Analysis of Factors Influencing Overall Survival of MDS Patients Transplanted with HSCs / 中国实验血液学杂志

OBJECTIVE@#To analyze the effect of clinical features, routine laboratory examination and related gene mutation on the OS of patients with myelodysplastic syndrome (MDS) after hematopoietic stem cell transplantation (HSCT).@*METHODS@#121 patients diagnosed as MDS and underwent hematopoietic stem cell transplantation in the First Affiliated Hospital of Soochow University from October 2013 to August 2018 were selected. Basic information of the patients was collected, and blood cells, bone marrow blasts at initial diagnosis, chromosomal karyotypes and gene mutations of the patients were detected.The effect of different factors on overall survival (OS) was analyzed by statistical method.@*RESULTS@#Kaplan-Meier univariate analysis shows that OS was significanly different among different age groups. The 3-year OS rate of patients aged 0-29 years was (83.3±7.7) %, the 3-year OS rate in patients aged 30-49 years was (58.1±7.7 %), and the 3-year OS rate of patients aged 50-69 years was (31.0±22.6) %, which was statistically different (P＜0.05) between different groups. There were also significant differences in OS among patients with different transplantation types. 3-year OS rate: HLA-matched sibling HSCT＞unrelated HLA-matched HSCT＞haploidentical HSCT＞micro HSCT. The OS rate of patients with bone marrow blasts≥10% seems lower than blasts＜10%, but there was no statistical difference.The 3-year OS rate of patients with chromosomal karyotype complex abnormality was (47.7±11.5) %, and that of patients without complex abnormality was (80±4.2) % which was statistical difference (P＜0.05). Patients with DNMT3A, NRAS, TP53 and GATA2 mutations had shorter OS time compared with patients without mutation of these genes, which shows statistically significant (P＜0.05). COX multivariate analysis showed that age, chromosome karyotype, DNMT3A, TET2, GATA2 and NRAS were the independent factors influencing OS of patients after HSCT, with statistically significant difference.@*CONCLUSION@#age of patients, donor selection of HSCT, chromosome karyotype, DNMT3A, NRAS, TP53, GATA2 and TET2 gene mutations are all independent factors affecting the OS of patients after HSCT. Therefore, the assessment of the OS of MDS patients with transplantation requires comprehensive consideration.

Differentiation of K562 Cells Induced by Pulsatilla Saponin A into Erythroid Lineage / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the differentiation-inducing potentiality of Pulsatilla saponin A on K562 cells.</p><p><b>METHODS</b>Pulsatilla saponin A of different concentrations was used to treat K562 cells; the benzidine staining and the hemoglobinometry were applied to measure the change of hemoglobin content; the flow cytometry (FCM) was used to detect the expression of CD71 and GPA on K562 cells.</p><p><b>RESULTS</b>K562 cells treated with 4 µg/ml pulsatilla saponin A differentiated into the erythroid lineage. With the treatment of pulsatilla saponin A, the hemoglobin content in K562 cells increased significantly; CD71 and GPA expression on the K562 cell surface were up-regulated.</p><p><b>CONCLUSION</b>Pulsatilla saponin A can induce K562 cells to differentiate into erythroid lineage.</p>

Clinical Analysis on Treatment of Chronic Myelomonocytic Leukemia with Allogeneic Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze retrospectively the therapeutic efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for chronic myelomonocytic leukemia (CMML).</p><p><b>METHODS</b>The engraftment, graft versus host disease (GVHD), infection, relapse, and survival of 13 CMML patients received allo-HSCT were observed. The clinical outcome of allo-HSCT for CMML was analyzed.</p><p><b>RESULTS</b>Thirteen (10 males and 3 females) CMML patients with a median age of 38 years old received allo-HSCT including 4 from HLA-matched unrelated donors, 6 from HLA-matched sibling donors and 3 from haploidentical related donors. All 13 patients achieved engraftment, and the median time of neutrophil engraftment and platelet engraftment were 12 (11-18) days and 15 (10-55) days respectively, acute GVHD occurred in 8 patients. After the median follow-up of 13 (6-29) months, the overall survival, disease free survival and relapse were 53.8%, 53.8%, 7.7%, respectively.</p><p><b>CONCLUSION</b>Allo-HSCT can improve the survival of patients with CMML, and is a effective method for treatment of CMML.</p>

