MicroRNA-10a expression in FAB different subtype of acute myeloid leukemia and its relationship with drug resistance / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was to investigate the expression of miR-10a in the different FAB subtype of acute myeloid leukemia (AML) and its relationship with drug resistance.</p><p><b>METHODS</b>Forty de novo patients with AML, 16 patients with non-malignant hematologic disease and three AML cell lines HL-60, U937 and HL-60/ADR were enrolled in this study, the MiR-10a expression in bone marrow mononuclear cells of above-mentioned patients and 3 AML cell lines was detected by TaqMan RT-PCR. The correlation of miR-10a with clinicopathological factors of AML patients was analyzed.</p><p><b>RESULTS</b>The miR-10a expression level in HL-60 cell line was higher than that in U937 cell line (P = 0.039). And its expression level in de novo AML patients was higher than that in patients with non-malignant hematologic disease (P < 0.01). FAB-AML-M3 patients exhibited higher expression of miR-10a than that in M1, M2 and M4 (P < 0.05); HL-60/ADR cell line showed higher miR-10a expression than that in HL-60 cell line (P < 0.01) . Except M3, the patients without CR (non-CR) after the first cycle of chemotherapy showed a higher level of miR-10a as compared with CR patients (P < 0.01).</p><p><b>CONCLUSION</b>The high expression of miR-10a may be closely related to over-proliferation of promyelocyte and drug resistance of acute myeloid leukemia cells, except M3.</p>

Comparison and analysis of SNV results: detected by transcriptome sequencing technology on initial diagnosis and remission stage of a patient with AML-M2 / 中国实验血液学杂志

This study was aimed to further clarify the pathogenesis of acute myeloid leukemia(AML), and to forecast new somatic mutations related with leukemia. The peripheral blood samples on initial diagnosis and remission stage of a patient with partial differentiated AML(AML-M2) were sequenced by high throughput transcriptome sequencing technology. The single nucleotide variation (SNV) which possibly related with pathogenesis of leukemia was screened through comparison of the expressed genes on initial diagnosis and after remission. The results showed that the Reads distributed uniformly in genome and covered completely, detecting most expression genes. by screening the SNV, a total of 29881 mutations were discovered, including 28113 germline mutations and 752 individual mutations. Among them, 11 acquired mutations (P < 0.05) in coding regions were got, including ZRSR1, MLXIP, TLN1, LAP3, HK3. It is concluded that the high throughput sequencing as an unbiased new method can find new tumor-related mutations. MLXIP may be a new molecular marker of AML-M2.

Significance of miR-155, miR-34a and miR-30a expression in diffuse large B-cell lymphoma / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the expressions of miR-155, miR-34a and miR-30a in diffuse large B-cell lymphoma (DLBCL) and to explore their potential correlation with clinicopathological characteristics.</p><p><b>METHODS</b>The expression level of miR-155, miR-34a and miR-30a in 46 DLBCL samples were determined with TaqMan real-time polymerase chain reaction. Interphase fluorescence in situ hybridization (I-FISH) was performed to detect MYC and p53 genes' status, and immunohistochemistry (Envision method) was used to evaluate the expression of CD3, CD10, CD20, BCL-6 and MUM-1 in DLBCL. The DLBCLs were classified into germinal center B cell-like (GCB) and non germinal center B cell-like (non-GCB) subtypes according to Hans' criteria.</p><p><b>RESULTS</b>Compared with normal controls, miR-155 expression level was significantly higher in DLBCL. The expression level of miR-155 in non-GCB type was higher than that in GCB type. It was shown that the patients with MYC rearrangement had lower expression level of miR-155 than the negative controls. Compared with p53 normal group, the expression level of miR-34a was significantly lower in p53 deletion group. It was also shown that the patients with BCL-6 protein expression had lower expression of miR-30a compared with the negative group.</p><p><b>CONCLUSION</b>miR-155 expression level is different in normal controls, DLBCL and patients with subtype DLBCL. It therefore has a diagnosis value for DLBCL. miR-34a is of great prognostic significance. miR-155, miR-34a and miR-30a may be potential therapy targets for DLBCL.</p>

Influence of 1,25(OH)(2) vitamin D(3) on maturation of human dendritic cells and DC-mediated immune tolerance / 中国实验血液学杂志

