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Background and Objective: Clinical studies indicate that combination chemotherapy with mitoxantrone (Mitox) and a purine analog can improve the response rate in indolent lymphoproliferative disorders. We explored the in vitro Mitox- fludarabine (FAMP)- and pentostatin (Pento)-induced cytotoxicity and their interactions in CLL., Methods: The peripheral lymphocytes of 24 CLL patients were tested at different drug concentrations, with Mitox, FAMP or their combinations in 22 cases, and with Mitox, Pento or their combinations in 20 cases, 18 of which were the same from the FAMP group. The MTT assay was chosen for the drug-induced cell cytotoxicity and flow cytometry analysis of the DNA hypodiploid peak for the study of the apoptotic process. Drug interactions were calculated in the MTT assay according to both multiplicative and maximum models., Results: According to the lethal dose (LD) 50 values, when the three drugs were tested alone, 11 out of 22 and 8 out of 20 samples were sensitive to Mitox in the FAMP and Pento groups, respectively; on the other hand, 2 out of 22 and 0 out of 20 samples appeared sensitive to FAMP or Pento alone, respectively. Analyzing the MTT assay data with the multiplicative and maximum model, the combinations of Mitox+FAMP and Mitox+Pento at different drug concentrations were synergistic in 28.2% and 39.3%, respectively. At leukemic cell survival < or = 50%, 11.7% and 11.1% of all combinations were synergistic in the Pento and FAMP group, respectively. The number of synergistic interactions at a therapeutically achievable plasma-drug concentration was an inverse function of the Mitox concentration. In the FAMP group, a direct correlation was found between the LD50 values of both FAMP and Mitox and the number of synergistic interactions, while the Pearson correlation coefficient was not significant in the Pento group. Finally, as measured by the DNA hypodiploid peak, Mitox (0.25 microgram/mL) plus Pento (0.16 microgram/mL) showed a significantly enhanced apoptosis in comparison to each single drug, while Mitox failed to demonstrate an additive effect with FAMP (1 microgram/ml)., Interpretation and Conclusions: This experience demonstrates the extent of the in vitro synergism of Mitox with FAMP and Pento in inducing cell cytotoxicity; it also shows an adjunctive apoptotic effect for the Mitox-Pento association only.

Background and Objective: Bcl-2 oncogenic protein expression plays a major role in blocking the apoptotic mechanism. p53 gene mutations have also been suggested to account for the chlorambucil resistance in CLL. Thus we studied the relationship between bcl-2 protein expression, p53 gene mutations and in vitro drug sensitivity in CLL., Methods: Fifty-three samples from untreated CLL patients in early disease stages were tested in vitro for chemosensitivity to chlorambucil (CLB), fludarabine (FAMP) and 2-chlorodeoxyadenosine (2-CDA) using the MTT assay. Intracellular bcl-2 protein expression was evaluated by flow cytometry analysis. p53 gene mutations were detected by using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis., Results: The median LD50 values were 1.55 microM, 4.41 microM and 58.2 microM for 2-CDA, FAMP and CLB, respectively. About 23%, 41% and 11% of samples were defined as being sensitive to FAMP, 2-CDA and CLB, respectively, when samples were clustered for LD50 threshold values corresponding to the plasmatic levels of the drug. No statistically significant difference in bcl-2 protein expression was noted between sensitive and resistant samples for each drug. A p53 gene mutation was detected in 4 of the 30 cases studies and all of them were among samples resistant to CLB., Interpretation and Conclusions: Bcl-2 expression is not an indicator of in vitro response to drugs in CLL; similarly, although the four cases showing a p53 gene mutation were associated with CLB resistance, drug resistant samples were also observed in the group of patients showing wild type p53, suggesting multiple mechanisms of drug resistance in CLL.

Background: Chlorambucil (CLB), 2-chlorodeoxyadenosine (2-CDA) and fludarabine (FAMP) are among the most widely used drugs in chronic lymphocytic leukemia (CLL). Therefore we evaluated in vitro sensitivity to these drugs and cross-resistance of purine analogs. In addition, we correlated the in vitro data with the main clinico-hematological variables and surface markers., Patients and Methods: Eighty CLL samples obtained from 63 untreated and 17 treated CLL patients were tested in vitro with the MTT assay. Lethal dose (LD)50 values were calculated to determine sensitivity to CLB, 2-CDA and FAMP., Results: Samples were clustered either for a one-log increase of LD50 values or for LD50 threshold values of 3 microM for FAMP, 0.3 microM for 2-CDA and 7 microM for CLB, which correspond to the therapeutically achievable plasmatic levels of these drugs. A higher number of samples sensitive to 2-CDA were identified by the first approach; with the second method the relative order of sensitivity was FAMP > 2-CDA > CLB. Concerning 2-CDA and FAMP cross-resistance, out of 61 samples resistant to 2-CDA, 29.5% were sensitive to FAMP. Conversely, 13.9% out of 43 samples resistant to FAMP were sensitive to 2-CDA. No correlation was found between the main clinico-hematological features and the LD50 values of each drug either considering the whole series or only the untreated cases. In vitro drug sensitivity was also evaluated during the steady-state of the disease and at disease progression in six untreated cases. We observed a mean increase in the LD50 values of about 13, 38 and 22 times for CLB, FAMP and 2-CDA, respectively. Among the treated cases, the LD50 values of both purine analogs and CLB correlated with bone marrow histology. CLL cells expressing CD14, CD11c, CD11b, and FMC7 were more resistant in vitro to purine analogs but not to CLB., Conclusions: This study suggests that i) the purine analogs exert a greater cytotoxic effect on CLL cells; ii) 2-CDA and FAMP are not cross-resistant in vitro in a percentage of CLL samples, iii) a possible change in LD50 values may be related to modification of the disease status, and iv) the expression of certain surface markers, which are CLL-unrestricted, identifies samples with higher in vitro resistance to purine analogs.