Comparision of Allogeneic Hematopoietic Stem Cell Transplantation between Children with Thalassemia of Different Ages / 中国实验血液学杂志

OBJECTIVE@#To investigate the difference of therapeutic effects on children with thalassemia at different age after hematopoietic stem cell transplantation.@*METHODS@#The clinical data of children with thalassemia treated in our hospital were retrospectively analyzed. The children were divided into 2-5 years old group and 6-12 years old group. The success rate of implantation, transplant-related mortality, GVHD incidence, and other transplant-related complications, as well as thalassemia-free survival (TFS) were compared between the two groups.@*RESULTS@#The incidence of GVHD, hemorrhagic cystitis and severe oral mucositis after transplantation in the 2-5 years old group were significantly lower than those in the 6-12 years old group, while there was no statistically significant difference in the TFS between the two groups.@*CONCLUSION@#Children in the low age (2-5 years old) group show fewer complications and higher quality of life after transplantation, therefore, stem cell transplantation at 2-5 years old is more conducive to rehabilitation of the children with thalassemia.

The Clinical Efficacy of Haploidentical Hematopoietic Stem Cell Transplantation by Using Parental Donors in Patients with Thalassemia / 中国实验血液学杂志

OBJECTIVE@#To analyze the clinical efficacy of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) by using parental donors on thalassemia patients.@*METHODS@#The 13 thalassemia patients treated by haplo-HSCT using parental donors in our hospital from July 1, 2016, to July 1, 2020 were retrospectively reviewed. Hematopoiesis reconstitution, the incidence of GVHD, infections and the long-term survival of the patients were analyzed.@*RESULTS@#Twelve of the 13 patients were successfully implanted, the success rate of implantation was 92.3%. The median time of neutrophil and platelet engraftment was 12.5 days (range, 9-22 days) and 21 days (range,12-34 days), respectively. One patient achieved primary graft failure. Three (25%) patients developed to acute GVHD (aGVHD) and achieved complete remission after treatment. Chronic GVHD developed in three (25%) patients, one of them was extensive and under treatment, while one patient developed to severe bacterial infection (7.7%). CMV viremia was diagnosed in two patients (15.4%). There were no patients developed to CMV disease. Three (23.1%) patients achieved EB viremia after transplantation, one of them developed to EBV-related lymphocytic proliferative disease, while there were no patients showed invasive fungal infection. At the last follow-up, all patients survived, twelve of them were free from transfusion dependency. There were no transplant-related deaths. Projected overall and thalassemia-free survival at three years was 100% and 92.3%, respectively.@*CONCLUSION@#The transplant protocol of haplo-HSCT by using parental donors in patients with thalassemia has reliable source of donors, high incidence of successful implantation and low incidence of GVHD, which can be used as an effective way to increase the source of donors in children with thalassemia.

Plasma MiR-181b and MiR-194 As Biomarkers for Acute Graft- Versus-Host Disease and Significance of Their Changes / 中国实验血液学杂志

OBJECTIVE@#To investigate the clinical correlation of expression level changes of miR-181b and miR-194 to the pathogenesis of acute graft-versus-host disease (aGVHD), and determine plasma miR-181b and miR-194 as the potential biomarkers for aGVHD.@*METHODS@#The plasma samples were collected from 31 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) at before HSCT, 15 days after HSCT and onset of aGVHD. The expression levels of miR-181b and miR-194 were detected by quantitative real-time PCR. Receiver-operating characteristic (ROC) curves and the area under the ROC curve (AUC) were used to assess the sensitivity and specificity of miRNA biomarkers for the diagnosis of aGVHD.@*RESULTS@#MiR-181b and miR-194 downregulated after treatment were significantly upregulated in the plasma at onset of aGVHD (P0.05). The expressions of plasma miR-181b and miR-194 collected on day 15 after HSCT were significantly upregulated in the patients with aGVHD in comparison with non-GVHD patients (P<0.05). Moreover, these elevated miRNAs were detected before aGVHD. The AUC of miR-181b predicting aGVHD was 0.91±0.05 (specificity was 0.94, sensitivity was 0.69). The AUC of miR-194 predicting aGVHD was 0.91±0.06 (specificity was 0.94, sensitivity was 0.77).@*CONCLUSION@#MiR-181b and miR-194 may serve as early biomarkers for the diagnosis and prognosis of aGVHD.

