The Determination Of Leukemia Stem Cell In Childhood Acute Myeloid Leukemia And Its Clinical Significance

BackgroundAcute myeloid leukemia(AML)is one of the most common types of hematopoietic malignancies in children.With the understanding of AML biological features,patients with AML can be treated effectively with chemotherapy based on the combination of anthracyclin and cytarabine.Chemotherapeutic treatment of AML has resulted in a high percentage of complete remission.However,drug resistance and relapse remain the two major obstacles to cure this disease,which have been now found to be derived from the existence of leukemia stem cells(LSC).LSC is a rare subpopulation of leukemia cells in patient with leukemia,which accounts for about 0.1～1.0%of total leukemia cells.This population is not composed of homogeneous leukemia cells,but of heterogeneous cells that are organized in a hierarchical fashion, analogous to normal hematopoietic stem cells(HSC).Several studies have demonstrated that LSC is the only subpopulation with the capacity to initiate and to maintain the leukemia proven by long-term culture in vitro and by injecting LSC into the severe combined immunodeficient mice(SCID/NOD).LSCs have some similar biological features to normal HSC in terms of self-renewal, multi-lineage commitment and extensively proliferative potential.In addition,several studies have showed that HSC and LSC share some cell-surface markers but not others.For instance, HSC and LSC both express CD34 but not CD38,CD71 and HLA-DR.However,Thy-1(CD90), c-kit(CD117)and GM-CSF Rα(CD116)are expressed on HSC but not on LSC.However, CD123 is only expressed on AML LSC but not on normal HSC.The concept that the immunophenotype for AML LSC is CD34+/CD38-/CD123+ and that for normal HSC is CD34+/CD38-/CD123- is generally accepted.If the cells whose immunophenotype as CD34+/CD38-/CD123+ are found in non-AML,we name them AML LSC immunophenotype-identical cells(AML LSC-IPIC).The presence of residual malignant cells among normal cells in patient with leukemia after complete remission(CR)is termed minimal residual disease(MRD).These malignant cells are often below the limits of detection by cytomorphological techniques.Several reports have showed that the detection of MRD is useful in clinical practice due to its important prognostic value in ALL.There are different views about prognostic value with regard to detection of MRD in AML.Although the existence of LSC is the origin of relapse and resistance to drugs, the correlation between LSC concentrations and MRD levels after remission has not been reported in the literature both at home and abroad.Although the presence of human AML LSC was confirmed in AML cases at diagnosis,one can imagine that the difference of AML LSC concentrations may also exist in AML both at diagnosis and at remission.Whether the presence of AML LSC in AML cases both at diagnosis and at remission predicts the outcome remains unclear.In order to address this question,by multi-parameter flow cytometer(FCM)and combination of monoclonal antibodies,we examined the AML LSC concentrations in childhood leukemia both at initial diagnosis and at remission and correlated it to the MRD levels at different time points after remission,which might provide clinician a new parameter for monitoring the proper treatment of childhood AML in the future.Material and Methods1.Patient samples:The consecutive patients from our hospital from July 2007 through February 2008 were enrolled into this study.A total of 113 samples from patients with acute leukemia,76 samples from boys,37 samples from girls with a male to female ratio is 2.1:1 were analyzed.The median age was 7.0 years(range 2～15 years).Bone marrow samples from 12 children(the median age was 5.5 years(range 1～12 years))with non-malignancy were selected as control(6 boys and 6 girls with a male to female ratio of 1:1).2.Separation and preparation of mononuclear cells(MNC):All heparinized bone marrow MNCs were separated by Ficoll-Hypaque gradient centrifugation.After wash with phosphate buffered saline(PBS)containing 0.1%fetal calf serum(FCS),MNC suspension was prepared.3.Staining of MNC with fluochrome labeled monoclonal antibodies:1×10~6 cells were aliquoted into each tube was incubated with 1ml human AB plasma for 10 min to prevent nonspecific antibody binding.In order to examine the LSC concentrations precisely,all samples was analyzed