This study was aimed to investigate the effect of 1,25(OH)(2) vitamin D(3) [1,25(OH)(2) Vit D(3)] on the differentiation, maturation and function of human dendritic cells (DC) in vitro and its mechanism. Human peripheral blood mononuclear cells were induced to differentiate to DC in vitro. The DC in test group were cultured with 1,25(OH)(2) Vit D(3) 1 nmol/L for 9 d, while the DC in control group were cultured with the equivalent of absolute alcohol. The expression of co-stimulatory molecules on DC were analyzed by flow cytometry. T cell proliferation induced by DC was assessed by MTT method. The expression of indoleamine 2, 3-dioxygenase (IDO) protein was determined by Western blot. The results showed that compared with the control group, the expression of CD80, CD83 and CD86 on DC in test group was significantly down-regulated (P < 0.05), while the CD1a was up-regulated (P < 0.05). The expression rate of CD80, CD83, CD86, CD1a in test group were (40.43 ± 9.83)%, (20.04 ± 4.73)%, (14.45 ± 5.38)%, (58.48 ± 10.72)% respectively, while in control group were (29.36 ± 13.34)%, (35.91 ± 10.19)%, (27.15 ± 11.64)%, (72.20 ± 12.79)% respectively. Compared with the control group, 1,25(OH)(2) Vit D(3)-treated DC exhibited a markedly reduced ability to stimulate allogenic T cell proliferation and up-regulated IDO protein expression.It is concluded that 1,25(OH)(2) Vit D(3) efficiently inhibits the maturation of DC and DC-mediated T cell proliferation, which may be related to the up-regulation of IDO protein expression.

Prognostic power of abnormal cytogenetics for multiple myeloma: a multicenter study in China / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Chromosomal abnormalities have been shown to play an important prognostic role in multiple myeloma (MM). Interphase fluorescence in situ hybridization (i-FISH) has been much more effective to identify cytogenetic aberrations in MM than conventional cytogenetic technique (CC). To clearly determine the cytogenetic features of Chinese MM patients and identify their prognostic implications, we designed a multicenter study based on i-FISH including 672 patients from 52 hospitals in China.</p><p><b>METHODS</b>All 672 patients were systematically screened for the following genomic aberrations: del(13q), IgH rearrangement, del(p53) and 1q21 amplifications.</p><p><b>RESULTS</b>The analysis showed that the chromosomal changes were detected in 22.1% patients by CC and in 82.3% patients by i-FISH. The most common abnormalities by CC were chromosome 1 aberrations (48.4%), -13/13q- (37.6%), hyperdiploidy (36.6%), hypodiploidy (30.1%) and IgH rearrangements (23.7%). The most frequent abnormalities by FISH was del(13q), which was found in 60.4% patients, whereas IgH rearrangement, 1q21 amplification and p53 deletions were detected in 57.6%, 49.0% and 34.7% cases, respectively. By statistical analysis, -13/13q- by CC was associated with low level of platelet (P = 0.015), hyperdiploidy was associated with low level of serum albumin (P = 0.028), and IgH rearrangement by FISH was associated with high level of β2 microglobulin (P = 0.019). Moreover, 1q21 amplification and del(p53) by FISH conferred a high incidence of progressive disease (PD) after initial therapy. Metaphase detection of IgH rearrangements and chromosome 1 aberrations concurrently was associated with a short progression free survival (PFS) (P = 0.036). No significant prognostic implications of other cytogenetic abnormalities were found associated with overall survival and PFS.</p><p><b>CONCLUSIONS</b>Chinese MM patients had similar cytogenetic abnormalities compared with the previous reported studies. However, the prognostic significance of FISH aberrations were not clearly determined and further study is required.</p>