Notch1 Gene Silenced by RNAi Inhibits Oncogenicity of Myeloma Cell Line / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of Notch1 gene silencing by RNA interference on oncogenicity of multiple myeloma cells in NOD/SCID mice.</p><p><b>METHODS</b>Targeting-silenced Notch1 gene was target-sotenced by transfection of Notch1-shRNA in multiple myeloma RPMI8226 cells of NOD/SCID mouse myeloma models, and the change of the volume and speed of oncogenicity in myeloma mouse models were evaluated after Notch1 gene silencing, and ELISA was used to detect the serum expression level of IL-6 and VEGF in the tumor-bearing mice.</p><p><b>RESULTS</b>After Notch1 gene was silenced by Notch1-shRNA, the speed of tumor formation was significantly inhibited and the tumor volume was reduced in the tumor-bearing mice, as compared with the control group, and the difference was statistically significant (P<0.05). The serum level of IL-6 and VEGF in the tumor-bearing mice significantly decreased in comparison with the control group (P<0.05 ).</p><p><b>CONCLUSION</b>The oncogenicity of myeloma cells in the models NOD/SCID mouse myeloma is significantly inhibited by Notch1 gene-silencing, and its mechanism may relate with the decreased secretory level of IL-6 and VEGF after Notch1 gene silencing. Notch1 gene silencing can be used as a new strategy to treat multiple myeloma.</p>

Targeting Notch1 Gene Inhibits the Proliferation of Multiple Myeloma Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of Notch1 gene silencing on the proliferation and apoptosis of multiple myeloma cells, and to find the new targets for the treatment of multiple myeloma.</p><p><b>METHODS</b>Notch1-shRNA targeted silencing Notch1 gene was transfected into multiple myeloma RPMI8226 cells, the CCK-8 and flow cytometry were used to detect the proliferation and apoptosis of myeloma cells after Notch1-shRNA transfection, the real-time fluorescence quantitative PCR was used to analyze expression level of Notch1 mRNA, and the Western blot were used to detect the expression level of Notch1 signaling pathway-related protein, such as Hes-1, Jagged-1, Jagged-2, BCL-2, PTEN, AKT and P-AKT.</p><p><b>RESULTS</b>The mRNA and protein expression levels of Notch1-shRNA transfected cells were significantly inhibited in the experimental group assayed by real time fluorescence quantitative PCR and Western blot, the mRNA and protein expression level were down-regulated to 66% + 0.1%, 88% + 3.4% respectively, as compared with the control group(P<0.05). CCK-8 results confirmed that the cell proliferation rate was significantly decreased in the experimental group 48 hours after transfection. Flow cytometry results showed that the cell apoptosis rate was significantly higher in the experimental group than that in the control group. The expression levels of downstream protein Hes1, p-AKT and BCL-2 were decreased, the level of PTEN increased significantly after Notch1 gene silencing.</p><p><b>CONCLUSION</b>Notch1 gene silencing by transfection of Notch1-shRNA can inhibit the proliferation of myeloma cells and induce their apoptosis, and its mechanism is related to the activation of PTEN gene and p-AKT signaling. Notch1 signal can be used as a potential target for multiple myeloma therapy.</p>

Research Progress on the Drug Resistance Mechanism of Acute Myeloid Leukemia Mediated by the MicroRNA -Review / 中国实验血液学杂志

The acute myeloid leukemia is a very common malignant tumor of hematopoietic system, which is seriously harmful to human health. The long-term survival rate of routine chemotherapy is not satisfactory, especially in the high risk patients. Resistance to chemotherapy drugs is one of the major reasons, which causes the refractorines and relapse of the acute myeloid leukemia. The microRNA found in eukaryote is an endogenous non-coding micromolecular signle-stranded RNA of 19 to 25 nt. The miRNA can degrade the specific RNA or inhibit its translation through combining to 3' untranslated regions of the specific RNA, which contributes to regulate the expression of the target gene. There are already some evidences showing that the disorder of miRNA plays an important role in the genesis and progress of the tumors. Recently, more and more researches suggest that the miRNA is also important in the genesis of drug resistance to tumors. This review summarizes the research progress and the major mechanism of drug-resistance mediated by the miRNA in AML, including multiple drug resistance, cell cycle and the tolerance to apoptosis, all of which are associated with ABC transport protein.

