Study On The Differentiation Abnormalities Of Bone Marrow Hematopoietic Cells And Its Related Mechanisms In Myelodysplastic Syndrome

ObjectiveThis study was to detect the differentiation abnormalities of bone marrow hematopoietic cells in myelodysplastic syndrome (MDS), its related mechanisms, MDS malignant clone cells and their malignant characteristics of excessive proliferation, abnormal differentiation and apoptosis escape.MethodsSixty-two cases of MDS patients,8 acute myeloid leukemias preceded by MDS (MDS-AML),7 acute myeloid leukemias and 16 case-controls, as well as 32 normal controls were enrolled in this study. Part?The differentiation antigens on the membrane of bone marrow granulocytes, monocytes and erythroblasts, the subset of stem cell and bone marrow cell cycle were measured by flow cytometry. Part?The expressions of stem cell factor receptor(SCF-R), erythropoietin receptor(EpoR), granulocyte colony-stimulating factor receptor (G-CSFR) and thrombopoietin receptor (TpoR) on hematopoietic stem/progenior cells and the expressions of GATA-1 and GATA-2 in nuclear of stem/progenitor cells were measured by flow cytometry. The expression of GATA-1 mRNA in the bone marrow cells of cases with MDS and normal controls were measured by RT-PCR. Part?The expression of interleukin-3 receptor?(CD123) on the bone marrow stem cells of cases with MDS, AML and normal controls were measured by flow cytometry. Further to investigate the characteristics of proliferation and differentiation of CD123+ cells.ResultsPart?The granulocytic differentiation was analyzed with the combinations of CD13/CD11b, CD13/CD16 and CD11b/CD16. The "right hook", "sickle" and "retroflex 7" shape expressions were found in normal and case controls while there were various changes in MDS groups. The ratios of CD11b-/CD11b+(0.32±0.29) and CD16-/CD16+(1.32±0.77) were significantly higher in high-risk MDS group than those in case-control group(0.05±0.03 and 0.33±0.32 respectively) and control group (0.07±0.05 and 0.39±0.31 respectively) (P<0.05). The MFI (mean fluorescence index) of SSC (side scatter) in the granulocyte gate of high-risk MDS group was significantly lower while MFI of CD13 was significantly higher than the case-control and control groups. The mean percentages of CD11b-HLA-DR+(4.08%±2.97%), CD11b-HLA-DR-(14.90%±15.44%), CD16-HLA-DR-(38.98%±21.59%), CDllb+CD16-(32.57%±16.52%) and CD13+CD16-(43.97%±20.31%) granulocytes of high-risk MDS group were significantly higher than those of low-risk, case-control and control groups (P<0.05). The monocytic differentiation was analyzed with the combinations of CD13/CD16 and CDllb/HLA-DR. There were various changes in MDS group comparison with case-control and control groups. The mean percentages of CD11b"HLA-DR+(12.38%±8.97%), CD11b+HLA-DR-(38.33%±18.43%) monocytes of high-risk MDS group were significantly higher than those of case-control and control groups (P<0.05). The mean percentage of CD14+ (56.74%±17.73%) on monocytes of high-risk MDS group was significantly lower than those of control group(P<0.05). The mean percentages of CD56+(32.45%±20.07%), CD34+(13.54%±13.73%) and CD7+(15.31%±12.76%) of high-risk MDS group were significantly higher than those of case-control and control groups (P<0.05). The erythroid cell lineage differentiation was analyzed with CD71/glycophorin A combination. Double positive expression was found in all case controls and controls, but asynchronous expression of CD71/glycophorin A was found in some MDS cases. The mean percentages of CD71+and GlyA+cells in CD45" cell population were significantly lower in low-risk and high-risk MDS groups. The mean percentages of CD71+GlyA+double positive cells in CD45", CD71+and GlyA+cell population were significantly lower in low-risk and high-risk MDS groups. The abnormal numbers of the antigen expression of MDS cases per case correlated directly with their IPSS (international prognostic scoring system) (r=0.662, P=0.000), WPSS (WHO adapted prognostic scoring system) (r=0.602, P=0.000) scores and malignant clone burden(r=0.477, P=0.001). The mean percentages of CD34+cells in bone marrow karyocyte of high risk(2.29%±2.17%) and MDS-AML groups(18.69%±17.47%) were significantly higher than that of control group (0.36%±0.49%, P<0.05). The mean percentages of CD34+CD38+cells were significantly lower in low risk, high risk and MDS-AML groups(86.09%±7.79%, 81.68%±11.82%and 82.88%±2.60% respectively) than that in control group (92.21%±3.85%, P<0.05), thus the percentages of CD34+CD38" cells were significantly higher in either MDS (low-risk and high-risk) or MDS-AML groups (13.91%±7.79%,18.32%±11.82%or 17.13%±2.60% respectively) than that in control group (7.79%±3.85%, P<0.05). The percentages of CD34+CD38'cells of MDS cases correlated directly with their IPSS (r=0.493, P=0.001) and WPSS (?