Clinical Characteristics and Survival Analysis of Patients with Chronic Myelomonocytic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate the clinical characteristics, prognostic factors and efficacy of hypomethylating agent (HMA) in patients with chronic myelomonocytic leukemia (CMML).@*METHODS@#The clinical data of 37 newly diagnosed patients with CMML was analyzed retrospectively, and their clinical characteristics and the efficacy of HMA were summarized. Kaplan-Meier and Log-rank test were used for univariate survival analysis, and Cox proportional hazards regression model was used for multivariate analysis.@*RESULTS@#The median age at diagnosis was 67 years old. Their common manifestations included fatigue, bleeding, abnormal blood routine and fever. Most patients had splenomegaly. According to FAB classification, there were 6 cases of myelodysplastic CMML and 31 cases of myeloproliferative CMML, while according to WHO classification, 8 patients belonged to CMML-0, 9 patients to CMML-1 and 20 patients to CMML-2. At the time of diagnosis, the median white blood cell count was 32.84×109/L, median hemoglobin (Hb) was 101 g/L, median platelet count was 65×109/L, median absolute monocyte count was 9.53×109//L, median absolute neutrophil count (ANC) was 11.29×109//L and median lactate dehydrogenase (LDH) was 374 U/L. Cytogenetic abnormalities were found in 4 cases among the 31 patients who underwent karyotype analysis or fluorescence in situ hybridization detection. There were 12 patients who had analyzable results and gene mutations were identified in 11 cases, including ASXL1, NRAS, TET2, SRSF2 and RUNX1. Among the 6 patients who were treated with HMA and could be evaluated for efficacy, 2 patients achieved complete remission, 1 patient achieved partial remission and 2 patients achieved clinical benefit. Compared with the non-HMA treatment group, overall survival (OS) time was not significantly prolonged in the HMA treatment group. Univariate analysis showed that Hb<100 g/L, ANC≥12×109/L, LDH≥250 U/L and peripheral blood (PB) blasts ≥5% were significantly associated with poor OS, while WHO classification CMML-2, Hb<100 g/L, ANC≥12×109/L, LDH≥250 U/L and PB blasts≥5% were significantly associated with poor leukemia-free survival (LFS) (P<0.05). Multivariate analysis showed that ANC≥12×109/L and PB blasts≥5% were significantly associated with poor OS and LFS (P<0.05).@*CONCLUSION@#CMML has high heterogeneity in clinical characteristics, genetic changes, prognosis and treatment response. HMA can not significantly improve the survival of CMML patients. ANC≥12×109/L and PB blasts≥5% are independent prognostic factors of OS and LFS in patients with CMML.

Effect of Different Protein Expression and Clinical Features on the Prognosis of Patients with Diffuse Large B-Cell Lymphoma / 中国实验血液学杂志

OBJECTIVE@#To investigate the efficacy of multiple protein expressions and clinical features on the threapeutic effect and prognosis of patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL).@*METHODS@#The clinical data of 68 DLBCL patients were collected and analyzed retrospectively. The clinical staging was performed according to Ann Arbor staging; the risk grading was performed by IPI index; the DLBCL typing (germinal center and non-germinal center) was performed according to B cell source; the expression of Ki67，BCL-2, BCL-6, C-MYC, MUM1 and CD10 protein was detected by immunohistochemistry method; the patients were divided into R-CHOP group(50 cases) and CHOP group(18 cases) according to chemotherapy regimen of using rituximab or not; finally, the related factors affecting the prognosis of patients(PFS and OS) were analysed statistically by using SPSS 22.0 software according to sex, age, erythrocyte sedimentation rate (ESR), lactate dehydrogenase(LDH) and use of rituximab or no, as well as the above-mentioned clinical indicators.@*RESULTS@#IPI grade high-risk, elevated LDH, positive expression of BCL-2 protein and negative expression of BCL-6 protein were independent prognostic factors for progression-free survival (PFS); elevated LDH and negative expression of BCL-6 protein were independent prognostic factors for overall survival time (OS); multivariate analysis showed that elevated LDH and positive expression of BCL-2 protein were independent prognostic factors for progression-free survival (PFS). The overall survival time (OS) associated with ESR, IPI classification and BCL-6 protein expression.@*CONCLUSION@#The expression of BCL-2 and BCL-6 protein and some clinical features can be used as predictors of clinical efficacy for DLBCL. Choosing the treatment regimen combined with ritu-ximab can further improve the survival and prognosis of DLBCL patients.