Effect of Hedgehog Signaling Pathway Abnormality on Chemothe-rapeutic Resistance of Multiple Myeloma / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the correlation between the excessive activation of Hedgehog signal and the drug resistance of multiple myeloma.</p><p><b>METHODS</b>The resistant cell line RPMI 8226/R of multiple myeloma was established by an ascending concentration gradient method. The experiment consisted of 4 groups: RPMI8226/R, RPMI8226/S, GANT61+RPMI8226/R and GANT61+RPMI8226/S. The CCK-8 (cell counting kit-8) assay was used to detect the cell proliferation inhibition rate in 4 groups; the RT-PCR was used to detect the expression of Gli1, Gli2, Shh, Ihh, Smo and Sufu in the RPMI8226/S and RPMI8226/R cells. The Western blot was used to detect the expression of the resistant protein Cyclin D1, P21 and BCL-2 and MDR-related signaling pathways protein p-Akt, p-MAPK and STAT3 in the RPMI8226/S and RPMI8226/R cells. After adding different concentration of GANT61, the Western blot was used to detect the expression of Gli2 in RPMI8226/R and RPMI8226/S cells.</p><p><b>RESULTS</b>The expression of Shh, Ihh, Smo Gli2 was enhanced significantly in the RPMI8226/R cells, but the expression of Sufu inhibitor was reduced, the expression level of related protein in Hedgehog signaling pathway was significanly higher in RPMI8226/R than that in RPMI8226/S. After theatment of GANT61 in vitro, the expression level of Gli2 in multiple myelom cells obviously decreased, the decreasing effect of GANT61 on Gli2 expression in RPMI8226/R cells was more significant than that in RPMI8226/S cells. The sensitivity of RPMI8226/R cells to DOX after treatment with GANT61 (IC) was risen from 7.11±0.061 µmol/L to 0.99±0.053 µmol/L, the corresponding cell resistance index decreased from 5.51 to 1.69.</p><p><b>CONCLUSION</b>the activation of Hedgehog signaling pathway is closely related with the resistance of multiple myeloma cells, and GANT61 can block the Hedgehog signaling pathway, thus Hedgehog signaling may be used as a new target for multiple myeloma treatment.</p>

Circulating Serum MicroRNA as Diagnostic Biomarkers for Multiple Myeloma / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the diagnostic value of circulating serum miRNA for multiple myeloma.</p><p><b>METHODS</b>Forty blood samples from patients with multiple myeloma were collected from July 2013 to June 2014 in Department of Hematology, Zhongshan Hospital Affiliated to Xiamen University. The real-time quantitative PCR was performed to detect the serum expression levels of miRNAs (miR-29a, miR-155, miR-16 and miR-92a) circulating in the different stages of patients with multiple myeloma and evaluate the diagnostic value for patients with multiple myeloma.</p><p><b>RESULTS</b>The serum level of miR-29a significantly increased in newly diagnosed patients as compared to healthy donor (P<0.01), serum miR-155 levels were significantly lower as compared with healthy donor(P<0.001); The ratio of miR-29a and miR-155 was an effective biomarker for distinguishing multiple myeloma from healthy donor, their sensitivity and specificity were 80.8% and 83.3% respectively for myeloma diagnosis. the change of miR-29a expression was consistent with the changes of bone marrow plasma cells and M protein levels.</p><p><b>CONCLUSION</b>These circulating serum microRNA, such as miR-29a, miR-155 and miR-16, may serve as potential diagnostic biomarkers for multiple myeloma, and the ratio of miR-29a/miR-155 may serve as a most useful biomarker for myeloma diagnosis.</p>

