Establishment and Validation of Immune Risk Score for Predicting Survival of Patients with Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To establish an immune gene prognostic model of acute myeloid leukemia (AML) and explore its correlation with immune cells in bone marrow microenvironment.@*METHODS@#Gene expression profile and clinical data of TCGA-AML were downloaded from TCGA database. Immune genes were screened by LASSO analysis to construct prognosis prediction model, and prediction accuracy of the model was quantified by receiver operating characteristic curve and area under the curve. Survival analysis was performed by Log-rank test. Enriched pathways in the different immune risk subtypes were evaluated from train cohort. The relationship between immune prediction model and bone marrow immune microenvironment was verified by flow cytometry in the real world.@*RESULTS@#Patients with low-risk score of immune gene model had better prognosis than those with high-risk score. Multivariate analysis showed that the immune gene risk model was an independent prognostic factor. The risk ratio for AML patients in the training concentration was HR=24.594 (95%CI: 6.180-97.878), and the AUC for 1-year, 3-year, and 5-year overall survival rate was 0.811, 0.815, and 0.837, respectively. In addition, enrichment analysis of differential gene sets indicated activation of immune-related pathways such as cytokines and chemokines as well as autoimmune disease-related pathways. At the same time, real world data showed that patients with high immune risk had lower numbers of CD8+T cells and B lymphocytes compared with low immune risk patients.@*CONCLUSION@#We constructed a stable prognostic model for AML, which can not only predict the prognosis of AML, but also reveal the dysregulation of immune microenvironment.

Clinical Analysis of CMV Infection after Allogeneic Hematopoietic Stem Cell Transplantation in Severe Aplastic Anemia / 中国实验血液学杂志

OBJECTIVE@#To investigate the clinical characteristics and risk factors of cytomegalovirus (CMV) infection after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with severe aplastic anemia (SAA).@*METHODS@#Clinical data from 270 SAA patients with allo-HSCT were retrospectively analyzed, including 108 sib congruence patients and 162 substitute donors (68 unrelated donor congruence patients and 94 related haploid patients). Different pretreatment schemes were selected for different transplantation modes. The HLA-identical sibling and haploid grafts were all bone marrow and peripheral blood stem cells, and the grafts from unrelated donors were peripheral blood stem cells. After granulocyte implantation, blood CMV-DNA was regularly monitored. Flow cytometry was also used to determine the absolute number of CD3@*RESULTS@#CMV infection occurred in 229 of 270 patients with an incidence of 84.8%. Among them, 18 patients developed giant cell disease. Univariate analysis showed that alternative donors (unrelated total and haploid donors), mycophenolate mofetil and acute graft-versus-host disease were statistically significantly associated with CMV infection (P<0.05). Multivariate analysis showed that alternative donors were associated with CMV infection. The recovery of CD3@*CONCLUSION@#After allo-HSCT, substitute donors are more easily to develop CMV infection than full-sibling donors, and the reconstruction of immune function is delayed after transplantation.

Effect of Competitive Antagonist of Transmembrane Glutamine Flux V-9302 on Apoptosis of Acute Myeloid Leukemia Cell Lines HL-60 and KG-1 / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of V-9302 (an antagonist of transmembrane glutamine flux) on the proliferation and apoptosis of acute myeloid leukemia cells HL-60 and KG-1.@*METHODS@#HL-60 and KG-1 cells at logarithmic phase were treated by different concentrations of V-9302. CCK-8 assay was used to detect the proliferation of the cells. Annexin V-FITC / PI double staining flow cytometry was used to detect the apoptosis of HL-60 and KG-1 cells. The expressions of BAX, BCL-2 and Caspase3 were detected by RT-qPCR and Western blot.@*RESULTS@#V-9302 could significantly inhibit the growth of HL-60 and KG-1 cells. The concentration of V-9302 at 10, 20 μmol/L could significantly promote the apoptosis of HL-60 and KG-1 cells(P<0.05). The results of apoptosis related gene detection showed that when V-9302 was applied to HL-60 and KG-1 cell lines at 10 and 20 μmol/L, the expression levels of Pro-apoptotic protein genes BAX and Caspase3 in HL-60 and KG-1 were significantly higher than those in control group (P<0.05), while the expression level of anti-apoptotic protein gene BCL-2 was significantly lower than that in the control group (P<0.05). The results of Western blot were basically consistent with that of RT-qPCR.@*CONCLUSION@#Competitive antagonist of transmembrane glutamine flux V-9302 can significantly promote the apoptosis of acute myeloid leukemia cell lines HL-60 and KG-1.

