中国实验血液学杂志最新文献

Objective: To harvest the primary Philadelphia chromosome-positive (Ph⁺) cells of B-acute lymphoblastic leukemia (B-ALL) and to establish the B-ALL mouse model.

Methods: The plasmid carrying BCR-ABL P210 fusion gene was transferred into the bone marrow(BM) cells of C57BL/6J mice by retrovirus. Syngeneic mice irradiated with 9 Gy of ⁶⁰Co γ-ray were injected with the transfected BM cells as the first generation (G1), and then the primary cells from the spleen and BM of the diseased mice were obtained and frozen. Sublethal γ-ray irradiated C57BL/6J mice were inoculated with the first generation of Ph⁺ cells for in vivo passage, which were named as the second generation (G2). The third and fourth generations (G3 and G4) of Ph⁺ cells and B-ALL mouse model were established by successive passages. Flow cytometry, H &E staining and peripheral blood smear were used to analyze the immunophenotypes and detect the pathological changes of the model mice.

Results: After infusion of P210-NGFR retrovirus infected BM cells, the mice exhibited significant symptoms including weight loss, lower limbs paralysis, and arched back. Primitive and immature lymphocytes were observed in peripheral blood smears of the leukemia mice. The results of H &E staining showed obvious infiltration of leukemic cells around the central vein of the hepatic lobule and at the edge of the liver in the diseased mice. The results of flow cytometry showed that the percentages of CD19⁺NGFR⁺ cells in spleen of the model mice were gradually increased with passage, which was 19.0%, 47.3% and 61.0% in G1, G2 and G3 mice, respectively. Immunophenotypic analysis indicated that Ph⁺ cells were stably passaged in B lymphocytes, and the purity of Ph⁺ B lymphocytes was obviously elevated with the increase of passage frequency.

Conclusion: In the present study, the primary Ph⁺ cells were successfully obtatined and passaged in vivo, and the B-ALL mouse model was successfully established.

Objective: To explore the mutation of PTPN11 gene in patients with myelodysplastic syndromes (MDS), and explore their correlation with mutations of other genes, clinical features and prognostic of patients.

Methods: High throughput DNA sequencing was used to identify mutations in common blood tumor genes. The mutational characteristics of the PTPN11 gene and the correlation between gene mutations and patients clinical characteristics and prognosis were retrospectively analyzed.

Results: The incidence of PTPN11 mutations in 131 MDS patients was 9.16%. The genes with a mutation rate greater than 10% were RUNX1 (24.43%), U2AF1 (20.61%), ASXL1 (19.85%), DNMT3A (15.27%), TP53 (14.50%) and TET2 (11.45%). The most common co-mutation gene of PTPN11 mutations was RUNX1 (50%, 6/12). There was no significant difference between the PTPN11 mutation and the wild-type groups in sex, peripheral leukocytes, hemoglobin, platelet levels, MDS subtype, karyotype, and bone marrow blast counts (P >0.05). The transformation rate in PTPN11 mutation group was higher than that in wild-type group ［54.55%(6/11) vs . 25.29%(22/87), P < 0.05］. The median OS of patients with PTPN11 mutation was significantly low than that in the wide-type group.

Conclusion: PTPN11 mutation had a modest incidence in MDS patients, which was often coexists with RUNX1 mutation. Patients with PTPN11 mutations were more likely to progress to AML than the wild-type group.

Objective: To investigate the expression of miR-383-5p in newly diagnosed multiple myeloma (MM) and its correlation with clinical features and prognosis.

Methods: 115 MM patients diagnosed and treated in the Department of Hematology, the First Affiliated Hospital of Bengbu Medical University from January 2013 to January 2017 and 115 non-tumor controls were enrolled in this study. Clinical characteristics, pathological data and therapeutic responses of the patients were collected. The expression of miR-383-5p in MM patients and non-tumor controls was detected by RT-qPCR, and the relationship between the expression level of miR-383-5p and the clinical characteristics and prognosis of MM patients was further analyzed.

Results: The results of RT-qPCR showed that the relative expression levels of miR-383-5p in bone marrow tissues of 115 non-tumor controls and 115 MM patients were 1.89±0.11 and 1.48±0.13, respectively, and the difference was statistically significant (P < 0.05). Chi-square analysis showed that the expression of miR-383-5p was correlated with bone injury, β₂-microglobulin and globulin (all P < 0.001), but not with other factors including age, sex, hemoglobin and light chain. Univariate and multivariate Cox regression analysis indicated that low expression of miR-383-5p was an independent risk factor for poor OS in MM patients (P < 0.001).

Conclusion: The expression of miR-383-5p is down-regulated in MM patients, and low expression of miR-383-5p is a risk factor for poor prognosis and can be used as a new prognostic biomarker.