Biocompatibility of polyethylene imine (PEI)-coated magnetic Fe₃O₄ nanoparticles in SHI-1 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the feasibility of magnetic resonance cell imaging technology by using polyethylene imine (PEI)-coated magnetic nanoparticles of Fe₄O₄ (PEI-Fe₄O₄-MNPs) to track cell biology behavior.</p><p><b>METHODS</b>Endocytic PEI-Fe₄O₄-MNPs in SHI-1 cells were observed by transmission electron microscopy (TEM) . Iron contents of nano-labeled cells were analyzed by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and Prussian blue staining. The proliferation ability of labeled cells was detected by cell counting kit-8 (CCK-8) assay; the differentiation and colony-forming abilities were also observed. SHI-1 cells without endocytosing PEI-Fe₄O₄-MNPs were used as control.</p><p><b>RESULTS</b>Our data showed that PEI-Fe₄O₄-MNPs could label SHI-1 cells. The labeling efficiency depended on the nanoparticles' concentration and the duration of cells treating. Inhibition rates of SHI-1cells labeled by 60-100 μg Fe/ml PEI-Fe₄O₄-MNPs were much higher than of 5-50 μg Fe/ml ones following treating by 5-100 μg Fe/ml PEI-Fe₄O₄-MNPs for 48 hrs. The expressions of CD11b and CD14 were (78.4±18.5)% and (18.7±2.9)% in control vs (83.3±14.2)% and (20.4±2.1)% in cells fractions treated by 30 μg Fe/ml PEI-Fe₄O₄-MNPs. Clony-forming rates of SHI-1 cells labeled by 0, 20 , 50 μg Fe/ml PEI-Fe₄O₄-MNPs were (25.20±7.22)%, (25.93±13.15)%, (23.37±9.33)%, respectively. Differentiation and colony-forming potentials of labeled cells were similar with control in the certain range of PEI-Fe₄O₄-MNPs concentration.</p><p><b>CONCLUSION</b>SHI-1 cells were efficiently labeled by PEI-Fe₄O₄-MNPs with well biocompatibilities in proper range of concentration, the latter could be coupled with magnetic resonance imaging (MRI) to track cells in vivo.</p>

Significance of interplay between Rap1 and cadherin to the development of myelodysplastic syndrome / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the hematopoietic pathophysiology of myelodysplastic syndrome (MDS) at stem/progenitor cell level by analyzing the gene expression profiles associated with hematopoiesis.</p><p><b>METHODS</b>The differentially expressed genes which were involved in the hematopoiesis were screened by microarray using CD34(+) cells from MDS patients firstly. RQ-PCR was then applied to validate the screened genes using CD34(+) cells from MDS-RA patients who had normal karyotype. The linkages with hematopoiesis among these validated genes were analyzed.</p><p><b>RESULTS</b>Among the differentially expressed genes in CD34(+) cells of MDS-RA patients, Rap1GAP was up-regulated significantly (P < 0.01). Cadherins, which can interplay with Rap1, including N-cadherin and E-cadherin, were down-regulated significantly (P < 0.01). β-catenin, a downstream effector of cadherins, was highly expressed in MDS-RA patients (P < 0.01). c-myc binding protein was down-regulated (P < 0.01), and c-myc promoter binding protein was up-regulated (P < 0.01). Rac1, Rac2 and Cdc42, which belong to RhoGTPases family and are associated with the cell morphology and hematopoiesis, were all expressed highly in MDS-RA patients (P < 0.01).</p><p><b>CONCLUSION</b>The abnormal expression of cadherin, β-catenin and c-myc associated genes were closely related to the dysplastic hematopoiesis of MDS. The down regulation of cadherin was associated with the positive feedback mechanism between Rap1 and cadherin. The aberrant expression of Rac1, Rac2 and Cdc42 may contribute to the morphological dysplasia of MDS.</p>