Detection of intron 22 inversion of factor VIII gene in severe hemophilia A patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the incidence of intron 22 inversion (INV22) of factor VIII (FVIII) gene in severe hemophilia A (HA) patients, clarify its pathological mechanism, and identify INV22 carrier in the female family members.</p><p><b>METHODS</b>One-stage method was used to assay the FVIII activity (FVIII:C)in 126 severe HA patients with a median age of 14 years old (range: 4 months-63 years). INV22 was analyzed by long-distance polymerase chain reaction (LD-PCR) and pulsed field gel electrophoresis (PFGE), and pedigree were conducted in 3 involved HA families.</p><p><b>RESULTS</b>Of all the 126 severe HA, 52 (41.3%) cases had the INV22. Four females including 3 mothers and 1 sister of probands were diagnosed as INV22 carriers among 11 suspected carrier mosaicisms from 3 INV22 positive HA families. In 8 females from one family without HA history, the patient's mother was a INV22 carrier, but her maternal grandmother, 2 maternal aunts, 2 female siblings and 1 elder female cousin were negative.</p><p><b>CONCLUSION</b>LD-PCR and PFGE could be used to diagnose severe HA patients with INV22 and identify the carriers.</p>

Effects of decitabine on biological behavior of U266 cells / 中国实验血液学杂志

This study was aimed to explore the effects of decitabine on the biological behaviour of U266 cells in vitro so as to provide a new thinking and experiment basis, as well as new evidences for the pathogenesis of multiple myeloma. MTT and colony formation assays were used to evaluate the impact of decitabine on the ability of proliferation of U266 cells; flow cytometry was used to analyze the cell distribution in cell cycle; transwell chamber and matrigel assays were used to observe the ability of migration and invasion. The results indicated that decitabine could significantly suppress the proliferation of U266 cells in time-and dose-dependent manners. The flow cytometric analysis demonstrated that the cells in G(0)-G(1) phase significantly increased while the cells in S and G(2)/M phase decreased. The migration and matrigel invading tests showed that the number of cells moving into under chamber of transwell decreased after U266 cells treated with decitabine. It is concluded that decitabine may act as an effective drug for MM by inhibiting the proliferation, migration and invasion ability, and the specific mechanism needs to be deeply explored.

Significance of B cell activating factor of TNF family promoter polymorphisms in patients with immune thrombocytopenic purpura / 中国实验血液学杂志

The study was aimed to examine the B cell activating factor promoter polymorphism of the TNF family (BAFF)-871 C/T in patients with immune thrombocytopenic purpura (ITP) and to explore its correlation with ITP and the relationship between the blood platelet count of newly diagnosed patients with ITP and genotypes of BAFF-871 C/T polymorphisms. Alleles specific polymerase chain reaction (ASP-PCR) and agarose gel electrophoresis were used to identify polymorphisms -871 C/T of BAFF promotor in 133 ITP patients and 117 healthy controls, and determine the genotype of subjects. Meantime, the frequency of genotype and alleles were analyzed. The results indicated that out of 133 patients with ITP, 33.1% patients exhibited C/C, 42.1% patients were heterozygous C/T, and 24.8% patients were homozygous T/T. The corresponding frequencies in 117 healthy controls were 55.6% C/C, 33.3% C/T and 11.1% T/T. The allele frequency of T in ITP patients and healthy controls were 45.9% and 27.4% respectively. There was significant difference in the BAFF-871 C/T genotypic frequency between the ITP patients and healthy controls (p < 0.05). The allele frequency of T in ITP patients was higher than that in healthy controls. There was no significant difference in the blood platelet counts between the various genotype (p > 0.05). It is concluded that the polymorphism -871 C/T of BAFF promoter is correlated with the pathogenesis of ITP. However, there is no significant difference in blood platelet counts between the various genotype, so the polymorphism -871 C/T of BAFF promoter can not be referred as the analysis index for evaluating the severity of ITP.