Objective: To explore the characteristics of the immunophenotypic expression of bone marrow monocytes (M) and its clinical significance in patients with multiple myeloma (MM).

Methods: The monocyte immunophenotypes expression of 67 MM and 30 anemic patients (control group) were detected by flow cytometry. The immunophenotypes that exhibited statistical differences from the control group were screened out. Further univariate and multivariate regression was used analyze the risk factors affecting the prognosis. The effect of monocyte immunophenotype on the prognosis of MM was analyzed. The correlation of CD38⁺ monocytes with clinical features was explored.

Results: The percentages of CD138⁺ monocytes (CD138⁺ M%), CD27⁺ monocytes (CD27⁺M%), and CD56⁺ monocytes (CD56⁺M%) in the MM group were significantly higher than that in the control group(P <0.05), but the percentages of CD38⁺ monocytes (CD38⁺ M%) and HLA-DR⁺ monocytes (HLA-DR⁺M%) were significantly lower than that in the control group (P < 0.01). The median progression-free survival (PFS) was shorter in the low CD38⁺ monocyte proportion (LCD38⁺ M%) group compared to the high CD38⁺ monocyte proportion (HCD38⁺M%) group. Additionally, the median overall survival (OS) was significantly shorter in the low CD138⁺ monocyte proportion (LCD138⁺M%), low CD27⁺ monocyte proportion (LCD27⁺M%), low CD38⁺ monocyte proportion (LCD38⁺M%), and low HLA-DR⁺ monocyte proportion (LHLA-DR⁺M%) groups. Cox regression analysis showed that the low CD38⁺M% was an independent risk factor for OS. The LCD38⁺M% group had significantly higher proportions of involved/uninvolved free light chain ratios ≥100 and 1q21⁺ compared to the HCD38⁺M% group (P < 0.05). Moreover, the proportion of CD38^- myeloma cells was significantly higher in the LCD38⁺M% group than that in the HCD38⁺M% group (P < 0.05).

Conclusion: The expression of CD38⁺ monocytes in bone marrow of MM patients is closely related to the prognosis and clinical characteristics. CD38⁺ monocytes maybe used to predict prognosis and guide treatment decisions.

Objective: To explore the diagnosis and treatment of acquired hemophilia A (AHA) based on the analysis of clinical data.

Methods: A retrospective analysis was conducted on the clinical manifestations, laboratory characteristics, treatment, and outcomes of 25 patients diagnosed with AHA who were admitted to the Second Hospital of Hebei Medical University.

Results: Among all patients, 11 cases had secondary factors, including 5 cases of autoimmune diseases, 3 cases of pregnancy-related disease, 1 case of pemphigoid, 1 case of Graves' disease, and 1 case of monoclonal gammaglobulinemia of unknown significance (MGUS). The bleeding sites include the skin, mucous membrane, muscle, joint cavity and brain tissue. Twenty-three patients were treated with prednisone combined with cyclophosphamide (CP regimen), one patient with rituximab combined with cyclophosphamide because of femoral head necrosis, and one case with rituximab monotherapy because of gastrointestinal bleeding after prednisone treatment. Among them, 23 cases achieved complete remission (CR), 2 cases were partial remission (PR), and 8 cases relapsed after CR. All of 10 patients including 2 cases with PR and 8 relapsed cases after CR were treated with rituximab. At last, 8 patients achieved CR, and 2 patients (both were patients with recurrence after first CR) achieved PR. These two patients achieving PR were treated with low-dose rituximab combined with bortezomib (RB regimen). One patient reached CR after 4 cycles and the other reached CR after 6 cycles of RB regimen. After CR, 4 of the 10 patients treated with rituximab received maintenance therapy with rituximab monotherapy for 1.5 to 2 years, in which, none of them relapsed. Among the 6 patients who did not receive maintenance therapy, 4 patients relapsed after CR, and the median time to relapse was 15 months. Eight patients treated with CP regimen developed common infections, and two patients treated with rituximab developed severe pneumonia. All 25 patients survived until the end of follow-up.

Conclusion: Skin ecchymosis, mucous hemorrhage and muscle hematoma are the most common hemorrhagic manifestations in AHA, and joint hemorrhage and cerebral hemorrhage can also occur. CP regimen is the preferred option of first-line therapy for AHA. Rituximab can be used for patients with steroid contraindication or who failed to respond to the above therapy or relapsed after effective treatment, and maintenance therapy is recommended to reduce the risk of recurrence. Meanwhile, close attention should be paid to the occurrence of infection events during rituximab treatment. Rituximab in combination with bortezomib can also be used in patients with refractory or relapsing AHA.