Relationship Between the Change of Microbial Diversity and Gastrointestinal Graft-Versus-Host Disease Following Allogeneic Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

OBJECTIVE@#To investigate the change of microbial diversity and its relation with gastrointestinal (GI) graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation (allo-HSCT).@*METHODS@#Fecal samples were collected at the time point of 2 weeks before transplantation (pre-transplant group), 1 month after transplantation (post-tranplant group) and onset of GI GVHD in 65 hematonosis patients, which were also collected in 26 donors and 10 healthy subjects (control group). 16S rRNA was extracted from fecal microbiotas whose V4 variable region was amplified. The amplification products were sequenced in Illumina HiSeq 2500 platform, and the sequencing results were analyzed and compared.@*RESULTS@#The microbial diversity was 5.70（3.74, 10.60）in pre-transplant group, 7.30（4.89, 11.41）in control group, and the differences between them were not statistically significant. The microbial diversity was 3.88（2.39, 6.49）in post-transplant group, lower than that in control group and pre-transplant group. After transplantation, the microbial diversity was 4.24（2.47, 7.16）in the patients without GI GVHD, while the microbial diverosity was 2.90 (1.48, 5.64) in patients subsequently suffered from GI GVHD, but the differences between them were not statistically significant. The microbial diversity was 2.13（1.76, 3.75）onset of GI GVHD, which was lower than that without GI GVHD.@*CONCLUSION@#Intestinal microbial diversity decreases after allo-HSCT, and is associated with with Gl GVHD.

Value of CD3CD4T Cell Count in Prediction of Viral Infection after Hematopoietic Stem Cell Transplantation in Patients Severe Aplastic Anemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the value of CD3CD4T cell count in prediction of viral infections after allogeneic hematopoietic stem cell transplantation(allo-HSCT) in the patients with severe aplastic anemia(SAA).</p><p><b>METHODS</b>A total of 78 SAA patients with allo-HSCT in Guangzhou First People's Hospital from January 2014 to July 2016 were enrolled in this study. The absolute numbers of CD3CD4T cells were measured by flow cytometry at 1,2,3,6, and 12 month after allo-HSCT. According to the cell counts, the patients were divided into 3 groups: i.e. <50/µl (n=120), 50-100/µl(n=48) and >100/µl(n=123)groups. The infection incidences of human cytomegalovirus (HCMV) and Epstein-Barr virus(EBV) within 2 weeks around each time point were compared between different groups. According the counts of CD3CD4T cells at 3 months after-transplant, these patients were divided into 2 groups, i.e.>100/µl (n=30) and ≤100/µl (n=48). The incidences and duration of HCMV and EBV infection, overall survival rate were compared between 2 groups.</p><p><b>RESULTS</b>The incidences of CMV and EBV infection significantly decreased in CD3CD4T cell >100/µl group as compared with <50/µl and 50-100/µl groups. At 3 months after-transplant, there was lower incidence rates of CMV disease, EBV infection, shorter durations of CMV infection and better survival in CD3CD4T cell >100/µl group as compared with ≤100/µl group.</p><p><b>CONCLUSION</b>CD3CD4T cell count is a good predictor for CMV and EBV infection after allo-HSCT in SAA patients. There are low risk of infe-ctions from CMV and EBV when CD3CD4T cell count >100/µl in any time after transplant, which means lower occurrence of CMV and EBV infection and better survival when CD3CD4T cell counts is >100/µl in 3 months after transplant in SAA patients.</p>

