Ph. Bernard

SYNTHESIS OF BCL-2 IN RESPONSE TO ANTHRACYCLINE TREATMENT MAY CONTRIBUTE TO AN APOPTOSIS-RESISTANT PHENOTYPE IN LEUKEMIC CELL LINES

BACKGROUND Some forms of chemoresistance in leukemia may start from failure of tumour cells to successfully undergo apoptosis and Bcl-2 may play a role in this defect. Therefore, we evaluated the Bcl-2 content and synthesis in relation with the apoptotic potential in leukemic cell lines after anthracycline treatment. METHODS U937, HL60, and K562 cells and their drug resistant (DR) variants were treated with varying concentrations of Idarubicin (IDA). Apoptosis was evaluated by fluorescence microscopy after acridine orange staining. Bcl-2 and Bax content were evaluated either by flow cytometry after indirect immunolabelling or by Western blot. RESULTS High Bcl-2 contents were not related to a poor ability to undergo apoptosis in U937, HL60, K562 and their DR variants. IDA induced a concentration-dependent increase in Bcl-2 content in all cell lines as long as they do not perform apoptosis. Enhanced Bcl-2 expression was inhibited by cycloheximide, actinomycin D, or antisense oligonucleotide directed against bcl-2 mRNA. Bcl-2 expression was also increased in the resistant U937 variant after serum deprivation or C2-ceramide treatment. The synthesis of Bcl-2 led to an increased Bcl-2/Bax ratio solely in the cells with an apoptosis-resistance phenotype. CONCLUSIONS These data suggest that exposure to IDA induces Bcl-2 expression in leukemic cell lines, and that this mechanism could contribute to apoptosis resistance and participate in the acquisition of chemoresistance. They also confirm that the evolution of the Bcl-2/Bax ratio reflects apoptotic ability better than the steady state level of Bcl-2 expression.

Fluorescent in situ hybridization on flow‐sorted cells as a tool for evaluating minimal residual disease or chimerism after allogeneic bone marrow transplantation

We studied the feasibility and the sensitivity of fluorescent in situ hybridization (FISH) using leukemic or host/donor-specific probes on flow-sorted cells to assess minimal residual disease (MRD) or chimerism in transplanted patients in complete remission. We first performed experimental models of MRD and chimerism by mixing HL60 cells and normal lymphocytes in different proportions. Over 80% HL60 cells were obtained from mixtures of 5% HL60 cells in peripheral blood mononuclear cells (PBMC). We then evaluated MRD and mixed chimerism in a chronic myelogenous leukemia patient in relapse after allogeneic sex-mismatched bone marrow transplantation (BMT), who had received a donor lymphocyte infusion (DLI). Three months after DLI, mixed chimerism was observed in each bone marrow (BM)-sorted lineage (CD13+, CD14+, CD20+, and CD3+), with the highest level of recipient cells in the granulocytic lineage (CD13+). Five months after DLI, host cells were at a low level but remained detectable in the granulocytic lineage. In the same sample, the bcr-abl gene was detected in the granulocytic lineage and not in the lymphocytes. We also studied chimerism in an aplastic anemia sex-mismatched transplanted female patient. We determined the proportion of recipient total lymphocytes, CD4+ and CD8+ lymphocytes, and CD14+ monocytes under cyclosporin A therapy on five peripheral blood samples and one BM sample over 5 months. Results showed a regular decrease in recipient total lymphocytes (26.6% to 10.6%) and monocytes (20.7% to 8%). CD8(+)-recipient cells decreased rapidly, while CD4+ remained stable (17%). This work demonstrates the feasibility of FISH after cell sorting, combining the sensitivities of both flow cytometry and FISH and the specificities of both immunophenotyping and genotype analysis.

Treatment of Relapsed Acute Myeloid Leukemia Using GM-CSF Before Intensive Chemotherapy

Ten patients with acute myeloblastic leukemia (AML) in first relapse were treated with high-dose cytosine-arabinoside (ara-C, 3 g/m2/12 hours x 4) and amsacrine (150 mg/m2/day x 5). In order to prime the cells, the patients were given rh-GM-CSF (3 micrograms/kg/d) for four days, the first infusion starting 48 hours before chemotherapy. Two patients died during the aplastic phase, seven patients achieved a second complete remission (CR2) and one patient remained leukemic. The median duration of aplasia was 17 days (14-21). These results were comparable to those obtained in our previous series of 27 patients treated for AML in first relapse with the same chemotherapy but without GM-CSF (66% CR2). After 48 hours of GM-CSF infusion, (before chemotherapy was started), seven patients had an increase in the white blood cell count with an increase in the absolute number of blast cells in five of these cases. Marrow blast cells percentages increased in 3 of the 8 patients analysed. Six of seven patients tested showed an increase in the percentage of cells in S-phase (studied by flow cytometry using the bromodeoxyuridine (BrdU/DNA) labelling technique and BrdU incorporation). GM-CSF used to prime leukemic cells may be safely administered but its clinical usefulness needs to be further evaluated.

Philadelphia negative BCR-ABL positive chronic myeloid leukemia mimicking juvenile chronic myeloid leukemia in a 2-year-old child.

We present the case of a two-year-old child with an atypical presentation of chronic myeloid leukemia. At diagnosis, he showed clinical and biological features of juvenile chronic myeloid leukemia (CML). However, eosinophilia was observed in blood and bone marrow. The bone marrow karyotype did not demonstrate the Philadelphia chromosome but BCR-ABL rearrangement was shown to be present by reverse transcriptase polymerase chain reaction (RT-PCR) analysis and confirmed by fluorescent in situ hybridization (FISH) analysis. Discussion centres on the differentiation between juvenile CML and childhood chronic myelogenous Leukemia and the importance of carrying out RT PCR for all juvenile CML cases.

Autologous Blood Stem Cell Transplantation Followed by Recombinant Alpha Interferon as Treatment for Patients with High-Risk Chronic Myelogenous Leukemia. A Report of 32 Cases

Autologous blood stem cell transplantation (ASCT) was performed in 32 patients with high risk chronic myelogenous leukemia (CML). Prior to ASCT, the patients were given Busulfan and high-dose Melphalan. Peripheral blood stem cells collected at diagnosis were used to rescue hematopoiesis. Recombinant Interferon was administered after ASCT. In 24 patients transplanted in transformation, 23 achieved a complete hematological response and nine are still alive 9 to 73 months after ASCT. Eight other patients were transplanted in chronic phase for either the presence of bad prognostic factors (Sokals classification) or no response to IFN. Seven are alive without transformation 16 to 48 months after ASCT. Although few patients presented a cytogenetical response (10/28), the survival observed in this series of patients compares favorably with that of patients treated conventionally. Thus, the place of ASCT in CML could now be tested prospectively.