Objective: To evaluate the clinical characteristics and risk factors of infections occurring during hospitalization in patients with multiple myeloma(MM) treated with new generation therapies (including immuno- modulatory drugs, proteasome inhibitors and monoclonal antibodies).

Methods: The clinical data were collected from 155 patients with multiple myeloma who were treated in Shanxi Bethune Hospital from March, 2017 to March, 2022 and were retrospectively analyzed. For this study, the following therapies were considered to be new generation therapies: lenalidomide, pomadomide, bortezomib, ixazomib, daratumumab. The clinical characteristics and risk factors of infection were analyzed.

Results: A total of 155 patients were included in this study. The median follow-up time was 20 months. Of 155 patients with MM, 242 infection episodes were identified. Among the 242 infections, the incidence of clinically defined infection (CDI) was the highest (186, 76.86%), followed by microbiologically defined infection (MDI) in 50 cases (20.66%), and fever at unknown focus (FUF) in 6 cases (2.48%). 35 cases (14.46%) of bacteria, 10 cases (4.13%) of viruses, and 5 cases (2.07%) of fungi were clearly infected. The most common site of infection was the lower respiratory tract in 90 cases (37.19%), the upper respiratory tract in 83 cases (34.30%), and the digestive tract in 27 cases (11.16%). All-cause mortality was 8.39%(13/155). In univariate analysis, there was a higher correlation between ISS stage III, the number of treatment lines ≥2, frail and infected patients with multiple myeloma. In multivariate analysis, ISS stage III（OR =2.96， 95%CI ： 1.19-7.40， P =0.02), the number of treatment lines ≥2 （OR =2.91， 95%CI ： 1.13-7.51， P =0.03） and frail （OR =3.58， 95%CI ： 1.44-8.89， P =0.01）were risk factors for infection in patients with multiple myeloma in the era of new drugs.

Conclusion: Patients with multiple myeloma treated with new agents are prone to bacterial infection during hospitalization. ISS stage III, lines of therapy（≥2） and frail were associated with high risk for infection.

Objective: To analyze the prognostic value and threshold effect of serum C3, C4 in patients with multiple myeloma (MM).

Methods: The clinical data of 146 patients with MM who visited Suqian First People's Hospital from October 2016 to October 2019 were collected. The patients were divided into deceased group (42 cases) and survival group (104 cases) according to their prognosis and survival. The risk factors affecting the prognosis of the patients were analyzed. The correlation of serum C3 and C4 with prognosis was analyzed by threshold effect. The predictive value of serum C3/C4 on the prognosis of MM patients was evaluated by the receiver operating characteristic (ROC) curve. Nomogram model was constructed, and the discrimination and accuracy of the model were evaluated. The nomogram model was internally validated by bootstrap resampling.

Results: Durie-Salmon (DS) stage Ⅲ, decreased low density lipoprotein cholesterol (LDL-C) and apolipoprotein B (Apo B), elevated homocysteine (Hcy), uric acid (UA), and serum C3 and C4 were independent risk factors affecting the prognosis of MM patients (P < 0.05). Curve fitting showed that the mortality probability of MM patients increased with the increase of serum C3 and C4 levels. The threshold effect analysis showed that when serum C3 was higher than 1.2 g/L or serum C4 was higher than 0.37 g/L, the mortality rate of MM patients increased with the increase of the index levels; When serum C3 was lower than 1.2 g/L or serum C4 was lower than 0.37 g/L, the mortality rate of MM patients had no significant correlation with the indexes. Serum C3 and C4 had a good predictive value for the prognosis of MM patients, and the combination of C3 and C4 had a higher predictive value. The validation results showed that the nomogram model constructed in our study had good discrimination and high accuracy.

Conclusion: Elevated levels of serum C3 and C4 are independent risk factors for mortality in patients with MM. The combination of serum C3 and C4 is more valuable in predicting mortality in MM patients than C3 or C4 alone, which can be used as a sensitive index to evaluate the prognosis of MM patients.

Objective: To investigate the correlation of the clinical characteristics, fever characteristics, serum biomarkers with cytokine release syndrome (CRS) in patients with relapsed/refractory multiple myeloma (R/R MM) treated with chimeric antigen receptor T cell (CAR-T) immunotherapy.

Methods: 104 R/R MM patients who received CAR-T cell therapy at the Affiliated Hospital of Xuzhou Medical University from June 2017 to November 2021 were included, and the correlations of their clinical characteristics, fever characteristics, serum biomarkers with the severity of CRS were analyzed.