Correlation of BCL-6, MYC and p53 gene abnormalities with immunological subtypes and prognosis of diffuse large B-cell lymphoma / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate BCL-6, MYC and p53 genes abnormalities in diffuse large B-cell lymphoma (DLBCL) and correlate the result with immunosubtypes and prognosis.</p><p><b>METHODS</b>Interphase fluorescence in situ hybridization (I-FISH) was performed to detect the BCL-6, MYC and p53 genes. Immunohistochemistry (Envision method) was used to measure the expressions of CD3, CD10, CD20, BCL-6, MUM -1, BCL-2 and Ki-67 genes in DLBCL. The patients were classified into germinal center B cell-like (GCB) and non-GCB subtypes according to Hans' algorithm.</p><p><b>RESULTS</b>BCL-6 rearrangement was detected in 10 of 46 DLBCL cases. The presence of gene rearrangement had no correlation with BCL-6 protein expression (P= 0.245). Overall survival (OS, P= 0.138) and progression-free survival (PFS, P= 0.095) were not influenced by BCL-6 rearrangement. All MYC rearrangements were detected in GCB type DLBCL. Deletion of p53 gene was detected in 14 cases and was significantly associated with shorter OS (P= 0.046) and PFS (P= 0.043).</p><p><b>CONCLUSION</b>I-FISH is a rapid, accurate and sensitive method for detecting BCL-6, MYC and p53 abnormalities. No correlation was found between BCL-6 gene rearrangement and BCL-6 protein expression. MYC translocation was more common in GCB type DLBCL compared with non-GCB type ones. Patients with p53 deletion had a poorer prognosis. The p53 gene may provide a useful indicator for the prognosis of DLBCL.</p>

Effects of simvastatin on PI3K/AKT signaling pathway in human acute monocytic leukemia cell line SHI-1 / 中国实验血液学杂志

To investigate the effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin (SV) on proliferation, apoptosis and the PI3K/AKT signaling pathway in human acute monocytic leukemia cell line SHI-1. SHI-1 cells were incubated with different concentrations of SV (5, 10, 15 µmol/L). Otherwise, SHI-1 cells without any treatment were used as control. Cells in different groups were collected at 24, 48 and 72 h after incubation for further detection. MTT method was used to assay the growth inhibition rate and flow cytometry was used to detect the early stage apoptosis ratio. The human PI3K-AKT Signaling Pathway RT(2) Profiler(TM) PCR Array was used to detect the expression of 84 genes involved in PI3K-AKT signaling. The results indicated that the SV inhibited the proliferation and inducted the apoptosis of SHI-1 cells in time- and dose-dependent manners significantly. The growth inhibition rates of SHI-1 cells treated with 15 µmol/L SV for 24, 48 and 72 h were 26.82, 47.09 and 63.92, respectively; and their early stage apoptosis ratios were 5.75, 13.25 and 15.59, respectively. Compared with the control group, expression levels of 39 genes were changed in the group of 15 µmol/L SV at 48 h, among them 26 genes were down-regulated and 13 genes were up-regulated. It is concluded that the SV can inhibit proliferation and induce apoptosis of SHI-1 cells, and the mechanism may be associated with the changes of gene expression level in PI3K-AKT signaling pathway regulated by SV.

Clinical study on platelet engraftment by thrombopoietin in patients with hematological malignancies after allogeneic hematopoietic stem cell transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of thrombopoietin (TPO) on platelet engraftment in hematological malignancies patients after allogeneic haematopoietic stem cell transplantation (allo-HSCT).</p><p><b>METHODS</b>One hundred and twenty patients were enrolled in a multicenter, open-label, randomized, controlled clinical trial, and were randomized into 4 treatment groups following allo-HSCT. Group A was the control arm without TPO, while group B, C and D were trial arms with received 300 U×kg(-1)×d(-1) of TPO starting from day +1, +4 and +7, respectively. A total of 89 cases were evaluated, of which 22 cases in group A, 23 in group B, 20 in group C and 24 in group D. Efficacy evaluation (the time of platelet engraftment, the number of platelet transfusion) and safety evaluation \[adverse events, routine blood tests, liver and renal function, coagulation function and occurrence of graft-versus-host disease (GVHD)\] were observed.</p><p><b>RESULTS</b>The median platelet engraftment time in experimental groups (groups B, C and D) were on day (13.17 ± 2.89), day (12.15 ± 2.08), day (12.33 ± 1.76), respectively, and that in control group was on day (14.82 ± 5.05). There was statistically significant difference between two groups (P = 0.029), There were no statistically significant difference in the average amount of platelet transfusion, platelet engraftment time, and platelet nadir value among the 3 experimental groups. No significant adverse events were observed in experimental groups.</p><p><b>CONCLUSIONS</b>TPO administration following allo-HSCT for patients with hematologic malignancies appears to shorten platelet engraftment time. TPO given starting from day +7 is effective and safe.</p>

Analysis of risk factors for overall survival at 5 years in 96 patients after allogeneic hematopoietic cell transplantation / 中国实验血液学杂志

