An effective,direct immunomagnetic procedure for purging acute lymphoblastic leukemia cells from human bone marrow

The AB4 monoclonal antibody, which recognizes an HLA-DR epitope, was found to bind to a high percentage of malignant blast cells in samples obtained from 27 patients with ALL. These included 11 of 11 cases with c-ALL, 3 of 7 with pre-pre-B, and 8 of 9 cases with pre-B ALL. AB4 was used together with anti CD10 and anti CD19 antibodies and super-paramagnetic particles for developing a direct immunomagnetic procedure for purging human bone marrow of leukemic cells. In model experiments with KM3 cells admixed to mononuclear bone marrow cells, the individual antibodies each removed 2.8–3.1 logs and 3.6–4.1 logs of tumor cells with one and two purging cycles, respectively. In comparison, the efficacy of a mixture of the three antibodies was 4.4 logs with one treatment cycle, and > 5 logs with repeated treatments. Whereas the use of a commercially available anti-HLA-DR antibody resulted in a 90% reduction in the survival of CFU-GMs and normal blast colonies, AB4 had only a moderate effect on the progenitor cells (46% and 30% reduction). In conjunction with autologous transplantation, bone marrow from a patient was purged with the antibody mixture and 50% of the CFU-GMs and 47% of the CD34⁺ cells remained after treatment. The patient showed a normal engraftment, reaching a level of 0.5 × 1071 neutrophils by day 20 and 20 × 107 9/1 platelets by day 30. It is concluded that the antibody cocktail may safely and effectively be used for rapid autograft purging in patients with c-ALL, and also in phenotypically selected cases with other subtypes of ALL. This work was supported by the Norwegian Cancer Society     

Magnetic affinity colloid (MAC) cell separation of leukemia cells from autologous bone marrow aspirates

A colloidal suspension of Co₂B with avidin irreversibly adsorbed to the surface has been used with biotinylated antibodies and lectins to eliminate specific cell populations from mixtures with peripheral blood or bone marrows. Using the monoclonal antibodies CF-1 and PM-81 with this magnetic affinity colloid (MAC), we can eliminate five logs of K562 cells from mixtures with peripheral blood or marrow cells as determined by a linear limiting dilution clonogenic assay. We have also used this separation to eliminate clonogenic leukemia cells from fresh samples of peripheral blood and bone marrow from relapsed acute leukemia patients. Using CF-1 alone or in combination with PM-81, we eliminated two logs of colonies and clusters of leukemia cells from the fresh samples. The same antibodies used with MAC separation of hematologically normal marrows allow recovery of greater than 30% of the hematopoietic progenitors.     

Monitoring the remission induction therapy of acute nonlymphoblastic leukemia by bone marrow cell culture criteria

In 31 cases of acute nonlymphoblastic leukemia, bone marrow cells were serially cultured in semi-solid agar during the remission induction therapy. A normal in vitro cell growth pattern returned in 15 out of 22 patients up to 77 days before a complete remission was established by clinical and hematological criteria. In 6 cases the return of normal colonies coincided with clinical and hematological evidence of a complete remission. Nine patients failed to attain a remission and died from complications of bone marrow aplasia. Only one had a normal number of colonies and a normal cluster/colony ratio in cultures prepared 11 days after the completion of the first course of chemotherapy. At this time, his platelet count increased to normal level, possibly indicating a developing remission. Bone marrow cell culture criteria are useful in monitoring the remission induction therapy in patients with acute nonlymphoblastic leukemia. An early return of normal in vitro cell growth pattern suggests an approaching remission, which may be achieved several weeks later.     