Results: Among 104 R/R MM patients receiving CAR-T treatment, no CRS was observed in 8 cases (7.7%), and 96 cases (92.3%) developed CRS. Patients with high-risk cytogenetics had a higher risk of developing CRS (P =0.040), while patients who had previously received autologous hematopoietic stem cell transplantation (ASCT) had a lower risk of developing CRS (P =0.004). There was a significant difference in the duration of fever between patients with grade 1-2 and grade 3-5 CRS (P =0.006). The highest body temperature varied among patients with different treatment regimens (P =0.001). The decrease in total protein in patients with CRS was more significant than in patients without CRS (P =0.002). Within one month after CAR-T cell infusion, the degree of albumin recovery in patients with grade 3-5 CRS was lower than that in patients with grade 0-2 CRS (P =0.037). Compared to patients with grade 1-2 CRS, patients with grade 3-5 CRS showed a significant increase in heart rate after CAR-T cell infusion (P =0.013), while IL-6, C-reactive protein (CRP), and serum ferritin (SF) also showed significant increases (P =0.007, P < 0.001, P =0.003).

Conclusion: High-risk cytogenetics is a risk factor for severe CRS. Long duration of fever is a clinical characteristic of severe CRS. CRP can better reflect the severity of CRS.

Objective: To analyze the genes related to platelet activation in essential thrombocythemia (ET) based on transcriptome sequencing technology (RNA-seq), and to explore the potential targets related to ET thrombosis.

Methods: Blood samples from ET patients and healthy individuals were collected for RNA-seq, and differentially expressed lncRNAs, miRNAs, and mRNAs were selected to construct a lncRNA-miRNA-mRNA regulatory network. Differential mRNAs in the regulatory network were enriched and analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The real-time PCR method was applied to validate differential mRNAs on crucial signaling pathways.

Results: A total of 32 lncRNAs (3 up-regulated, 29 down-regulated), 16 miRNAs (8 up-regulated, 8 down-regulated), and 35 mRNAs (27 up-regulated, 8 down-regulated) were identified as differentially expressed. Among them, 5 lncRNAs, 12 miRNAs, and 19 mRNAs constituted the regulatory network. KEGG enrichment analysis showed that the differential mRNAs were related to the platelet activation signaling pathway, and there were 6 differential mRNAs related to platelet activation, namely F2R, ITGA2B, ITGB1, ITGB3, PTGS1, and GP1BB, which were all up-regulated in their expression. RT-PCR results showed that the expression of five mRNAs including F2R,ITGA2B,ITGB1,ITGB3, and GP1BB were upregulated in ET patients compared with healthy subjects, and consistent with RNA-seq results, while PTGS1 expression was not significantly different.

Conclusion: Differential mRNAs in ET patients are related to the platelet activation pathway, and F2R, ITGA2B, ITGB1, ITGB3, and GP1BB mRNAs may serve as novel targets associated with platelet activation in ET.

Objective: To investigate the reversal effect and mechanism of asiatic acid (AA) on multidrug resistance in human adriamycin (ADR) chronic myeloid leukemia K562/ADR cells.

Methods: CCK-8 assay was used to detect the resistance of K562 cells and K562/ADR cells to ADR. CCK-8 assay was used to detect the effect of AA on K562/ADR cell viability and adriamycin sensitization. After K562/ADR cells were treated with non-toxic doses of AA(10, 20 μmol/L), the average fluorescence intensity of ADR was detected by flow cytometry. Real-time quantitative PCR was used to detect the expression levels of MRP1, P-gp, β-catenin, C-myc and cyclinD1 mRNA. Western blot was used to detect the expression levels of MRP1, P-gp, β-catenin, C-myc and cyclinD1 proteins. Western blot assay was used to detect the expression levels of MRP1, P-gp, β-catenin, C-myc and cyclinD1 proteins in K562/ADR cells treated with 20 μmol/L AA and Wnt/β-catenin pathway agonist WAY-262611 (5 μmol/L).

Results: The CCK-8 assay showed that the drug resistance of K562/ADR cells was 56.57 times that of K562 cells, showing stable drug resistance, and the difference was statistically significant (P < 0.05). AA inhibited the proliferative activity of K562/ADR cells in a concentration-dependent manner（r =0.9666）. Compared with 0 μmol/L AA group, the 10 and 20 μmol/L AA groups could significantly enhance the average fluorescence intensity of intracellular ADR (P < 0.05), and reverse the cell resistance to ADR (P < 0.05). The mRNA and protein expressions of MRP1, P-gp, β-catenin, C-myc and cyclinD1 in cells were down-regulated (P < 0.05). Compared with 20 μmol/L AA group, the expression levels of MRP1, P-gp, β-catenin, C-myc and cyclinD1 protein in 20 μmol/L AA+WAY group were significantly increased (P < 0.05).

Conclusion: AA inhibits K562/ADR cell proliferation in a concentration-dependent manner and reverse their resistance to ADR, the reversal mechanism may be related to the down-regulation of MRP1 and P-gp expression after inhibiting Wnt/β-catenin signaling pathway.