The aim of this study was to analyze the risk factors for overall survival at 5 years in 96 patients undergoing allogeneic hematopoietic stem cell transplantation by retrospective analysis. 11 clinical parameters including age, sex, disease status, HLA locus, donor type, donor-recipient blood type, conditioning regimen, aGVHD, HC, VOD and IP were selected for univariate analysis by using a Cox regression. Factors have statistic significance at the 0.1 level on univariate analysis were evaluated by multivariate analysis by a Coxs regression. The cumulative incidence of aGVHD and survival rate of patients were calculated by the method of Kaplan and Meier. The results showed that 95 patients achieved sustained donor engraftment except 1 patients. The median time of leukocyte engraftment (ANC > or = 0.5 x 10(9)/L) was 13 days. The aGVHD of I - IV grade was observed in 42 out of 96 patients (43.75%), in which 11 patients were with aGVHD of I grade (11.46%), 19 patients were with aGVHD of II grade (19.79%), 12 patients were with aGVHD of III - IV grade (12.50%). Out of 96 patients 10 relapsed and 38 dead, the overall survival at 5 years was 60.42%. The Cox regression analysis showed that aGVHD and disease status before transplant were main factors affecting long-term survival of patients, relative risks of which were 2.996 and 2.619 respectively. It is concluded that the main factors affecting long-term survival of patients are aGVHD and disease status. The key to improve the outcome of allo-HSCT is to reduce the incidence and severity of aGVHD, meanwhile to select the CR1 for allo-HSCT to treat the patients in advanced refractory and relapsed situation should be considered as important risk factors.

Gene KIR in match with HLA-Cw impacts on NK cell cytotoxicity / 中国实验血液学杂志

The aim of this study was to investigate how the killer immune globulin-like inhibition receptor (KIR) in match with HLA-Cw impacts on NK cell activity. Mononuclear cells were isolated in 20 ml peripheral blood from 27 healthy persons by Ficoll-Hypaque and purified by NK cell isolation kit. Target cells were mononuclear cells isolated from bone marrow of 30 de novo AML patients. The KIR expression were detected by flow cytometry with antibodies against CD158a, CD158b. The 2 ml of peripheral blood from healthy persons and AML patients were collected, the DNA was extracted by using PROTRANS method, the HLA-Cw and KIR gene were detected by PCR-SSP typing with sequence specific primers. The NK cell cytotoxicity against AML cells was determined by MTT after combination of KIR with HLA-Cw gene. The results indicated that the purity of NK cells was (90.8 +/- 6.08)%. The less the KIR/HLA-Cw matched, the more activity was shown in NK cells. When no match of NK cell/target cell (KIR/HLA-Cw) there was, the cytotoxicity was (50.66 +/- 8.40)%, 1 or 2 matches showed cytotoxicity of (38.28 +/- 6.71)% and (19.74 +/- 4.15)% (p < 0.001). Expression level of KIRs on NK cells also was related with cytotoxicity level (p < 0.001). It is concluded that the interaction between inhibitory KIR and HLA ligands, and also expression level of KIRs on NK cells both impact significantly on NK cell function, that the less match of KIR/HLA-Cw, and the less expression of KIRs on NK cells result in the stronger NK cell cytotoxicity.

Alteration and biological significance of peripheral dendritic cells in patients with chronic idiopathic thrombocytopenic purpura / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the quantity and function of circulating dendritic cells (DC) in patients with chronic idiopathic thrombocytopenic purpura (ITP).</p><p><b>METHODS</b>High dose dexamethasone (HD-DXM) at a dose of 40 mg orally per day for four consecutive days was the initial treatment for chronic ITP patients. Flow cytometry was used to analyze the number of myeloid DC (mDC), plasma cytoid DC (pDC) and CD4+FOXP3+ T cells in patients before and after the treatment, meanwhile the co-stimulatory molecules on circulating DCs were assayed as well. Monocyte-derived DCs and CD4+ T cells were co-cultured with autologous or allogeneic normal fresh platelets and after 6 days of incubation H-TdR was used to assay the proliferation of CD4+ T cells.</p><p><b>RESULTS</b>The absolute numbers of circulating mDC and pDC were not significantly different between pre-treatment patients and healthy controls (P > 0.05 and P >0.05). However, percentage of CD4+ FOXP3+ T cells was decreased (P < 0.01), and their percentage was inversely correlated with the number of pDC and mDC (r = -0.396, P =0.045 and r = -0.410, P =0.037). The initial response rate to HD-DXM was 92.3%. After 4-days treatment, CD4 FOXP3+ Treg cells increased (P <0.01) while pDCs decreased (P <0.01). Although mDCs increased after HD-DXM (P <0.05), their CD11c expression level was decreased (P < 0.01), the mean fluorescence intensity (MFI) decreased from 340 +/- 30 before treatment to 199 +/- 21 after treatment. The inverse correlation between pDCs and CD4+ FOXP3+ Treg cells remained (r= -0.524, P =0.006) while that between mDCs and Treg cells disappeared (r = - 0.360, P =0.071). The MFI of CD86 on DCs was higher in ITP patients than in healthy controls (P <0.05), while the proportions of CD86, CD40, CD80 and the MFI of CD40, CD80 in ITP patients were normal (P > 0.05). DCs from chronic ITP patients co-cultured with autologous or allogeneic platelets were highly efficient in stimulating autologous CD4+ T cells proliferaton as compared to those derived from healthy donors (P < 0.05 and P <0.05).</p><p><b>CONCLUSION</b>DCs may play a role in the pathogenesis of chronic ITP in relation with CD4+CD25+ Treg cells.</p>