Reinfusion of leukemic cells with the autologous marrow graft: preclinical studies on lodging and regrowth of leukemia

To evaluate in quantitative terms the contribution of leukemic cells present in the autologous marrow graft to the occurrence of leukemia relapse after autologous bone marrow transplantation, preclinical studies were performed in a rat model for human acute myelocytic leukemia (BNML). Firstly, the number of leukemic cells which--after intravenous transfer--cause death from leukemia in 50% of the recipient rats proved to be 24.7 cells. Secondly, it appeared that the regrowth of leukemic cells in rats heavily pretreated with high-dose cyclophosphamide and total body irradiation was significantly hampered as compared with non-pretreated controls as judged by survival times (37 and 31 days, respectively after 10(3) BNML cells i.v.). The most likely explanation is treatment-induced damage to the microenvironment. Differences in patterns of lodging of infused leukemic cells were ruled out by comparing the uptake of 51Cr-labeled BNML cells in various organs. Finally, extrapolated from the available rat data on log leukemic cell kill induced by high-dose chemoradiotherapy, an hypothesis is presented relating the total tumor load in man to the clinical outcome of autologous bone marrow transplantation. From this hypothesis it is derived that the minimal number of leukemic cells that causes leukemia upon intravenous transfer varies between 10(4) and 10(6).     

Differences in uptake of adriamycin and daunomycin by normal BM cells and acute leukemia cells determined by flow cytometry

Flow cytometry was used to measure the uptake of adriamycin (AM) and daunomycin (DM) by various cell types of the haemopoietic organs and by acute myeloid leukemia (AML) cells. For this purpose, the spontaneous fluorescence of the drugs upon excitation with laser light at 488 nm was measured. The difference in the uptake of these two drugs correlates with their cytotoxicity. Myeloid progenitor cells from mouse bone marrow (CFU-C) are ten times more sensitive for DM than AM [ED₁₀ = 0.85 μg/ml (DM); 6.40 μg/ml (AM)]. The cytotoxicity of DM is correlated with a higher fluorescence intensity of blast cells, lymphocytes and granulocytes when treated with DM as compared to AM. In contrast to this, the fluorescence of AML blast cells is significantly lower after treatment with DM than with AM. The uptake of both drugs seems to occur following active membrane transport, although to a different extent.     

漆姑草醇提物对人急性早幼粒白血病细胞的诱导分化作用

目的： 探讨漆姑草醇提物（HSJ）对人急性早幼粒白血病细胞（NB4）的诱导分化作用。方法： 以NB4细胞为研究对象,设HSJ低（30 μg/mL）、中（60 μg/mL）、高（120 μg/mL）剂量组和阴性对照组,共4组;HSJ作用NB4细胞48 h后,采用透射电镜法观察NB4细胞形态;硝基四唑氮（NBT）还原实验检测NB4细胞分化能力;流式细胞术检测NB4细胞周期分布;流式细胞术检测NB4细胞表面分化抗原CD14和CD11b细胞的表达率;实时荧光定量PCR （qPCR）检测c-fos mRNA表达;Western blot检测c-fos蛋白表达。结果： 与对照组比较,3个不同剂量HSJ处理组NB4细胞胞核均缩小,胞浆呈空泡状,核形呈肾形或蚕豆形;各HSJ处理组NB4细胞的NBT还原率均高于对照组,差异有统计学意义（P < 0.05）;HSJ处理组NB4细胞中G2期细胞比例均高于对照组,而S期细胞比例均低于对照组,差异有统计学意义（P < 0.05）;HSJ处理组CD14和CD11b阳性细胞表达率均高于对照组（P < 0.05）;HSJ低剂量组cfos mRNA和蛋白表达与对照组比较,差异均无统计学意义（P > 0.05）,HSJ中、高剂量组c-fos mRNA和蛋白表达均高于对照组,差异均有统计学意义（P < 0.05）。结论： HSJ可能诱导NB4细胞向成熟粒细胞方向分化。     

Unexpected absence of a myeloid surface antigen (3-fucosyl-N-acetyllactosamine) in promyelocytic leukemia

According to the criteria of the FAB classification we diagnosed acute promyelocytic leukemia in 19 out of 191 AML blast cell populations. The reaction pattern with the anti-myeloid monoclonal antibodies VIM2, MCS2 and MY9 and the MHC-class II antibody VID1 in all cases showed the characteristics which were expected from the normal granulopoiesis. The monoclonal antibody VIM-D5, which recognizes the myeloid cell surface structure 3-fucosyl-N-acetyllactosamine (3-FAL), was not reactive with the majority of promyelocytic blast cells, however. In 13 cases 3-FAL was not detectable and in 6 cases only low or moderate reactivity of VIM-D5 with blast cells was observed. The phenotype of leukemic promyelocytes thus differs from the majority of normal promyelocytes in this respect.     