Construction of nonsense-mutated eukaryotic expression vector of factor IX gene and its expression in COS-7 cells / 中国实验血液学杂志

The purpose of this study was to construct 4 types of nonsense-mutated eukaryotic expression plasmids of fIX gene, using pcDNA3.1 plasmid containing fIX cDNA as template, and to identify, then to perform their expression in COS-7 cells. These stop mutants constructed by site-directed mutagenesis based on PCR, and further confirmed by DNA sequencing. COS-7 cells were transfected with either the wild-type or mutated fIX expression constructs, then the relative expression levels of fIX mRNA were detected by real time fluorescent quantitative PCR. The result showed that except the designed sites, there were no other nucleotide mutation in the sequences of four nonsense mutants. The results of real time PCR proved that the nonsense-mutated vectors can be effectively expressed in COS-7 cells. It is concluded that the nonsense-mutated eukaryotic expression vectors of fIX gene have been successfully constructed and can express in COS-7 cells, which provides the material basis for further researches on mechanism and treatment of FIX deficiency and the function defects caused by nonsense mutation.

Expression and gene polymorphisms of B cell activating factor in patients with idiopathic thrombocytopenic purpura / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the polymorphisms of B cell activating factor (BAFF) gene and the plasma levels of BAFF in patients with idiopathic thrombocytopenic purpura (ITP), and to investigate their roles in the pathogenesis of ITP.</p><p><b>METHODS</b>Alleles specific polymerase chain reaction (AS-PCR) and agarose gel electrophoresis were used to identify polymorphisms -871C/T of BAFF promotor in 133 ITP patients and 117 healthy controls. The plasma levels of BAFF were assayed by ELISA.</p><p><b>RESULTS</b>In ITP group, the frequency of C/C, C/T and T/T was 33.1%, 42.1% and 24.8%, respectively, the corresponding frequency in control group was 55.6%, 33.3% and 11.1%, respectively. The allele frequency of T in ITP and control groups was 45.9% and 27.4%, respectively. There was a significant difference in the BAFF -871C/T genotypic frequency between the ITP and control groups (P < 0.05). BAFF antigen in untreated ITP, treated patients and controls was 875.86 pg/ml, 502.59 pg/ml and 736.88 pg/ml, respectively, being also a significant difference among the three groups (P < 0.05). BAFF antigen in homozygous T/T was higher than that in homozygous C/C and heterozygous C/T, but the difference was not statistically significant (P > 0.05).</p><p><b>CONCLUSIONS</b>Over expression of BAFF may be a risk factor for ITP patients. There is a correlation of the BAFF promotor polymorphism -871C/T with ITP, but the polymorphism does not affect the expression of BAFF.</p>

Significance of detection of anti-GPIIb/IIIa antibody secreting B cells and platelet-specific antibody in patients with idiopathic thrombocytopenic purpura / 中华血液学杂志