Effects of DNMT1 Gene Silencing on Methylation of SOCS-1 Gene in Myeloma Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of DNA methyhransferase l (DNMT1) gene silencing on methylation of suppressor of cytokine signaling (SOCS-1) in multiple myeloma RPMI 8226 cells.</p><p><b>METHODS</b>Recombinant plasmid pshRNA-DNMTl was transfected into multiple myeloma RPMI 8226 cells by lipofectamine 2000. RT-PCR and Western blot were used to detect the mRNA and protein expression of DNMTl in RPMI 8226 cells respectively before and after transfection. Methylation-specific polymerase chain reaction was used to detect the methylation level change of SOCS-1 gene in RPMI8226 cells transfected.</p><p><b>RESULTS</b>DNMTl targeted short hairpin RNA(shRNA) was successfully inserted into the plasmid vector pshRNA. RT-PCR results showed that the relative mRNA expression level of DNMTI gene in RPMI 8226 cells transfected with pshRNA was 0.176±0.004 which was significantly lower than that in cells transfected by empty vector (0.956±0.033, P<0.01). Western blot analysis showed that the relative expression level of DNMT1 protein of RPMI 8226 cells transfected by pshRNA was 0.065±0.014, which was significantly lower than that in transfected cells by empty vector(0.415±0.027) (P<0.05). These results indicated that the recombinant plasmid pshRNA could effectively knock down the expression of DNMT1 gene in RPMI 8226 cells. Methylation analysis showed that the methylation level of SOCS-1 gene was obviously reduced after transfection.</p><p><b>CONCLUSION</b>DNMT1 gene in RPMI 8226 cell can be silenced by shRNA. DNMT1 gene silencing can significantly induce SOCS-1 gene hypomethylation, which indicates that DNMT1 may play an important role in the process of SOCS-1 hypermethylation.</p>

Expression of SLC25A38 in leukemic cells from children with acute lymphoblastic leukemia / 中国实验血液学杂志

This study was aimed to investigate the SLC25A38 expression in pediatric patients with acute lymphoblastic leukemia (ALL) and its clinical significance. A total of 23 newly diagnosed ALL pedictric patients were enrolled in test group, 10 pediatric patients with non-hematologic malignancies were selected as control group. The expression in protein and mRNA levels of SLC25A38 were detected by Western blot and real-time PCR respectively. The results showed that the SLC25A38 protein was positive in 8 of 23 pediatric ALL patients (34.78%), while no positive case was found in 10 controls. The relative expression level of SLC25A38 mRNA was 0.4673 ± 0.05344 in SLC25A38-protein positive group of ALL patients, while that was 1.296 ± 0.2517 in SLC25A38-protein negative group of ALL patients. The expression level of SLC25A38 mRNA in SLC25A38-protein positive group was significantly lower than that in negative group (P = 0.001) . No statistically significant difference was found in comparison of SLC25A38-protein negative group of ALL patients with the control group (P = 0.1097). The analysis of clinical data showed that there were significantly differences in sex, immunophenotype, initial peripheral white blood cell count and LDH between the SLC25A38-protein positive and SLC25A38-protein negative groups (P < 0.05). It is concluded that as a novel protein, SLC25A38 highly expressed in pediatric ALL patients, indicating that SLC25A38 may serve as a molecular marker and potential therapeutic target for acute lymphoblastic leukemia in children.

Molecular mechanism of Doxorubicin resistance in multiple myeloma cell line / 中国实验血液学杂志

This study was aimed to investigate the molecular mechanism of doxorubicin resistance in multiple myeloma cell line and certify the effect of Notch signal over-expression on drug resistance of myeloma cells. The doxorubicin RPMI 8226 cell line (RPMI8226/DOX) was established by culturing 8226 cells with continuous low concentration and intermittent gradually-increasing-concentration of doxorubicin in vitro, the mRNA expression of Notch2,Jagged1, Jagged2, HES1 were measured by RT-PCR and the P-170 protein expression was detected by Western blot in RPMI 8226 cell line; the changes of IL-6 and VEGF were tested by ELISA. The results showed that the Notch mRNA expression (Notch2, Jagged1, Jagged2 increased gradually along with the increase of chemotherapeutic drug resistance, but the expression of HESI mRNA gradually decreased along with the increase of drug resistance. The expression level of P-170 protein was upregulated gradually along with the increase of drug resistance. The level of VEGF and IL-6 in culture supernatants of RPMI8226/DOX was higher than that in RPMI 8226. It is concluded that the establishment of RPMI 8226/DOX cell line is a useful model to analyze the mechanism of chemotherapeutic drug resistance in multiple myeloma, Notch activation is closely correlated with the drug resistance of multiple myeloma and Notch signaling may to be used as a new target for multiple myeloma treatment.