Effect of 4-hydroperoxycyclophosphamide on leukemic and normal human myeloid progenitor cells

The sensitivity of myeloid progenitor cells from normal subjects (N-CFU-GM) and from leukemic patients in complete remission (LR-CFU-GM) to 4-hydroperoxycyclophosphamide (4-HC) were compared to the sensitivity of leukemic progenitor cells (L-CFU) to this drug. The results were expressed as the dose of 4-HC needed to kill 90% (TD 90) of the progenitor cells. The mean TD 90 were respectively for N-CFU-GM : 59 (+/- 11 S.E.M.) nM ml-1 and for L-CFU 79 (+/- 6 S.E.M.) nM ml-1. Thus, L-CFU were equally sensitive to 4-HC as N-CFU-GM. Moreover, the mean TD 90 for LR-CFU-GM was 87 (+/- 5 S.E.M.) nM ml-1. Thus, the sensitivity of N-CFU-GM and LR-CFU-GM did not differ significantly from that of L-CFU. These results are not encouraging for the use of 4-HC in vitro to eliminate the residual leukemic cells from autologous bone marrow of AML patients in complete remission. The sensitivity of L-CFU was modified neither by previous cytoreductive therapy (different from cyclophosphamide) nor by the time elapsed since diagnosis of AML.     

Increased in vitro radioresistance of bone marrow fibroblastic progenitors (CFU-F) from patients with acute non-lymphocytic leukemia

The proliferative potential following in vitro irradiation of bone marrow fibroblastic progenitors (CFU-F) derived from four patients with acute nonlymphocytic leukemia (ANLL) and seven nonleukemic subjects was compared. The CFU-F from the ANLL patients were significantly more radioresistant than the CFU-F from the nonleukemic subjects. The increased radioresistance in ANLL patients was evident in both the mean slope of the survival curve (control = ?0.385, ANLL = ?0.256) and in the D_o values (control = 2.68 Gy, ANLL = 4.61 Gy). Thus CFU-F derived from ANLL patients differ from those derived from nonleukemics in both radioresistance and in granulopoietic effects as suggested from previous studies.     

Ex vivo isolation,expansion and bioengineering of CCR7+CD95-/or CD62L+CD45RA+ tumor infiltrating lymphocytes from acute myeloid leukemia patients’ bone marrow

T cell based immunotherapies can be applicable to acute myeloid leukemia (AML). Therefore, the selection of optimal T cells, cell manufacturing, and therapeutic T cell engineering are essential for the development of effective adoptive T cell therapies for AML. Autologous tumor-infiltrating lymphocytes (TILs) have been in clinical trials to treat solid malignancies. Herein, we assessed whether TILs can be isolated from the bone marrow (BM) of AML patients, expanded ex vivo and utilized as a novel therapeutic strategy for AML. To this end, firstly we analyzed the immunophenotypes of a series of primary BM samples from AML patients (N = 10) by flow cytometry. We observed a variable amount of CD3+ TILs (range ∼2.3–∼32.6% of mononuclear cells) among BM samples. We then developed a novel protocol that produced a three-log ex vivo expansion of TILs isolated from AML patient BM (N = 10) and peripheral blood (PB) (N = 10), including from patients with a low number of CD3+ T cells, within 3, 4 weeks. Further, we identified previously described naïve T cells (CCR7+CD95-/or CD62L+CD45RA+) in AML BM and PB samples, which seemed to be required for a successful TILs ex vivo expansion. Finally, we showed that the expanded TILs could: (1) cause cytotoxicity to autologous AML blasts ex vivo (90.6% in control without T cell treatment vs. 1.89% in experimental groups with PB derived T cells and 1.77% in experimental groups with BM derived TILs, p < 0.01), (2) be genetically engineered to express CYP27B1 gene, and (3) infiltrate the BM and reside in close proximity to pre-injected autologous AML blasts of engrafted immunodeficiency mice. Altogether, these results provide a rationale for further studies of the therapeutic use of TILs in AML.