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No specific characteristics have been identified as predictors of peripheral blood stem cells (PBSC) mobilization in healthy donors. In this study, clinical characteristics and laboratory data for 122 healthy donors who underwent apheresis on day 5 of treatment with recombinant granulocyte colony-stimulating factor (G-CSF) were retrospectively analyzed for correlations with CD34(+) cell mobilization. The variables that were analyzed included age, sex, body weight, basal complete blood count, and maximum white blood count (WBC) before apheresis, G-CSF type, and dosage. Median age and body weight were 42.5 years (range 16-65) and 72.5 kg (range 47-121), respectively. By univariate analysis, male sex (P = 0.007), body weight (< or = 70 vs. >70 kg, P = 0.04), and donor's age (< or = 50 vs. > 50 years; P = 0.015) were correlated with the number of CD34(+) cells mobilized. By multivariate analysis, donor's age and male sex were the only two variables that significantly predicted a high CD34(+) cell level. In conclusion, our data suggest that male sex and younger age are the only factors that significantly affect CD34(+) mobilization in healthy donors.

Mobilization of CD34+ into peripheral blood is attained by either glycosylated (lenograstim) or non-glycosylated recombinant G-CSF (filgrastim). 101 donors, 57 males, median age 42 years (range 16-63) entered this retrospective study. Group I (55 cases) received filgrastim and group II lenograstim subcutaneously for 5-6 days. The peak number of CD34+ cells/microl blood observed on day 4 and 5 was not significantly different in the two groups. No differences were shown in terms of both circulating CFU-GM at the time of harvesting and CD34+ target of collection. The most frequent side effects were bone pain (18.2% grade I; 36.4% grade II, 7.3% grade III), headache (18.2%), nausea (9.1%), fever (5.5%) and a mild splenomegaly (> 2 cm) (5.5%) in filgrastim group, and bone pain (37.0% grade I, 26.1% grade II, 2.2% grade III), headache (17.4%), nausea (15.2%), fever (4.4%) and a mild splenomegaly (4.3%) in lenograstim group, respectively. CD34+ collection was associated with thrombocytopenia, which was not significantly different between the two groups. No donor in either group developed long-term adverse effects. We conclude that both G-CSFs are comparable in terms of CD34+ cell collection, safety and tolerability., (Copyright 2005 Wiley-Liss, Inc)

Hematological and extra-hematological toxicity of mitoxantrone-containing regimens with autologous stem cell rescue was evaluated in 32 metastatic breast cancer patients. The schedule was the final part of two high-dose chemotherapy programs, including an induction phase with three courses of conventional chemotherapy with epirubicin (120 mg/m2) and cyclophosphamide (600 mg/m2) plus three courses of docetaxel (100 mg/m2) and a first high-dose chemotherapy consisting of cyclophosphamide (6000 mg/m2), thiotepa (500 mg/m2) and carboplatin (800 mg/m2) or melphalan (160 mg/m2) plus thiotepa (600 mg/m2). The final second autograft phase included mitoxantrone (60 mg/m2) associated with melphalan (160 mg/m2) and autologous stem cell rescue infusion. The median duration of severe neutropenia and thrombocytopenia was 9 (range, 7-13) and 11.5 (range, 9-29) days. The median number of units of erythrocytes and platelets transfused was 1 (0-11) and 4 (1-9), respectively. Fever for a median of 2 (0-8) days developed in 71.8% of the patients. Mucositis was observed in 81.2% (WHO grade 3-4 in 25%). No acute or late cardiac toxicity was observed. One patient died because of a transplant-unrelated cause. The response according to the program phase showed an increased rate of complete response, from 12.5% at the end of conventional chemotherapy to 21.9% after the first high-dose chemotherapy course, to increase to 43.9% after the treatment with mitoxantrone-melphalan. We conclude that a conditioning regimen with high dose mitoxantrone-melphalan fits well within the high-dose chemotherapy program.

We investigated the possible value of an in vitro drug-induced cytotoxicity assay in predicting clinical response to fludarabine (FAMP) in chronic lymphocytic leukaemia (CLL). The median FAMP-LD50 values for cases designated as complete response (CR), partial response (PR), no response (NR) and progressive disease (PD) were 1.43, 2.05, 24.8 and 77.9 microg/ml, respectively (P=0.013). A significant lower mean FAMP-LD50 value was observed in the in vivo responding cases as compared to non-responding ones (3.0 +/- 0.82 SEM v 42.3 +/- 12.6 SEM, P=0.001). Only FAMP-LD50 < or = 2.5 microg/ml and more than four FAMP courses significantly influenced the response rate in univariate and multivariate analyses. The MTT assay may be of value in predicting clinical response to FAMP in CLL patients.

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.

The decrease in cell viability observed in vitro from the effect of chlorambucil (CLB), fludarabine (FAMP) and 2-chlorodeoxy-adenosine (CDA) on peripheral lymphocytes from 49 untreated CLL patients was investigated by the MTT colorimetric assay. The effects of recombinant-interleukin (r-IL)-2 and alpha-interferon (alpha-IFN) on drug-induced cell death were evaluated. r-IL-2 significantly increased in vitro resistance to CLB, while purine analog cytotoxicity was slightly reduced by the cytokine. The potential in vivo significance of r-IL-2, acting as a survival signal on CLB-induced cell death, is supported by the correlation between the lowest IL-2 serum levels, the highest in vitro sensitivity to CLB and a major clinical response after CLB treatment in six out of eight CLL patients. Using 25 samples, alpha-IFN enabled CLL cells to increase resistance to CLB, CDA and FAMP in 14, eight and seven samples, respectively; conversely, alpha-IFN showed a synergism with both CLB and FAMP in six samples and with CDA in four. These results correlate with immunoenzymatic assay data showing that alpha-IFN either up- or down-regulates tumor necrosis factor and IL-1 levels in supernatants of some CLL samples. Apparently, alpha-IFN plays a dual role in regulating drug-induced cell death, while IL-2 seems to solely favor cell survival in CLL.