| ObjectiveTo explore diagnostic indexes correlated with the malignant clone burden of myelodysplastic syndrome (MDS) involving immunophenotyping, cytogenetics, morphology and histochemistry, and further analyze the curative effects of various regimens on MDS in vitro and in vivo.MethodsSeventy-three cases of MDS patients as well as twelve normal controls were enrolled in this study. First Section The expression of interleukin-3 receptor a (CD123) and granulocyte colony stimulating factor (G-CSF) receptor (CD114) on the bone marrow cells of cases with MDS and normal controls were measured by FACS, in order to identify the characteristic immunophenotypes of malignant clone in MDS. Second Section The expression of delta-like 1 (dlk1) mRNA in the bone marrow cells of cases with MDS, AML and normal controls were measured by RT-PCR, aiming to search for the cytogenetic marker of malignant clone. Third Section Statistic methods such as t-test,χ2 test and Logistic regression analysis were adopted to calculate morphological, histochemical and iron metabolic indexes which were significantly correlated with typical karyotypes, malignant clone burden and cytogenetic evolution of MDS. Fourth Section Hematopoietic stem/progenitor cell culture of MDS was conducted in this section. Decitabine, homoharringtonine, daunorubicin and cytarabine were administered to the cells after fourteen days' culture. The apoptosis ratio, expression of caspase 3 mRNA, expression of CD11b and HLA-DR of the cells were measured by FCM and RT-PCR to compare the effects and mechanisms of different regimens on MDS. Fifth Section Seventy-three patients with MDS were classified into lower-risk and higher-risk groups according to IPSS scores, and the curative effect of stratified therapy on MDS was analyzed.ResultsFirst Section The ratio of CD34+CD38-/CD34+ in the bone marrow cells of MDS patients was〔(14.03±5.27)%〕, significantly higher than that of normal controls〔(7.70±4.36)%〕(P<0.01); The ratio of CD123+CD34+CD38-/CD34+CD38- in the bone marrow cells of MDS patients〔(48.39±28.15)%〕was significantly higher than that of normal controls〔(8.75±11.71)%〕(P<0.01), and was significantly positively correlated with the proportion of bone marrow blasts (r=0.457, P<0.05) and malignant clone burden(r=0.357, P<0.05). The expression of CD114 on CD123+CD34+CD38-cells〔(34.82±29.58)%〕was significantly lower than that on CD123'CD34+CD38-cells〔(53.48±27.41)%〕(P<0.05). CD123+CD34+CD38-cells were probably the malignant clone cells in MDS. Second Section The expression of dlkl mRNA in BMMNC of MDS patients (0.7342±0.3652) was significantly higher than that of normal controls (0.4801±0.1759) (P<0.05), and was significantly positively correlated with the proportion of bone marrow blasts (r=0.467, P<0.05). Patients with higher expression of dlkl (≥0.8) presented significantly higher malignant clone burden (0.4134±0.3999) than those with lower expression (<0.8) of dlkl (0.1517±0.3109) (P<0.05). Third Section Significant correlations were found between double-nucleus granulocytes and -7, erythrocyte rouleaux formation and -7, double-nucleus granulocytes and -5/5q-, hypolobated neutrophils and-20/20q-(P<0.05). The indexes of high malignant clone burden included group A (odd number-nucleus erythrocytes, blasts in peripheral blood) and group B (double-nucleus granulocytes, hypolobated neutrophils, dysplasia in three myeloid lineages, positive PAS, serum ferritin level>220ng/mL, and UIBC<40umol/L).Patients who fulfilled both criterion of A, or one of A and at least two of B, or at least four criterion of B presented significantly higher malignant clone burden (P<0.01). The risk factors of cytogenetic evolution of MDS include:odd number-nucleus erythrocytes, megaloblastic granulocytes and high myeloid differentiation index (DI) (P<0.05). Fourth Section The apoptosis ratios of cells administrated with 1.6mmol/L,3.2mmol/L and 6.4mmol/L decitabine were (21.969±10.187)%, (24.246±9.053)% and (26.438±10.742)% respectively, significantly higher than that of negative control (13.965±3.503)% (P<0.05). The expression of CDllb on cells administrated with 3.2mmol/L and 6.4mmol/L decitabine were (43.546±11.222)% and (46.854±10.347)% respectively, significantly higher than that of negative control〔(31.196±12.839)%〕(P<0.05). The apoptosis and death ratio of cells administrated with HA were (32.048±9.525)% and (31.550± 9.514)%〕respectively, both significantly higher than that of negative control (P<0.05). The death ratio of cells administrated with DA〔(55.396±16.063)%〕was significantly higher than those of all the other groups (P<0.05), while the apoptosis ratio〔(4.664±4.074)%〕and expression of CD11b〔(14.422±14.921)%〕were significantly lower than those of all the other groups (P<0.05). Fifth Section Stratified therapy based on different risk grades was the main strategy in MDS treatment. IPSS low and intermediate patients treated with hematopoietic stimulating factors (HSF) presented significantly higher overall effective rate (74.4%) and marked effective rate (65.1%) than those who did not receive HSF treatment (36.4%,18.2%) (P<0.05). IA was the first-line regimen for high-risk MDS patients. Decitabine exerted good effects on MDS patients.Conclusions(1)The indexes of high malignant clone burden of MDS include high expression of CD 123, high expression of dlkl, odd number-nucleus erythrocytes in bone marrow aspirate and blasts in peripheral blood.(2)Significant correlations were found between double-nucleus granulocytes and-7, erythrocyte rouleaux formation and -7, double-nucleus granulocytes and -5/5q-, hypolobated neutrophils and -20/20q-.(3)The risk factors of cytogenetic evolution of MDS include odd number-nucleus erythrocytes, megaloblastic granulocytes and high myeloid DI (P<0.05).(4) Stratified therapy based on different risk grades is the main strategy in MDS treatment. IPSS low and intermediate risk patients could benefit from the use of hematopoietic stimulating factors; The regimen of IA is the first-line treatment for high-risk MDS patients; Decitabine exerts good effects on MDS patients both in vitro and in vivo.
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