Alterations of CD4+ CD25+ regulatory T cells in patients with idiopathic thrombocytopenic purpura / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the role of CD4+ CD25+ regulatory T (Treg) cells in patients with ITP.</p><p><b>METHODS</b>Flow cytometry was used to examine the number of CD4+ CD25+, CD4+ CD25 high, CD4+ Foxp3+ and CD4+ CD25+ Foxp3+ T cells. The level of Foxp3 mRNA expression was analyzed by realtime quantitative reverse transcriptase polymerase chain reaction (RT-PCR) . CD4+ CD25 high T cells was cocultured with CD4+ CD25 - T cells from patients or controls for assessing the regulatory properties of CD4+ CD25 Treg cells.</p><p><b>RESULTS</b>The proportion of CD4+ CD25+ T cells in the peripheral blood of patients with ITP [(15.64 +/- 5.82) %] was significantly higher than that in normal control group [(9.30 +/- 3.95)%] (P <0.01). There was no significant difference in the percentages of CD4+ CD25 high T cells between ITP patients and controls [(1.53 +/- 0.66)% versus (1.36 +/- 0.55)% (P = 0.317)]. But the number of CD4 Foxp 3+ T cells and CD4 + CD25+ Foxp3+ T cells in patients were significantly lower than that in control group (P <0.01). The expression of Foxp3 mRNA reduced (P < 0.01) and the suppressive activity of CD4+ CD25 high T cells is lower than that of healthy controls (P <0.01).</p><p><b>CONCLUSION</b>In patients with ITP, both the number and immuno-regulative function of CD4+ CD25+ Treg cells are reduced.</p>

Detection of WT1 expression in bone marrow of acute leukemia patients with real-time quantitative RT-PCR / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate Wilms' tumor gene (WT1) expression levels in bone marrow (BM) of acute leukemia patients (ALs).</p><p><b>METHODS</b>A real-time quantitative reverse transcriptase polymerase chain reaction (RQ-RT-PCR) method was established for detecting WT1 and internal reference GAPDH expression levels in BM of 108 ALs and 23 non-leukemia controls by Light Cycler.</p><p><b>RESULTS</b>The median expression levels of WT1 in 70 newly diagnosed ALs and 11 relapsed ALs were statistically higher than those in 23 ALs in complete remission (CR) and 23 non-leukemic controls (75.10 and 89.56 vs 2.07 and 1.51 respectively). No statistic differences was found between the CR group and control group, nor between the newly diagnosed group and relapsed group. Of the 70 newly diagnosed ALs, median WT1 expression level of acute granulocytic leukemias was significantly higher than that of acute monocytic leukemias (M(5)), but there was no statistic differences among the M(1), M(2), M(3) and ALL subtypes. Furthermore the WT1 levels were not correlated to peripheral WBC counts, BM blast percentage and multidrug resistant gene (mdr1) expression at presentation, but correlated to chromosome karyotypes. Dynamic analysis of WT1 levels of 2 patients on treatment showed that WT1 expression levels predicted relapse.</p><p><b>CONCLUSION</b>WT1 expression levels in ALs were strikingly higher than that in non-leukemias. WT1 can be a marker for detecting MRD and evaluating therapy efficacy in leukemias.</p>