Effect of RYBP Gene Silencing on Sensitivity of HL-60 Cells to Chemotherapeutic Drugs / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of RYBP gene on sensitivity of HL-60 cells to chemotherapy drugs by using RNA interference.</p><p><b>METHODS</b>Plasmid expressing RYBP specific shRNA was constructed and then was used to establish the RYBP knockdown stable HL-60 cell line. Q-PCR and Western blot were used to confirm the efficacy of RYBP gene silencing at mRNA and protein level respectively; then the DNA ladder and Annexin V labeled flow cytometry were used to detect cell apoptosis; CCK-8 was used detect the sensitivity of HL-60 cells to the chemotherapeutic drug cytarabine or daunorubicin.</p><p><b>RESULTS</b>The lentiviral-RYBP-shRNA vector was succesfully and effectively inhibit the expression of RYBP at mRNA and protein in HL-60 cells. It was found that without chemotherapy drug treatment the apoptosis rate of RYBP shRNA group was lower than that of the empty vector control group (NC group). When treated with cytarabine, the apoptosis rate and inhibitive rate of RYBP shRNA group were lower than those of NC group. Besides, when treated with daunorubicin, the apoptosis rate of RYBP shRNA group was lower than that of NC group, while the inhibitive rate had no significant difference.</p><p><b>CONCLUSIONS</b>RYBP gene silencing can inhibitive the apoptosis of HL-60 cells and significantly reduce the sensitivity to cytarabine, but this gene silencing can't affect the sensitivity to daunorubicin.</p>

Effect of Compound Zaofan Pill on Bone Marrow MVD and VEGF of Patients with Chronic Aplastic Anemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the clinical efficacy of compound Zaofan pill combined with cyclosporine and androgen for treatment of patients with chronical aplastic anemia(CAA), and its effect on bone marrow microvessel density(MVD) and vascular endothelial growth factor(VEGF) of CAA patients.</p><p><b>METHODS</b>A total of 76 cases of CAA were randomly divided into group 1 and group 2, among them the group 1 (38 cases) was treated with cyclosporine and androgen alone, while the group 2(38 cases) was treated with compound Zaofan pill combined with cyclosporine and androgen. Samples of 20 cases with normal bone marrow were used as controls. The clinical effects of each groups were observed, and the mean bone marrow MVD and VEGF levels were detected before and after half a year's treatment.</p><p><b>RESULTS</b>The mean bone marrow MVD and VEGF levels in CAA group before treatment were significantly lower than that in normal control group, but bone marrow MVD and VEGF levels in 2 groups significantly increased after treatment. Group 1 showed the cure and response rate of 15.8%, while group 2 showed the cure and response rate of 44.7%, there was significant difference between the two groups (P<0.05)). There was no significant difference of bone marrow MVD and VEGF expression before treatment between group 1 and group 2. After treatment, bone marrow MVD and VEGF expression of group 2 were significantly higher than pre-treatment, moreover the bone marrow MVD and VEGF expression in group 2 were significantly higher than that in group 1.</p><p><b>CONCLUSIONS</b>The compound Zaofan pill combined with cyclosporine and androgen can obviously increase the levels of MVD and VEGF in bone marrow, and improve the efficacy of treatment in CAA patients.</p>

Analysis and significance of hematopoietic progenitor B cells in patients with acute leukemia / 中国实验血液学杂志

Normal hematopoietic B progenitor cells are similar with acute B lymphoblastic leukemia (ALL) cells in terms of morphology and immunophenotypes which easily result in misdiagnosis of diseases. This study was purposed to explore the importance of B progenitor cell (BPC) level in differential diagnosis of hematologic diseases. A total of 664 specimens including 87 specimens from patients with non-malignant hematologic diseases as control and 577 specimens from AL patients in different progressive stage were analyzed. Out of 577 specimens 26 were collected from ALL patients, 261 were collected from B-ALL, 290 were collected from AML. The relation of different clinical status (new diagnosis, remission, relapse), age and degree of leukemia cell involvement with hematopoietic BPC level were analyzed through identification of CD34/CD10/CD19/CD45 antibody combination and quantification of hematopoietic BPC. The results indicated that (1) CD45 distributed from positive to weak positive, and with very low side scatter. The early hematopoietic BPC expressed CD34⁺, along with increasing of cell maturation, the CD34 expression gradually disappeared, while CD19 and CD10 showed positive in whole stage of hemaropoietic BPC, and early CD10 highly was expressed. (2) the mean percentage of hematopoietic BPC was 1.36% in control group, 0.60% in T-ALL, 1.39% in B-ALL and 0.80% in AML; the detected rate of hematopoietic BPC in control, T-ALL, B-ALL and AML were 87.4%, 61.5%, 83.5%, 75.9%, respectively; the mean percentage of hematopoietic BPC was 0.37% at new diagnosis, 1.66% in remission and 0.55% in relapse. (3) along with increase of age, the hematopoietic BPC level generally disclined. (4) specimens >5% hematopoietic BPC were mainly found in remission stage of leukemia patients. It is concluded that the hematopoietic BPC are present in malignant and non-malignant hematologic diseases. The changes of hematopoietic BPC level correlate with disease state, age and leukemia cell involvement. The increased hematopoietic BPC level are observed most often in the patients with remission after themotherapy. It should be carefully to diagnose and discriminate between malignant and benign cells with double positive CD19 and CD10. Use of multiparametric flow cytometry and optimal antibody combination are important for discriminating hematopoietic BPC from minor residual disease and accuratly diagnosing diseases and evaluating curative effectiveness.

