Braun S, Gerhartz H H, Schmetzer H M
Charite, Virchow Klinikum, Exp. Chirurgie, University of Berlin, Germany.
Ann Hematol. 2000 Jul;79(7):363-73. doi: 10.1007/s002770000161.
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.
我们在一项为期14天的体外培养试验中研究了细胞因子混合物对急性髓系白血病（AML）骨髓（BM）细胞存活的影响。使用一组不同的探针结合光密度测定法和流式细胞术进行Southern印迹分析，以检测和比较培养前后克隆性或CD34阳性BM细胞的数量。在诊断时研究的所有16例具有CD34阳性原始细胞表型的病例中（P<0.001），10例复发病例中的3例，以及18例完全缓解病例中的8例，用细胞因子混合物[白细胞介素（IL）-1β、IL-3、IL-6、干细胞因子（SCF）、促红细胞生成素（EP）与粒细胞巨噬细胞集落刺激因子（GM-CSF，细胞因子1）]孵育后，CD34阳性细胞显著减少。在健康供体中，5个样本中有5个样本的CD34阳性细胞增加。在诊断时研究的5例（100%）病例、4例复发病例中的1例（25%）以及9例完全缓解病例中的7例（78%），通过与细胞因子1孵育实现了克隆性DNA的减少。含有GM-CSF的细胞因子混合物（细胞因子1）在减少（克隆性）CD34阳性细胞方面比不含GM-CSF的混合物（细胞因子2）更有效。如果AML患者诊断时和完全缓解时的CD34阳性或克隆性细胞能够通过细胞因子培养在体外减少，则其生存概率更高（P<0.05）。BM细胞的活力不受为期14天的细胞因子治疗影响；然而，细胞因子1和细胞因子2可使总细胞数增加55 - 174%，但对照培养基无此作用。我们的数据表明：（1）在诊断、完全缓解和复发期间可定期检测到克隆性细胞群体；（2）AML中的CD34阳性细胞可被证明是克隆性的、基因重排的细胞；（3）用细胞因子1孵育AML BM细胞可导致诊断时所有病例以及AML完全缓解病例中78%的病例CD34阳性克隆性细胞负荷减少，但复发病例中仅25%的病例减少；（4）在所有健康BM样本中，“健康”CD34阳性细胞的比例增加。此外，对AML诊断、复发或完全缓解时以及健康供体获得的细胞进行细胞因子孵育可增加绝对细胞计数，表明对健康而非白血病CD34阳性细胞有选择性刺激作用；（5）含有GM-CSF的细胞因子混合物在减少白血病细胞方面比不含GM-CSF的混合物更有效；（6）克隆性或CD34阳性BM细胞对细胞因子1的体外反应具有临床相关性。我们得出结论，Southern印迹分析和流式细胞术是检测和量化白血病疾病以及区分克隆性或非克隆性CD34阳性细胞的合适方法。细胞因子特定组合的体外或临床应用可能是AML免疫治疗的一种可行且成功的应用，值得进一步研究。