Research on NPM1 gene mutations in acute myeloid leukemia / 中国实验血液学杂志

Nucleolar phosphoprotein (nucleophosmin 1, NPM1), also known as B23, N038, is located in the nucleolar particles of a multifunctional protein widely expressed in various types of cells. At present, a number of studies found that the NPM1 gene mutation is the most frequent acquired molecular genetic abnormalities in acute myeloid leukemia (AML), especially in normal karyotype AML (nk-AML). NPM1 mutation is a special subgroup in AML, which has relatively unique clinical features, and is the independent prognostic indicators of AML. Research on NPM1 mutation has an important clinical significance in the diagnosis, treatment and prognosis judgment of AML patients. This article reviews the discovery of NPM1 gene mutation in AML in recent years, including structure and physiological functions of NPM1 gene, NPM1 gene mutation in AML, detection methods of NPM1 gene mutation, and so on.

Expression of pituitary tumor transforming gene in patients with acute lymphoblastic leukemia / 中国实验血液学杂志

The aim of this study was to explore the expression of pituitary tumor-transforming gene (PTTG) in acute lymphoblastic leukemia (ALL) and its relationship with the pathogenesis of ALL, as well as study the difference of the PTTG expression in ALL patients with Ph1 chromosome and without Ph1 chromosome. The mRNA expressions of PTTG in bone marrow from 28 patients with ALL and 28 normal controls were quantitatively detected by real-time quantitative polymerase chain reaction (real-time PCR). The results indicated that the expression of PTTG mRNA was significantly higher in ALL patients (1.9428E5 ± 1.8372E5) than that in normal controls (4.5766E3 ± 1.1817E3) (P < 0.05). The expression of PTTG mRNA was higher in Ph1 chromosome positive patients. The initial expression of PTTG mRNA was lower in patients achieved complete remission than that in patients with non-complete remission. It is concluded that the overexpression of PTTG may be related to the progression and genesis of ALL. Overexpression of PTTG may be intimately related to the progression and genesis of Ph1 chromosome positive ALL. It provides a new ideas to research the pathogenesis and genic target treatment of ALL.

Expression of FoxO3a in patients with acute myeloid leukemia and its clinical significance / 中国实验血液学杂志

This study was aimed to investigate the expression and clinical significance of forkhead box protein O3a (FoxO3a) in the patients with acute myeloid leukemia (AML). Western blot was used to detect the FoxO3a protein expression in bone marrow samples from 44 newly diagnosed AML patients and 5 healthy donors. Additionally, 14 patients' samples were reevaluated when they got complete remission (CR). The results showed that FoxO3a expression (FoxO3a/β-actin 0.43 ± 0.19) in newly diagnosed AML patients was much higher than that in healthy donors (FoxO3a/β-actin 0.19 ± 0.06) (P < 0.001). The FoxO3a level was down-regulated when CR was got and there was not significant difference between patients in CR and healthy donors (P > 0.10). The correlation analysis showed that the level of FoxO3a expression positively correlated with the white blood cell count of AML patients at the time of diagnosis. Although FoxO3a expression did not positively correlate with the CR rate, the higher FoxO3a expression in AML patients showed a shorter remission duration. It is concluded that FoxO3a may be a oncoprotein in AML, and the high FoxO3a expression is associated with poor prognosis.

