P-Glycoprotein Expression in Acute Myeloid Leukaemia Cells at Diagnosis: Its relationship to Daunorubicin or Idarubicin Induction Therapy and Survival; Malignancy.

We investigated the expression of P-glycoprotein (P-gp) in 50 adults with de novo diagnosed acute myeloid leukaemia (AML) and the relationship between presence of P-gp in leukaemic cells and efficacy, as remission induction and survival rate, of two different anthracyclines, daunorubicin (DNR) and idarubicin (IDR). We found that 30 out of 50 patients (60%) were negative (Group 1) and 20 (40%) were positive (Group 2) for P-gp expression evaluated by mean of MRK16 MoAb using a cut-off of 10% positive cells. Thirty-five out of 50 patients (70%) obtained complete remission (CR); depending on P-gp expression, the CR rate was 80% for group 1 and 45% for group 2 (p < 0.005). The median duration of overall survival was 20 months for patients in Group 1 as compared with 10 months for patients of Group 2 (p < 0.005). Regarding the anthracycline used, no significant difference in CR was observed in patients of Group 1 (75% of CR with DNR vs. 90% with IDR); Group 2 obtained 40% of CR with DNR vs. 70% with IDR (p < 0.005). The median duration of overall survival (OS) with the two regimens was comparable in Group 1, while it was significantly longer in patients of Group 2 treated with IDR compared with DNR regimen (p < 0.005). These results confirm the prognostic value of P-gp expression in AML at first appearance and we suggest that idarubicin could be a valid anthracycline drug in the treatment of AML to be evaluated as potential drug of choice in patients with primary or drug-induced multidrug resistance.

Sensitivity to the abl inhibitor STI571 in fresh leukaemic cells obtained from chronic myelogenous leukaemia patients in different stages of disease.

STI571 (CGP57148B) is an inhibitor of BCR/ABL, the cause of chronic myeloid leukaemia (CML). A difference exists between CML patients in chronic phase, in which responses to STI571are durable, and patients in blast crisis, who generally experience only transient responses. Leukaemic cells from six CML patients from whom samples could be obtained during chronic phase and at the time of blast crisis (BC) were compared for sensitivity to STI571, using an in vitro assay. BC samples showed a sensitivity similar to that obtained during chronic phase, suggesting that no substantial intrinsic resistance to STI571 was present in BC.

Differential recognition of a BCR/ABL peptide by lymphocytes from normal donors and chronic myeloid leukemia patients.

The BCR/ABL oncogenic fusion protein transforms normal bone marrow stem cells into neoplastic cells. It has been shown that peptides derived from the junctional region of this oncogenic fusion protein can be recognized by human T-lymphocytes obtained from normal donors. In this study, we investigated the immunogenicity in patients with chronic myeloid leukemia (CML) of a 17 mer b3/a2 Bcr/abl peptide (B/A1), which was shown to induce proliferative responses in lymphocytes from normal donors. A total of 56 CML patients in chronic phase were studied. Twenty-two patients were studied at diagnosis without any treatment (group I). Fourteen patients were receiving IFN (group II), 14 patients were being treated with hydroxyurea (group III), and 6 patients were on different regimens (group IV). Patients were initially assessed for general immunological competence using both in vivo and in vitro assays. Patients were also selected for the expression of HLA-DR0401, the HLA specificity known to present peptide B/A1 to CD4 lymphocytes. With the exception of the six patients in group IV, the results of all these assays (in vitro phytohemagglutinin/tetanus toxoid responses, in vivo skin reaction to ubiquitous antigens) in CML patients did not significantly differ from those obtained in normal donors, thus excluding the presence of generalized immunosuppression. Eight patients with HLA-DR0401 and a b3/a2 type of fusion were identified and further studied. In these eight patients dendritic cells were obtained from adherent peripheral blood mononuclear cells and used to stimulate CD4 lymphocytes. No patient developed a specific response to the bcr/abl peptide, although patients' lymphocytes proliferated in response to a promiscuous tetanus toxoid peptide in all but one case. In contrast, response to the bcr/abl peptide was observed in seven of eight HLA-DR0401 healthy donors tested. These data suggest that immunocompetent, HLA-DR0401+ CML patients are unable to respond to peptide B/A1, at difference from healthy donors. The implication of these results for the immunotherapy of CML is discussed.

P-Glycoprotein Expression in Acute Myeloid Leukaemia Cells at Diagnosis: Its relationship to Daunorubicin or Idarubicin Induction Therapy and Survival.

We investigated the expression of P-glycoprotein (P-gp) in 50 adults with de novo diagnosed acute myeloid leukaemia (AML) and the relationship between presence of P-gp in leukaemic cells and efficacy, as remission induction and survival rate, of two different anthracyclines, daunorubicin (DNR) and idarubicin (IDR). We found that 30 out of 50 patients (60%) were negative (Group 1) and 20 (40%) were positive (Group 2) for P-gp expression evaluated by mean of MRK16 MoAb using a cut-off of 10% positive cells. Thirty-five out of 50 patients (70%) obtained complete remission (CR); depending on P-gp expression, the CR rate was 80% for group 1 and 45% for group 2 (p lt; 0.005). The median duration of overall survival was 20 months for patients in Group 1 as compared with 10 months for patients of Group 2 (p < 0.005). Regarding the anthracycline used, no significant difference in CR was observed in patients of Group 1 (75% of CR with DNR vs. 90% with IDR); Group 2 obtained 40% of CR with DNR vs. 70% with IDR (p < 0.005). The median duration of overall survival (OS) with the two regimens was comparable in Group 1, while it was significantly longer in patients of Group 2 treated with IDR compared with DNR regimen (p < 0.005). These results confirm the prognostic value of P-gp expression in AML at first appearance and we suggest that idarubicin could be a valid anthracycline drug in the treatment of AML to be evaluated as potential drug of choice in patients with primary or drug-induced multidrug resistance.

Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL+ leukemic cells and induces apoptosis.

The BCR/ABL fusion protein transforms myeloid stem cells. Both chronic myelogenous leukemias (CML) and a subset of acute lymphoblastic leukemias (ALL) are associated with the expression of BCR/ABL proteins. This knowledge has not yet been translated into any specific tool to control ABL driven neoplastic cells growth. CGP57148B is an ATP-competitive inhibitor of the ABL protein kinase; it has been shown to inhibit the kinase activity of ABL both in vitro and in vivo and to inhibit the growth of v-abl and bcr/abl transfectants, as well as the in vitro formation of bone marrow (BM)-derived colonies in the presence of growth factors in some CML patients. These studies were performed to investigate the activity of CGP57148B on the spontaneous proliferation of both fresh and cultured, leukemic and normal, BCR/ABL positive and negative cells, and to study its mechanism of action. Six cell lines derived from BCR/ABL+ leukemias (K562, BV173, KCL22, KU812, MC3, LAMA84), thirteen BCR/ABL negative lines, both neoplastic (KG1, SU-DHL-1, U937, Daudi, NB4, NB4.306) and derived from normal cells (PHA blasts, LAK, fibroblasts, LCL, renal epithelial cells, endothelial cells, CD34(+) cells), and 14 fresh leukemic samples were tested using a tritiated thymidine uptake assay. The in vivo phosphorylation of the BCR/ABL protein was evaluated by western blot, while apoptosis was detected by the annexin V/propidium binding test. The induction of differentiation was assayed by immunofluorescence using multiple antibodies. All six BCR/ABL+ lines showed a dose dependent inhibition of their spontaneous proliferative rate, which was not accompanied by differentiation. The treatment caused, within minutes, dephosphorylation of the BCR/ABL protein, followed in 16-24 hours by a decrease in cycling cells and induction of apoptosis. No significant inhibition of DNA synthesis was observed in any BCR/ABL negative normal or neoplastic line at concentrations

Lack of T-cell-mediated recognition of the fusion region of the pml/RAR-alpha hybrid protein by lymphocytes of acute promyelocytic leukemia patients.

In previous studies, it was shown that the fusion region of the pml/RAR-alpha protein, expressed by acute promyelocytic leukemia (APL) cells, can be specifically recognized in vitro by donor (D. E. ) CD4 T cells in a HLA class II DR11-restricted fashion. We present here the results on the recognition of several pml/RAR-alpha peptides by APL patients expressing HLA DR11. The in vitro immunization of peripheral blood lymphocytes from four patients in remission (S. R., F. R., M. M., P. G.) with BCR1/25, a 25-mer pml/RAR-alpha, did not elicit either a polyclonal or a clonal immune response specific to the peptide. We then generated new donor anti-pml/RAR-alpha CD4(+) T-cell clones. These clones were tested for their recognition of BCR1/25. One clone (C3/5, CD3(+), CD4(+), CD8(-)) was selected for further analysis. Clone C3/5 showed specific proliferation, cytotoxicity, and cytokine (tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor) production when challenged with autologous lymphoblastic cell lines pulsed with peptide BCR1/25. C3/5 cells developed specific proliferation and cytotoxicity when challenged with peptide-pulsed lymphoblastic cell lines and peripheral blood lymphocytes from the four DR11(+) APL patients. APL blasts, available only from patients F. R. and P. G., were not lysed by C3/5 and were unable to present peptide BCR1/25. Incubation of APL cells with IFN-gamma failed to induce HLA class II molecules and recognition by the C3/5 clone. Since APL cells do not express HLA class II molecules, we tested in two donors (D. E. and C. H. R.) and in patients S. R. and P. G. whether the use of 9-mer peptides (BCR1/9) would generate a CD8/HLA class I-restricted response. No peptide-specific T-cell line or clone could be generated from both donors and patients. These findings are discussed in relation to possible therapeutic approaches to the immunotherapy of APL.

Idarubicin in combination with cytarabine and VP-16 in the treatment of post myelodysplastic syndrome acute myeloblastic leukemia (MDS-AML).

Fifteen patients with a primary myelodysplastic syndrome (MDS) transformed into acute myeloblastic leukemia (AML) were treated with an intensive chemotherapy regimen containing idarubicin. A complete response (CR) was obtained in 10 patients (66.6%). In five of them this was achieved after a single course of chemotherapy. The median time to achieve a CR was 32 days (range 16-42). A partial remission (PR) was obtained in 2 patients after two induction courses of chemotherapy. One patient died during the first induction course following acute respiratory distress syndrome (ARDS) complication, whereas the chemotherapy regimen failed in 2 patients. A short interval between MDS and transformation into AML was associated with a better chance of achieving a CR. Age, karyotype, type of MDS, peripheral blood or bone marrow findings appeared to have no influence on the response to treatment. The median event free survival for patients who achieved CR was 15 months and the median actuarial survival 18 months. These preliminary results need to be confirmed in a multicentre prospective study comparing idarubicin with other anthracyclines.