<p><b>OBJECTIVE</b>To detect the frequencies of anti-GPIIb/IIIa antibody secreting B cells and platelet-specific antibody in patients with idiopathic thrombocytopenic purpura (ITP) and non-immune thrombocytopenia, and to evaluate their roles in the diagnosis of ITP and their clinical significance.</p><p><b>METHODS</b>The frequencies of circulating B cells secreting anti-GPIIb/IIIa antibody and platelet-specific antibody in 58 ITP patients, 33 non-ITP patients and 31 healthy controls were tested by Enzyme-linked Immunospot Assay (ELISPOT) and modified monoclonal antibody immobilization of platelet antigens assay (MAIPA) respectively.</p><p><b>RESULTS</b>The frequencies of circulating B cells secreting anti-GPIIb/IIIa antibody in ITP patients \[(6.6 ± 4.2)/10(5) PBMNC\] were significantly increased (P < 0.05) than that of the controls \[(1.3 ± 0.5)/10(5) PBMNC\] and non-immune thrombocytopenic purpura patients \[(2.2 ± 2.0)/10(5) PBMNC\]. However there was no apparent difference between the latter two groups (P > 0.05). ELISPOT had a sensitivity of 70.69%, a specificity of 90.91% for the diagnosis of ITP, the sensitivity being higher than that of modified MAIPA's (43.10%) (χ(2) = 7.03, P < 0.05). The ROC curve showed the discriminative validity of cytometric bead array was 0.886.</p><p><b>CONCLUSION</b>The frequencies of circulating B cells secreting anti-GPIIb/IIIa antibody may reflect the pathogenesis of ITP. ELISPOT assay have high sensitivity and specificity than modified MAIPA for the diagnosis of ITP and the guidance for clinical therapy.</p>

Correlation analysis of tissue factor promotor polymorphism -1208I/D and venous thromboembolism / 中国实验血液学杂志

The study was aimed to investigate the correlation of tissue factor promotor polymorphism -1208I/D with the venous thromboembolism in patients. Tissue factor promotor polymorphism -1208 was detected by polymerase chain reaction (PCR) and DNA sequencing in 96 cases of DVT, 14 cases of PE and 59 nonthrombosis normal individuals. The results showed that the allele containing a 18-bp nucleotides insertion at -1208. 67.8% of normal individuals exhibited D/D, 25.4% were heterozygous I/D, and 6.8% were homozygous for I/I. DVT group and PE group exhibited a similar distributions (62.5%D/D, 29.8% I/D, 8.3% I/I and 57.1% D/D, 35.7% I/D, 7.1% I/I). The allele frequencies of D and the allele frequencies of I in the normal control, DVT and PE groups were 80.5%, 77.1%, 75.0% and 19.5%, 22.9%, 25.0% respectively. There was no significant difference in the TF-12081/D genotype frequency between the groups of patients and normal individuals. In conclusion, there is no correlation of the tissue factor promotor polymorphism -1208I/D in the patients with venous thromboembolism. The gene of promoter -1208I/D may not be a major susceptible gene of VTE in Chinese Han. Further investigations would be necessary to define accurately tissue factor gene polymorphisms.

Expression of insulin in the brain tissue of rats after scald / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe the expression of insulin in the brain tissue of rat after scald.</p><p><b>METHODS</b>Fifteen Wistar rats subjected to 30% TBSA scald were enrolled in the study. Zamboni fixating solution was infused into left ventricle and the brain tissue was harvested at 4, 12 and 24 post-scald hours (PSH) for the detection of insulin expression with fluorescent immunohistochemistry, with 5 rats at each time-point. Another group of 10 rats were enrolled as controls.</p><p><b>RESULTS</b>There exhibited no obvious insulin expression in the brain tissue of rats in the control group. Insulin immune responsive positive cells were detected in the olfactory bulb and cerebral cortex of rats at 4 PBH. These cells were big with oval and fusiform shape, big, round, transparent nuclei, and prominent processes. The positive insulin substance was mainly distributed in cytoplasm, and some in the processes of cells. No insulin immune-responsive cells were observed in rat brain tissue at 12 and 24 PSH.</p><p><b>CONCLUSION</b>The brain have the potentiality of self-biosynthesis of insulin, but very little of synthesized insulin exists in normal states, but the amount increases after scald.</p>