C-kit, NPM1 and FLT3 gene mutation patterns and their prognostic significance in 656 Chinese patients with acute myeloid leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the prevalence and distribution of C-kit, NPM1 and FLT3 gene mutations in patients with acute myeloid leukemia (AML), and to analyze the relationship between the gene mutations and their prognosis.</p><p><b>METHODS</b>Mutations in exon 8 and 17 of C-kit gene, exon 12 of NPM1 gene, exon 20 of FLT3-TKD gene, and exon 14/15 of FLT3-ITD gene were detected by direct sequencing. Clinical data was collected and followed up if the patient had accepted treatment in our hospital.</p><p><b>RESULTS</b>Among the 656 AML patients, mutations in C-kit exon 8 were found in 6 patients (0.9%), C-kit exon 17 in 33 (5.0%), NPM1 in 169 (25.8%), FLT3-TKD in 46 (7.1%), and FLT3-ITD in 178 (27.1%). Six subtypes of mutations were detected in C-kit exon 8, 8 in C-kit exon 17, 11 in FLT3-TKD, 15 in NPM1, of which 5 were not reported before. C-kit exon 17 mutations were more frequently detected in patients with t(8;21) and exon 8 in patients with inv(16) cytogenetic abnormality. No other gene mutations except FLT3 were detected in M(3) patients. NPM1 and ITD mutations were often detected in individuals with normal cytogenetics or M(5) and M(1) of FAB classification, and accompanied with high white blood cell counts in peripheral blood, high blast counts in bone marrow and low CD34 expression. The older the patients were when diagnosed, the more gene mutations and the higher white blood cell count were detected. More mutations were found in individuals with normal karyotype than that with other karyotypes. It appeared that FLT3-ITD was significantly associated with shorter overall survival (OS) (P = 0.004), NPM1 was not significantly associated with OS, but NPM1(+)/ITD(-) patients had the longest OS.</p><p><b>CONCLUSIONS</b>Our results showed that the mutation types and amounts had particular distribution in MICM subtypes, and were associated with white blood cell counts in peripheral blood, blast counts in bone marrow and prognosis. Especially for patients with normal karyotype, the genetic mutations could be new molecule marker.</p>

Monitoring the expression ratio of AML1-ETO9a isoform in t(8;21) acute myeloid leukemia and its significance / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the expression ratio of AML1-ETO9a (AE9a) isoform in t(8;21) acute myeloid leukemia (AML) and its clinical significance.</p><p><b>METHODS</b>Bone marrow samples from 44 newly diagnosed t(8;21) AML patients co-expressed AE9a and AE were screened by RT-PCR. The alteration of the AE9a expression ratio was monitored during follow-up by using quantitative real-time RT-PCR (qPCR).</p><p><b>RESULTS</b>The expression level of AE9a was markedly lower than that of AE in these patients. There was a positive correlation between the expression level of AE9a and AE in most of bone marrow samples. The transcript level of both AE9a and AE was decreased in the 44 patients after one course of standard chemotherapy, but the percentage of AE9a expression level was increased in comparison with that before treatment (P < 0.05). After one course of standard chemotherapy treatment, the percentage of AE9a in incomplete remission (ICR) patients was significantly higher than that in CR patients (P < 0.05). Relapsed patients had a higher AE9a ratio than the unrelapsed patients (P < 0.05). During the remission, the percentage of AE9a in 11/17 relapsed patients obviously elevated even while the expression of AE fusion gene at low level.</p><p><b>CONCLUSIONS</b>AE9a and AE co-expressed in most of AML patients with t(8;21) translocation. The expression level of AE9a was lower than that of AE, and there is a positive correlation between the expression level of these two isoforms. The sensitivity of AE9a gene to the standard chemotherapy is less than that of the AE fusion gene. Monitoring the AE9a to AE ratio during the CR can predict the early relapse of the disease compared to monitoring the AE alone.</p>

Construction of AML1-ETO eukaryotic expression vector and its effects on proliferation and differentiation of U937 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To construct a pcDNA3.1-AML1-ETO expression vector and investigate its effects on proliferation and differentiation of U937 leukemic cells.</p><p><b>METHODS</b>AML1-ETO gene was amplified by PCR from pCMV5-AML1-ETO and inserted into eukaryotic expression plasmid pcDNA3.1/V5-His-TOPO. The recombinant plasmid was transfected into U937 cells by Lipofectamin 2000. Individual clones selected with G418 were isolated. The integration and the expression levels of AML1-ETO in transfectants were determined by PCR, RT-PCR and Western blot analysis respectively. Trypan blue refusal staining method was used to detect the proliferation of U937 cells. Light microscope was applied to observe the morphologic changes of the cell. The expression of myeloid cell differentiation antigen was detected using flow cytometry.</p><p><b>RESULTS</b>The recombinant pcDNA3.1-AML1-ETO was confirmed by enzyme digestion and sequencing. The highly expressing AML1-ETO subclone was established. AML1-ETO was expressed in U937 cells transfected with pcDNA3.1-AML1-ETO. The growth of the monoclonal cells was inhibited evidently (P < 0.05). The expression of CD11b in transfected group \[(4.17 ± 0.31)%\] was lower than that in empty plasmid transfected group and non-transfected group \[(11.40 ± 0.17)% and (11.03 ± 0.15)%\] respectively (P < 0.001). Transfected cells displayed morphology of less differentiation. The expression level of CDl1b was unchanged in transfected cells treated with TPA (P > 0.05).</p><p><b>CONCLUSION</b>The eukaryotic expression vector for AML1-ETO gene was successfully constructed and expressed in U937. AML1-ETO inhibits the proliferation and differentiation of transfected cells. It provides the basis for further study of mechanisms of AML1-ETO in leukemogenesis.</p>