Establishment and application of a novel method for detecting MLL fusion genes of acute leukemia / 中国实验血液学杂志

This study was aimed to establish an efficient method to detect 10 common MLL fusion genes in patients with acute leukemia. Firstly, the relevant references and databases were searched to thoroughly investigate all fusion breakpoints; the primers and probes were designed according to nearly all the involved fusion types of gene. Then the multiplex real-time PCR system was established and optimized by using the established 16 positive plasmids and negative cell lines. Finally, the detection system was clinically evaluated by means of collected 54 samples of leukemia. The results indicated that the established detection system could efficiently detect all positive plasmids with sensibility to 10 copies. Four kinds of fusion gene types such as MLL-AF4, MLL-AF9, MLL-AF10, MLL-ELL could be detected in 54 samples, the sequencing of positive samples showed consistency of sequencing results with detection results. It is concluded that a novel multiplex real-time PCR detection method is established which can detect 10 common MLL fusion genes covering about 90% of the cases harboring MLL fusions. This method is fast, sensitive, specific and reliable, and should be an useful clinical tool for identification and management of leukemia patients with MLL fusions.

Establishment of a new method for screening of CBFB-MYH11 fusion gene in acute myeloid leukemia and its value in clinical use / 中国实验血液学杂志

This study was purposed to establish new method for detecting CBFB-MYH11 fusion gene in acute myeloid leukemia (AML) and to evaluate its value in clinical use. All fusion types of reported CBFB-MYH11 fusion gene were defined by search of references and databank, then the primers and probes were designed on this basis, and 3 positive plasmids and negative cell line as control were established. GUSB gene was also amplified as an internal reference. The primer/probe sets were tested with 3 positive plasmids and HL-60 cDNA using quantitative real-time PCR (qPCR) assays, which were then combined as a multiplex qPCR for simultaneous detection of CBFB-MYH11 and GUSB. After optimization, the multiplex qPCR assay demonstrated both high sensitivity (10 copies for all the 3 plasmids) and high specificity. Finally, the multiplex qPCR assay was clinically evaluated with 58 AML patients, and 4 CBFB-MYH11-positive cases (6.9%) were detected, involving A type (3 cases) and J type (1 case). By comparison, the multiplex qPCR assay showed results concordant with sequencing results, and detected one case that was missed by cytogenetic analysis. It is concluded that a novel qPCR method for screening of CBFB-MYH11 fusion gene in AML is established. This method is fast, comprehensive, sensitive, specific, reliable, and should consider to be a robust tool for identification and management of AML patients with CBFB-MYH11 fusion gene.

A preliminary study on efficacy of allogeneic hematopoietic stem cell transplantation by myeloablative conditioning regimen with fludarabine for high risk leukemia patients / 中国实验血液学杂志

The aim of this study was to explore the efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) by myeloablative conditioning regimen with fludarabine for high risk leukemia patients. 25 refractory and relapsed leukemia patients underwent allo-HSCT with new conditioning regimen consisted of fludarabine, busulfan and cyclophosphamide. Donors for 15 patients were sibling, but donors for the rest 10 patients were all unrelated. HLA matched and mismatched donors were for 15 and 10 patients respectively. The graft versus host disease (GVHD) prophylaxis included cyclosporine A and methotrexate, while mycophenolate mofetil and rabbit anti-T-lymphocyte globulin (ATG) were used in case of unrelated and HLA mismatched HSCT. The results showed that unrelated donor HSCT in 10 cases was successful (100%), 14 out of 15 patients with donors of sibling or parent also reconstructed their haematopoietic system. One mismatched patient (4/6) died of graft failure. The time from transplantation to ANC > 0.5 × 10(9)/L and Plt > 20 × 10(9)/L were 13 (11 - 19) days and 13 (12-20) days after transplantation respectively. The cumulative incidence of grade II-IV acute GVHD and chronic GVHD was 12.5% (3/24) and 47.4% (9/19), respectively. In a follow-up duration of 6-84 months, 12 patients were dead, out of which 8 died of relapse; 1 cases died of regimen-associated side effect. 3 cases died of serious infection. The other 13 patients remained alive and disease-free survival probability was 48.7%. It is concluded that allo-HSCT by myeloablative conditioning regimen with fludarabine is a safe and effective option for high risk leukemia patients, which reduces aGVHD incidence and regimen-associated side effect, but it should be modified for higher rate of relapse.

