abstract:  Objective: The anti‐tumour in vitro activity of proteasome inhibitor bortezomib (PS‐341, VELCADE) in combination with purine nucleoside analogues, cladribine (2‐CdA) and fludarabine (FA) was tested in lymphocytes derived from 26 patients with B‐cell chronic lymphocytic leukaemia (B‐CLL). Methods: Cell viability was assessed by propidium iodide staining, and apoptosis by annexin‐V and caspase activation flow cytometry assays. Additionally, expression of the apoptosis‐regulating proteins Bax, Bak, Bid, Bcl‐w, Bcl‐2, XIAP and Mcl‐1 was evaluated in B‐CLL lymphocytes. Results: Bortezomib alone induced significant, dose‐dependent cytotoxicity starting from the low concentration 2.5 nm, inducing apoptosis of B‐CLL cells. Combination of this agent with 2‐CdA or FA resulted in an increase of cytotoxicity when compared with that mediated by single drugs. The observed increase was especially evident when 5 nm of bortezomib were combined with suboptimal doses of 2‐CdA or FA. The combination index (CI) was 0.87 for bortezomib + 2‐CdA and 0.82 for bortezomib + FA, indicating an evident additive effect of these combinations. Moreover, B‐CLL cells were more sensitive to proteasome inhibitor used alone or combined with 2‐CdA or FA comparing to CD3+ lymphocytes. Corresponding to enhanced apoptosis, the expression levels of several apoptosis‐regulating proteins were altered. The most pronounced changes were down‐regulation of XIAP and up‐regulation of Bid proteins by the combination of bortezomib with either 2‐CdA or FA. Conclusions: This study suggest that the in vitro cytotoxic effect through proteasome inhibition by bortezomib can be increased substantially with low doses of the purine nucleoside analogues, 2‐CdA and FA, and that this effect on B‐CLL cell is selectively higher than on normal, CD3‐positive lymphocytes.

CD38 Gene Polymorphisms Contribute to Genetic Susceptibility to B-Cell Chronic Lymphocytic Leukemia: Evidence from Two Case-Control Studies in Polish Caucasians

Given the recent findings on the importance of CD38 signaling in the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL), we hypothesized that single nucleotide polymorphisms (SNP) in the CD38 gene may be related to B-CLL risk. We evaluated two potentially functional CD38 SNPs, intronic rs6449182 (184C>G) and missense rs1800561 (418C>T, Arg140Trp) in two hospital-based case-control studies (study A and validation study B). Genotyping was done using PCR-based assays in a total of 460 Polish Caucasian patients with B-CLL and 503 age-matched and gender-matched controls. We found that frequencies of both variant alleles (rs6449182 G and rs1800561 T) were significantly higher in B-CLL. In study A, logistic regression analysis revealed an association between B-CLL and genotypes: rs6449182 CG [odds ratio (OR), 3.57; 95% confidence interval (95% CI), 2.4-5.3], rs6449182 GG (OR, 5.2; 95% CI, 2.36-11.5), and rs1800561 CT (OR, 6.72; 95% CI, 1.5-30.1), although no homozygous rs1800561 TT genotype was detected in either study. These results were confirmed in study B, which showed an association between B-CLL and genotypes rs6449182 CG (OR, 4.00; 95% CI, 2.7-6.0), rs6449182 GG (OR, 12.84; 95% CI, 4.3-38.7), and rs1800561 CT (OR, 10.12; 95% CI, 1.3-81.6), and in the combined analysis of both studies. We also observed that rs6449182 G carriers had more advanced clinical stage (P = 0.002) and tended to be younger at diagnosis (P = 0.056). Furthermore, we found higher CD38 transcript levels and higher proportions of CD38-positive cells in carriers of rs6449182 G and rs1800561 T alleles (P < 0.05 for all comparisons). In conclusion, our data show that CD38 SNPs may affect CD38 expression and contribute to the increased risk of B-CLL carcinogenesis. (Cancer Epidemiol Biomarkers Prev 2009;18(3):945–53)

No influence of 3435C>T ABCB1 (MDR1) gene polymorphism on risk of adult acute myeloid leukemia and P-glycoprotein expression in blast cells

Inherited differences in xenobiotic transport and metabolism may play an important role in the development of adult acute myeloid leukemia (AML) and response to the chemotherapy. An ATP-binding cassette (ABC) family transporter P-glycoprotein (P-gp or ABCB1), encoded by ABCB1 (MDR1) gene, is involved in the protection against xenobiotics and multi-drug resistance. The aim of this study was to investigate the potential involvement of the ABCB1 gene exon 26 3435C>T single nucleotide polymorphism (SNP) in the genetic susceptibility to AML and regulation of P-gp expression and activity in AML cells. A total of 180 adult AML patients and 180 sex-matched controls were genotyped using PCR-RFLP method. Moreover, in 40 AML patients ABCB1 gene expression was studied by real-time RT-PCR and P-gp expression and activity were assessed by flow cytometry assays. The prevalence of 3435C>T ABCB1 polymorphism was similar in patient and control cohorts (P = 0.16). Furthermore, the carriers of different ABCB1 genotypes did not differ significantly according to ABCB1 gene expression (P = 0.99), P-gp expression (P = 0.42) and P-gp activity (P = 0.83) in leukemic cells. The authors conclude that isolated 3435C>T ABCB1 SNP is not a major factor of the genetic susceptibility to adult AML, and that genotyping of this polymorphism does not allow predicting P-gp expression or activity in AML cells.

Rapamycin, the mTOR kinase inhibitor, sensitizes acute myeloid leukemia cells, HL-60 cells, to the cytotoxic effect of arabinozide cytarabine

The mammalian target of rapamycin (mTOR) kinase is a key regulator of cell growth and proliferation. Overexpression of the mTOR signaling pathway has been described in several tumor cells, including the majority of acute myeloid leukemia (AML) cases. The anti-tumor efficacy of mTOR inhibitors was shown in several preclinical and clinical studies. In AML, however, the potential antineoplastic effect of mTOR inhibitors has received little attention thus far. In this in-vitro study of the human AML cell line, HL-60, we aimed to assess the antileukemic activity of rapamycin (RAPA), an mTOR inhibitor, alone and in combination with cytarabine (Ara-C). The study showed that RAPA in concentrations of 1–10 nmol/l arrested the cell cycle progression of Hl-60 cells in the G1 phase, without evident cytotoxic effect. This effect was associated with significant inhibition of cyclin E expression. At concentrations higher than 10 nmol/l, RAPA exerted a significant proapoptotic effect, with the collapse of mitochondrial potential and caspase-3 activation. The most prominent proapoptotic effect was observed for a combination of 1 nmol/l of RAPA and 50 nmol/l of Ara-C, especially when Ara-C was added at a 24-h interval after RAPA. In conclusion, these data indicate that RAPA might be effective in the treatment of acute leukemia patients, especially in combination with Ara-C, the drug routinely used in AML treatment. On the basis of these results, attempts to combine classical induction chemotherapy with an inhibitor of the mTOR kinase in AML treatment could be warranted.