freshly and four tubes were employed for one sample.Different monoclonal antibodies or mouse IgG1 isotype controls were added into four different tubes. Antibodies labeled with APC were added 5μl per tube,while those labeled with PerCP and PE were add 20μl per tube.MNCs(10~6/tube)were incubated with various combinations of monoclonal antibodies at room temperature for 20 min protected from light.After one wash with PBS to remove excess free antibodies,the red blood cells(RBC)were lysed in 2 ml lysing solution.MNC were washed again and suspended in a total of 400μl of PBS with 0.1%FCS before flow cytometrical analysis.4.AML LSC and AML LSC-IPIC determination and analysis:Cell acquisition and analysis were performed using a FACScalibur flow cytometer.For each specimen,100,000 events were acquired and saved in computer.The gating strategy was according to the method described in the literature.5.Minimal residual disease(MRD)detection:At diagnosis,leukemia-associated immunophenotypes(LAIP)were established.The rationale for the detection of MRD using LAIP was based on the idea that the MRD cells beard the same immunophenotype on blasts at diagnosis that survived chemotherapy.Results1.AML LSC or AML LSC-IPIC concentrations in newly diagnosed acute leukemia specimens The AML LSC or AML LSC-IPIC concentrations in AML,ALL and control were in average 166(range 14～1459)/100,000,7(range 0～560)/100,000 and 0(range 0～6)/100,000 at diagnosis,respectively.The AML LSC concentrations in AML at diagnosis were significantly higher than those either in ALL at diagnosis or in non-malignancy control group(P＜0.017). The AML LSC-IPIC concentrations in ALL at diagnosis was significantly higher than that in non-malignancy control group(P＜0.017).2.AML LSC or AML LSC-IPIC concentrations in leukemia specimens after remission(CR) The AML LSC or AML LSC-IPIC concentrations in AML,ALL and control were in average 6 (range 0～41)/100,000,10(range 0～105)/100,000 and 0(range 0～6)/100,000 after CR, respectively.No significant differences of AML LSC or AML LSC-IPIC concentrations were found between AML and ALL samples(P＞0.017).The concentrations of AML LSC or AML LSC-IPIC in AML and ALL were all higher significantly as compared to that of control (P＜0.017).3.AML LSC concentrations in AML at different stagesThe AML LSC concentrations in AML at diagnosis,AML at non-CR,AML after CR and control were in average 166(range 14～1459)/100,000,36(range 5～224)/100,000,6(range 0～41)/100,000 and 0(range 0～6)/100,000,respectively.No significant differences of AML LSC concentrations were found between AML at diagnosis and non-CR samples(P＞0.008). The AML LSC concentrations after remission were significantly reduced as compared to those at diagnosis(P＜0.008),but the concentrations after remission,was still higher than that.of the control(P＜0.008).4.AML LSC-IPIC concentrations in ALL at different stagesThe AML LSC-IPIC concentrations in ALL at diagnosis,ALL after CR and control were in average 7(range 0～560)/100,000,10(range 0～105)/100,000 and 0(range 0～6)/100,000, respectively.No significant differences of AML LSC-IPIC concentrations were found between at diagnosis and after CR in samples(P＞0.017).The concentrations of AML LSC-IPIC at diagnosis and after CR in ALL samples were all higher significantly as compared to that of control(P＜0.017).6.The correlation of AML LSC concentrations and MRD levels in AML after remission In order to clarify if there was any correlation between AML LSC and MRD levels,the AML LSC concentration from 33 AML samples were determined.The results showed that significant negative correlation were found between AML LSC concentrations and MRD levels(r=-0.466, P=0.006). Conclusions1.The AML LSC concentrations in newly diagnosed AML are significantly higher than those in newly diagnosed ALL and the control group.Those cells in AML patients are significantly reduced when complete remission has achieved after chemotherapy,but the low levels of these populations still remain;2.The phenotypically same(CD34+/CD38-/CD123+)AML LSC populations have also been found in ALL patients' bone marrow,but the concentrations are not significantly different when CR has achieved.No significant elevation of these phenotypically same populations has been found in non-malignancy group;3.The significant negative correlation between AML LSC concentrations and MRD levels in AML patients after remission has been found.