DNMT3A gene mutations in acute myeloid leukemia / 中国实验血液学杂志

Epigenetic changes, including abnormal DNA methylation, have been identified to play significant roles in tumor initiation and progression. Recently, mutations of DNMT3A were identified in acute myeloid leukemia (AML), which possibly caused changes in DNA methylation, and indicated a poor prognosis. Sequencing analysis showed that most of the mutations were single nucleotide variations, including a hotspot Arg882. DNMT3A mutations were detected in about 20% AML patients, and closely associated with the age over 60, the M(4), M(5) subtypes and intermediate-risk cytogenetics. Others showed that these alterations also present in myelodysplastic syndrome (MDS) and primary myelofibrosis (PMF) prior to development of the obvious leukemia, indicating that these mutations might contribute to leukemogenesis. However, its prognostic value of minimal residual disease and role of therapeutic targets are still unclear, focusing on a large cohort of AML patients will solve these issues. In this review, the achievement in studying DNMT3A gene mutation are summarized, and the latest research progress is briefly discussed.

Simultaneous detection of FLT3-ITD and NPM1 gene mutations in acute myeloid leukemia by double PCR / 中国实验血液学杂志

Objective of this study was to establish a method for simultaneous detection of FLT3/ITD and NPM1 gene mutations in AML. A double PCR was firstly designed and optimized to amplify both exon 12 of NPM1 and exon 14-intron 14-exon 15 of FLT3, with the aim of detecting almost all reported mutations. After optimization, a touchdown PCR was chosen for the multiplex PCR procedure, with the primer concentrations of NPM1 and FLT3-ITD being 200 nmol/L and 152 nmol/L respectively. The PCR amplicons were separated by capillary electrophoresis and the presence of mutants was recognized by the size difference between the mutants and wild-type products. The areas of mutant peak and wild-type peak were used to calculate the mutant/wild-type ratio. All the positive mutated samples were confirmed by sequencing. The results showed that 17 patients with NPM1 mutation, 15 patients with FLT3-ITD mutation, 6 patients with both NPM1 and FLT3-ITD mutations were found among 93 patents. 7 patients with M₂, 4 patients with M₄, 5 patients with M₅ and 1 patients with M₆ were found out of 17 patients with NPM1 mutation, in which 10 patients were male and 7 patients were female, 15 patients were with type A, 1 patients was with type B and 1 patients was with type Nm, strikingly 1 CML patient in blast crisis was found to carry a type A mutation. Among 15 patients with FLT3-ITD mutation 1 patient with M₁, 8 patients with M₂, 2 patients with M₂, 2 patients with M₃, 1 patient with M₄, 3 patients with M₅ were found, in which 5 patients were male and 10 patients were female. Sequencing results further confirmed the accuracy and reliability of this method. It is concluded that a novel method with the ability to detect both FLT3-ITD and NPM1 mutations has been developed when genomic DNA was templated. This method is fast, easy, accurate and capable to calculate the mutant/wild-type ratio.

Inhibitory effect of phenylhexyl isothiocyanate on notch signaling of multiple myeloma cells in vitro / 中国实验血液学杂志

In order to investigate the mechanisms of phenylhexyl isothiocyanate (PHI) inhibiting the proliferation of multiple myeloma cell RPMI8226 in vitro, the RPMI8226 cells were co-cultured with PHI of various concentrations. The inhibition of proliferation was measured by MTT test and the cell apoptosis was assayed by DAPI staining. The changes of Notch1, Jagged2, BCL-2 and p-Akt proteins in the PHI-treated cells were detected by Western blot. The results showed that PHI inhibited RPMI8226 cell proliferation in certain concentration range and induced their apoptosis. The inhibiting effect caused by PHI showed a concentration-and time-dependent manner. The PHI decreased expressions of Notch1 and Jagged2 proteins in a concentration-and time-dependent manners, the levels of BCL-2 and p-Akt declined at the same time. It is concluded that PHI can inhibit proliferation of RPMI8226 cells, and induce their apoptosis. The cell apoptosis is associated with the inhibition of Notch signaling and downstream targets BCL-2 and p-Akt proteins of RPMI8226 cells, PHI may be a new Notch signaling inhibitor and a promising therapeutic drug for multiple myeloma.