=0.586,P=0.000) scores. MDS patients with low percentages of CD34+CD38-(?12.0%) cells presented higher therapeutic efficacy than those with high percentages of CD34+CD38'(> 12.0%) cells (P> 0.05), but not reveal significant differences. The percentages of bone marrow mononuclea cells(BMMNCs) in Go/Gi phase of in low-risk, high-risk and MDS-AML groups (94.52%±4.32%, 96.07%±3.88%and 94.65%±4.55%respectively) were significantly higher than that of control group(88.94%±7.30%, P<0.01), thus the percentages of BMMNCs in S and G2/M phase were significantly lower in either MDS (low-risk and high-risk) or MDS-AML groups than that in control group (P<0.05).Part II In control group, the mean percentage of antigen expression of EpoR was significantly lower in CD34+CD38+cells (18.68%±18.34%) than that in CD34+CD38- cells (63.61%±19.98%, P<0.01).The expressions of SCF-R,G-CSFR and TpoR were not significantly different between the two cell populations. The expression of EpoR on CD34+CD38+cells of low-risk and high-risk MDS groups(8.30%±6.55%,7.82%±7.98%) were significantly lower than that of control group(18.68%±18.34%, P<0.05). The expression of G-CSFR on CD34+CD38+cells of low-risk and high-risk MDS groups(29.78%±19.14%,28.66%±21.14%)were significantly lower than that of control group(44.37%±23.43%, P<0.05). The amount of EpoR on CD34+CD38- cells of low-risk and high-risk MDS groups(42.19%±21.87%,25.67%±15.64%) were significantly lower than that of control group(63.61%±19.98%, P<0.01), The expression of TpoR on CD34+CD38' cells of low-risk and high-risk MDS groups(5.42%±4.72%,4.05%±3.95%) were significantly lower than that of control group(10.13%±8.31%, P<0.05). The incidence of cytopenia with low expression rates of hemopoietic cytokine receptors on CD34+ cells were higher than that of MDS with high expression rates of hemopoietic cytokine receptors. The expression of GATA-1 on CD34+CD38+cells of MDS groups were higher than that of control group, but not reveal significant differences.The expression of GATA-2 on CD34+CD38+cells of high-risk MDS group (12.47%±13.00%) was significantly higher than that of control group(5.64%±7.32%, P<0.05). The expression of GATA-1 on CD34+CD38- cells of high-risk MDS group (28.16?±13.71%) was significantly higher than that of control group(12.76%±14.02%, P<0.05). The expression of GATA-2 on CD34+CD38" cells of low-risk and high-risk MDS groups(17.12%±10.61%,29.38%±7.71%) were significantly higher than that of control group(10.07%±9.26%, P<0.05). The expression of GATA-1 mRNA in BMMNC of MDS patients (0.504±0.156) was significantly higher than that of normal controls (0.323±0.086, P<0.01).Part?The ratios of CD34+CD38"CD123+/CD34+CD38- in the bone marrow cells of low-risk, high-risk MDS and AML groups (42.48%±25.88%,51.62%±29.24%, 56.19%±32.20%) were significantly higher than that of normal control (9.06%±10.04%, P<0.01). The ratio of CD34+CD38"CD123+/CD34+CD38'in MDS groups was significantly positively correlated with the malignant clone burden(r=0.419,P=0.003) and the abnormal numbers of the differentiation antigen expression (r=0.462, P=0.001). The expressions of GATA-1, GATA-2 and CD71 in CD34+CD38-CD123+cells were significantly higher than those in CD34+CD38" CD123-cells of MDS and AML groups(P<0.01). The expression of EpoR on CD34+CD38-CD123+cells was higher than that on CD34+CD38-CD123- cells, but not reveal significant differences. The expressions of CD11b, CD114 and Annexin V on CD34+CD38-CD123+cells were significantly lower than those on CD34+CD38-CD123-cells of MDS and AML groups(P<0.05).Conclusions(1) There are abnormal expressions of differentiation antigens on bone marrow myeloied cells of MDS patients. And the severity is correlated with the IPSS, WPSS scores and malignant clone burden. The abnormal differentiation of myeloid cells is probably involved in the pathogenesis of MDS. So the examination of these antigenic expressions with flow cytometry might be helpful for diagnosis and prognosis of MDS.(2) There was abnormalitie of differentiation of CD34+bone marrow cells and high proportion of G0/G1 cells which indicated a G1 phase arrest in MDS. That indicated there were abnormal characteristics of differentiation and proliferation in hematopoietic cells of MDS. So the examination of CD34+bone marrow cells and cell cycle might be helpful for MDS diagnosis and assessment of prognosis and therapeutic effect. (3) There were abnormalities of some membrane hemopoietic cytokine receptors and transcription factors of CD34+bone marrow cells in MDS, which were associated with MDS cytopenia and might be useful for MDS diagnosis.(4) The percentage of CD34+CD38-CD123+cells in the bone marrow of MDS patients was increased. These cells manifest the malignant characteristics of excessive proliferation, abnormal differentiation and apoptosis escape. CD34+CD38" CD123+cells were probably the malignant clone cells in MDS.