Regulatory function and expression of rap1gap gene in hematopoietic cells-review / 中国实验血液学杂志

Rap1 is a small G protein belonging to the RAS superfamily. Rap1 signalling has effects on cell growth, cell proliferation and involves in regulation of the mitogen activated protein (MAP) kinase or ERK (extracellular signal regulated kinase) cascade. Rap1 will directly activate ERK through B-Raf. B-Raf is a member of Raf family, and presents in neuronal and hematopoietic cells. Oncogenic mutations of gene RAS are most frequent and detected in 20% - 30% of human leukemias and 10% - 15% of MDS cases. The review summarizes the regulatory function of Rap1 in development of hematopoietic cells and effect of Rap1 in hematologic malignancies.

Protein RAP1GAP in human myelodysplastic syndrome detected by flow cytometry and its clinical relevance / 中国实验血液学杂志

Previous study on the gene expression profile of human MDS by using microarray discovered that transcription of RAP1GAP was up-regulated, which was confirmed by quantitative RT-PCR in expanding cohort of MDS patients. This study was pourposed to investigate the expression of RAP1GAP in human MDS and its clinical relevance. The expression of RAP1GAP in bone marrow cells of 19 MDS patients was detected by flow cytometry and was compared with that in patients with non-malignant blood diseases and acute leukemias, meanwhile the relevance between expression level of RAP1GAP and hemoglobin, leukocytes, platelets, blasts percentage in bone marrow cells and IPSS score was analyzed. The results indicated that the expression level of RAP1GAp in MDS patients significantly increased as compared with patients with non-malignant blood diseases or AML (8.42 +/- 8.37% vs 2.97 +/- 4.75% or 2.26 +/- 4.24%). Among MDS patients, the expression level of RAP1GAP in MDS-RA was significantly higher than that in MDS-RAEB (11.64 +/- 9.07% vs 4.37 +/- 4.65%). However, no definitive correlation of expression level with above-mentioned clinical parameters was found in detected patients with DMS. In conclusion, the expression of RAP1GAP in MDS patients obviously increases, the relationship between expression level of RAP1GAP and laboratory hematological parameter and IPSS score does not be confirmed. The role played by RAP1GAP expression in the pathogenesis of MDS and its clinical significance during progression of MDS towards AML deserves further studies.

Detection of clonal immunoglobulin and T-cell receptor gene rearrangements in newly diagnosed adult patients with acute lymphoblastic leukemia by using multiplex PCR protocols / 中华血液学杂志

<p><b>OBJECTIVE</b>To provide the evidence of RQ-PCR-based assessment of minimal residual disease (MRD), the clonal immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements were identified in newly diagnosed adult patients with acute lymphoblastic leukemia (ALL) by multiplex PCR protocols.</p><p><b>METHODS</b>Forty newly diagnosed adult patients with B-lineage (B-) and T cell (T-) ALL were involved in this study. All DNA samples were obtained from the bone marrow (BM) mononuclear cells (MNC). IgH, IgK, TCRB, TCRG and TCRD gene rearrangements were detected by BIOMED-2 multiplex PCR protocols, which included 96 different primers and 14 multiplex PCR tubes.</p><p><b>RESULTS</b>The clonal immunoglobulin (Ig) rearrangements were found in 96% of B-ALL, 86% being IgH and 14% IgK. While in T-ALL, clonal TCR rearrangements were found in all of the patients, 83% being TCRB, 78% TCRG and 39% TCRD. More than two clonal markers were found in 91% of B-ALL and 89% of T-ALL patients.</p><p><b>CONCLUSIONS</b>The detection rate of clonal rearrangements using the BIOMED-2 14 multiplex PCR tubes is high, which can detect virtually all clonal B and T-cell proliferations. It can be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.</p>