Comparison and significance of DCDF probe and ES probe in the detection of BCR/ABL fusion gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the signal patterns of dual color dual fusion (DCDF) probe and extra signal (ES) probe in the detection of BCR/ABL fusion gene, and illustrate the relation between the fluorescence in situ hybridization (FISH) pattern and the karyotype.</p><p><b>METHODS</b>Sixty-five cases of chronic myelocytic leukemia (CML) and 50 cases of acute lymphoblastic leukemia (ALL) were detected by FISH with DCDF probe, the BCR/ABL positive samples were detected by FISH with ES probe. Among these cases, 47 cases of CML and 40 cases of ALL perform conventional cytogenetics simultaneously.</p><p><b>RESULTS</b>All 65 cases of CML were all BCR/ABL positive by FISH. 17 cases showed the atypical pattern by DCDF-FISH, and 12 cases showed the atypical pattern by ES-FISH. There were 7 cases of BCR/ABL positive in 50 cases of ALL by FISH. By ES-FISH, there were 5 cases in which the break-point of BCR gene was located in m-bcr, 2 cases in which the break-point of BCR gene was located in M-bcr. Conventional cytogenetics demonstrated that 43/44(98%) cases of CML and 7/32(22%) cases of ALL were Ph positive.</p><p><b>CONCLUSION</b>The features of DCDF-FISH, ES-FISH and conventional cytogenetic are different from each other. According to the features of these method, it can increase the precision of the adjustment of genetic feature to analyze these results comprehensively.</p>

Expression of histone demethylase lysine specific demethylase 1 in acute leukemia and its clinical significance / 中国实验血液学杂志

The aim of this study was to investigate the expression of histone demethylase lysine specific demethylase1 (LSD1) in patients with acute leukemia (AL) and its clinical significance. LSD1 protein expression level was detected by semi-quantitative Western blot in HL-60 and SHI-1 leukemia cell line, in bone marrow mononuclear cells of acute AL patients with different condition [new diagnosis, complete remission (CR) and relapse] and in patients with non malignant hematopathy (control). Clinical data of AL patient followed up was collected. The relationship of LSD1 expression level with clinical prognosis was analyzed. The results showed that in HL-60 and SHI-1 leukemia cell line, LSD1 expression was strong positive, relative amount (LSD1/β-actin gray level rate) was 4.647 ± 3.840 and 1.628 ± 0.185 (n = 4) respectively. In 72 AL patients, LSD1 expression levels were quite different. LSD1 positive rate was 56.9% (41/72), average relative amount was 1.053 ± 1.976. In 17 controls, LSD1 positive rate was 0%, relative amount was 0.004 ± 0.012. The LSD1 positive rate in newly diagnosed AML or ALL group (90.4%, 77.8%) and refractory/relapse AML or ALL group (100%, 100%) was higher than that in AML or ALL CR group (4.7%, 0%) (p = 0.000), relative amount of LSD1 showed no statistically difference between newly diagnosed AML and ALL groups (1.177 ± 1.646, 1.275 ± 1.845) or refractory/relapse group (2.050 ± 2.470, 4.107 ± 3.676) and CR group (0.029 ± 0.033, 0.019 ± 0.024) (p > 0.05). In all AL patients, LSD1 positive rate in newly diagnosed (84.6%) and refractory/relapse groups (100%) was higher than that in CR group (3.8%). LSD1 relative amount in newly diagnosed group (1.274 ± 1.760), refractory/relapse group (3.359 ± 3.319) and CR group (0.027 ± 0.031) was higher than that in control group (p < 0.01), and in refractory/relapse group was higher than that in newly diagnosed group and CR group (p < 0.01), in newly diagnosed group was higher than that in CR group (p < 0.01). It is concluded that overexpression of LSD1 is correlated with refractory or relapse in AL. LSD1 expression level can reflect disease status of AL patients and may be a predictive biomarker for unfavourable prognosis of AL.