Cytokines can reduce clonal, CD34-positive cells in acute myeloid leukemia in vitro.
We studied the influence of cytokine mixes on the survival of acute myeloid leukemia (AML) bone-marrow (BM) cells in a 14-day culture assay in vitro. Southern-blot analysis using a panel of different probes in combination with densitometry and flow cytometry were used to detect and compare the amount of clonal or CD34-positive BM cells before and after the culturing procedure. A significant reduction of CD34-positive cells after incubation with a cytokine mix [interleukin (IL)-1beta, IL-3, IL-6, stem cell factor (SCF), erythropoietin (EP) with granulocyte macrophage/colony-stimulating factor (GM-CSF, Cytok1) could be achieved in all 16 cases with a CD34-positive blast phenotype studied at diagnosis (P<0.001), in 3 of 10 cases at relapse, and in 8 of 18 cases in complete remission. In healthy donors, an increase of CD34-positive cells was demonstrated in 5 of 5 samples. A reduction of clonal DNA through incubation with Cytok1 was achieved in 5 of 5 (100%) cases studied at diagnosis, in 1 of 4 (25%) cases at relapse, and in 7 of 9 cases (78%) in complete remission. Cytokine cocktails with GM-CSF (Cytok1) were more efficient in reducing (clonal) CD34-positive cells than cocktails without GM-CSF (Cytok2). AML patients at diagnosis and in complete remission had a better survival probability if their CD34-positive or clonal cells could be reduced in vitro by cytokine cultivation (P<0.05). Vitality of BM cells was not influenced by 14-day cytokine treatment; however, the total cell count could be increased by Cytok1 and Cytok2 by 55-174%, but not by the control medium. Our data show that: (1) clonal cell populations can be regularly detected at diagnosis, during complete remission, and at relapse; (2) CD34-positive cells in AML can be demonstrated to be clonal, gene-rearranged cells; (3) incubation of AML BM-cells with Cytokl leads to a reduction of the CD34-positive, clonal cell load in all cases at diagnosis and in 78% of the cases in complete remission of AML, but in only 25% of the cases at relapse; (4) in all healthy BM samples, proportions of 'healthy' CD34-positive cells were increased. Moreover, absolute cell counts were increased by cytokine incubation of cells obtained at diagnosis, relapse, or complete remission of AML and from healthy donors indicating a selective stimulation of healthy, but not of leukemic CD34-positive cells; (5) cytokine cocktails containing GM-CSF are more efficient in reducing leukemic cells than cocktails without GM-CSF; and (6) in vitro reactivity of clonal or CD34-positive BM cells against Cytokl has clinical relevance. We conclude, that Southern-blot analysis and flow cytometry are suitable methods to detect and quantify leukemic disease and to distinguish between clonal or non-clonal CD34-positive cells. The ex vivo or clinical application of specific combinations of cytokines might be a feasible and successful application of immunotherapy in AML that merits further investigations.
Braun
S
S
Charite, Virchow Klinikum, Exp. Chirurgie, University of Berlin, Germany.
Gerhartz
H H
HH
Schmetzer
H M
HM
D000208:Acute Disease; D018952:Antigens, CD34 / Q000032:analysis*; D001854:Bone Marrow Cells / Q000187:drug effects / Q000276:immunology; D002452:Cell Count / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002999:Clone Cells; D016207:Cytokines / Q000494:pharmacology*; D004921:Erythropoietin / Q000494:pharmacology; D015321:Gene Rearrangement / Q000187:drug effects; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D007951:Leukemia, Myeloid / Q000175:diagnosis / Q000473:pathology*; D011379:Prognosis; D012008:Recurrence; D012074:Remission Induction; D019089:Stem Cell Factor / Q000494:pharmacology
D018952:Antigens, CD34; D016207:Cytokines; D007377:Interleukin-3; D015850:Interleukin-6; D019089:Stem Cell Factor; D004921:Erythropoietin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor