中国实验血液学杂志最新文献

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.

Objective: To summarize and analyze the clinical features of blastic plasmacytoid dendritic cell neoplasm (BPDCN), so as to enhance the understanding of this disease.

Methods: The clinical manifestations, immunophenotype, pathological features, treatment and prognosis of 11 cases of BPDCN were retrospectively analyzed.

Results: Among the 11 patients diagnosed with BPDCN, there were 8 males and 3 females, with a median age of 44 (6-81) years. The main clinical symptoms were rash and mass, accompanied by lymph node and bone marrow involvement. The neoplastic plasmacytoid dendritic cells (pDC) were positive for HLA-DR, CD4, CD56 and CD123, but negative for cCD3, cMPO and cCD79a; In some cases, they were also positive for CD38, CD99 and CD36. Patients who have underwent surgical resection and those who experienced multiple chemotherapy failures tend to have rapid recurrence and shorter survival time. Patients who achieved complete remission (CR) after the first chemotherapy exhibit no expression of CD56 on pDC cells, and tend to have a longer survival time after bone marrow transplantation.

Conclusion: The immunophenotype of BPDCN is heterogeneous. CD56 is a reliable marker to distinguish neoplastic pDC cells from reactive pDC cells. The BPDCN patients who underwent hematopoietic stem cell transplantation (HSCT) after achieving remission from initial chemotherapy tend to have a better prognosis.

Objective: To investigate the effect of genetic polymorphism of MTHFR C677T (rs1801133) on methotrexate (MTX) related toxicity in pediatric mature B-cell lymphoma patients.

Methods: Fifty-eight intermediate and high risk patients under 18 years of age with mature B-cell lymphoma who received 5 g/m ² MTX (24 h intravenous infusion) in Sun Yat-sen University Cancer Center from August 2014 to December 2021 were included, and their toxicity of high-dose MTX (HD-MTX) were monitored and analyzed.

Results: Among the 58 pediatric patients, the number of CC, CT, and TT genotypes for MTHFR C677T was 33, 19 and 6, respectively. A total of 101 courses of HD-MTX therapy were counted, of which plasma MTX level >0.2 μmol/L at 48 h post-MTX infusion were observed in 35 courses, ≤0.2 μmol/L in 66 courses. Inter-group comparison showed that plasma MTX level >0.2 μmol/L at 48 h post-MTX infusion increased the risk of developing oral mucositis (P <0.05). Compared with wild-type (CC genotype), patients in the mutant group (CT+TT genotype) were more likely to develop myelosuppression, manifested as anemia, leucopenia, neutropenia and thrombocytopenia. However, plasma MTX level at 48 h was not associated with MTHFR C677T gene polymorphism.

Conclusion: The risk of developing oral mucositis in children with mature B-cell lymphoma is associated with plasma MTX concentration. Polymorphism of MTHFR C677T gene is not related to plasma MTX concentration in children with mature B-cell lymphoma, but is related to grade III to IV hematological toxicity.

Objective: To determine the serum levels of CMTM6 mRNA and CCN1 in patients with acute leukemia (AL), and to analyze their relationship with the clinical efficacy and prognosis of the patients.

Methods: 103 AL patients admitted to our hospital from February 2015 to January 2019 were included as the study subjects. Additionally, 100 healthy subjects who underwent physical examinations during the same period were included as the control group. qRT-PCR method was applied to detect the serum CMTM6 mRNA level of the study subjects, the serum CCN1 level was measured by ELISA. The levels of serum CMTM6 mRNA and CCN1 between the control group and AL patients, as well as between patients at initial diagnosis and after one course of chemotherapy were compared, the correlation of CMTM6 mRNA and CCN1 levels at initial diagnosis with clinicopathological features and short-term efficacy in AL patients was analyzed. The correlation of the CMTM6 mRNA and CCN1 expression levels with prognosis of the patients was analyzed by Kaplan-Meier curves.

Results: Compared with the control group, the serum CMTM6 mRNA level in AL patients was significantly increased (P < 0.05), while the serum CCN1 level was significantly decreased (P < 0.05). There were no statistically significant differences in serum CMTM6 mRNA and CCN1 levels between patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) (P >0.05). Compared with those at the initial diagnosis, both the serum CMTM6 mRNA and CNN1 expression levels in AL patients were significantly altered after one course of chemotherapy, with CMTM6 mRNA significantly decreased (P < 0.05) and CCN1 significantly increased (P < 0.05). The 3-year recurrence-free survival (RFS) rate was lower in the CMTM6 high expression group and the CCN1 low expression group, compared with the CMTM6 low expression group and CCN1 high expression group, respectively (P < 0.05).

Conclusion: Serum CMTM6 is highly expressed and CCN1 is lowly expressed in AL patients. The expression levels of serum CMTM6 and CCN1 are associated with the 3-year prognosis of the patients.

Objective: To establish an efficient gene editing method of HLA-I gene to prepare HLA-I universal hematopoietic stem cells.

Methods: The easyedit small guide RNA(sgRNA) was designed according to the sequences of β2 microglobulin gene and synthesized by GenScript company. RNP complexes were formed by NLS-Cas9-NLS nuclease and Easyedit sgRNA according to different molar ratios (1∶1~1∶4). Control group and four transfection groups were performed respectively. HEK-293 cells and CD34⁺ hematopoietic stem cells were nucleotransfected with RNP complex by Lonza 4D Nucleofector system. The expression of HLA-I on the surface of HEK-293 cells was detected by flow cytometry after transfection for 72 hours, the cleavage effect was determined by T7E1 enzyme digestion reaction and the presence of nested peak in the DNA sequence was identified by direct sequencing.

Results: The transfection groups had different levels of HLA-I negative expression cell populations by flow cytometry after transient transfection of HEK-293 cells and CD34⁺ hematopoietic stem cells with different molar concentrations of RNP complex for 72 hours. There were nested peaks proximal to the sgRNA PAM sequence in the transfection groups by direct DNA sequencing, indicating that sgRNA had obvious editing effect. In the transfection of HEK-293 cells, the highest proportion of HLA-I negative expression cells was (87.69±0.83)% when the molar ratio of NLS-Cas9-NLS nuclease to Easyedit sgRNA was 1∶4. The cutting efficiency of T7E1 was the highest up to (38±2.0)% when the molar ratio was 1∶3. In the transfection of CD34⁺ hematopoietic stem cells, the proportion of HLA-I negative expression cells was (91.56±3.39)% when the molar ratio was 1∶2, and the cutting efficiency of T7E1 was (64±8.45)% when the molar ratio was 1∶1.

Conclusion: This study provides an efficient gene editing method for classical HLA-I molecules, which can effectively silence the expression of class HLA-I molecules on the cell surface, and is suitable for stem cell system with difficult transfection.

Objective: To investigate the cause of the production of anti-D and anti-E mixed antibody in an RhD positive patient.

Methods: The ABO/Rh blood group typing and irregular antibody specificity were identified by conventional serological methods, the RHD gene exon 1-10 and heterozygous analysis were performed by sequence-specific primer polymerase chain reaction (PCR-SSP), and the whole exon sequence was analyzed by first-generation sequencing.

Results: The patient's Rh blood group was weak D Type33, with the allele was RHD*01W.33, the patients was found to be RhD⁺/RHD^- heterozygous, with an Rh typing of Ccee, and the patient had developed anti-D combined with anti-E mixed antibodies.

Conclusion: The patient has A c.520G>A mutation in exon 4 of the RHD gene, to lead decreased expression of RhD antigen in red blood cells and the anti-D and anti-E mixed antibodies were produced by transfusion immunostimulation.

Objective: To investigate the expression of superoxide dismutase 1 (SOD1) in tumor tissue of patients with diffuse large B-cell lymphoma (DLBCL) and in DLBCL cell lines, to explore the effect of SOD1 inhibitor LCS-1 on proliferation and apoptosis of DLBCL cell lines and analyze its possible mechanisms of action.

Methods: Immunohistochemistry assay was used to detect the expression level of SOD1 in DLBCL tissues and reactive lymph node hyperplasia tissues. The expression levels of SOD1 protein in DLBCL cell lines (TMD-8, OCI-Ly10, OCI-Ly18, OCI-Ly19) were detected by Western blot. After the DLBCL cell lines were treated with different concentrations of LCS-1, the cell proliferation activity was detected by CCK-8 assay, the expression levels of SOD1 protein was detected by Western blot, and the cell apoptosis was detected by TUNEL method. The genes enrichment of the SOD1 high expression group were analyzed by the KEGG database.

Results: The expression levels of SOD1 in the tumor tissues of DLBCL patients and DLBCL cell lines TMD-8, OCI-Ly18, and OCI-Ly19 were significantly increased. SOD1 inhibitor LCS-1 showed a certain inhibitory effect on the activity of DLBCL cell lines TMD-8, OCI-Ly18, and OCI-Ly19 in a concentration- and time-dependent manner (r =0.730, r =0.929,r =0.976). After being treated with different concentrations of LCS-1, the expression level of SOD1 protein in OCI-Ly18 and OCI-Ly19 cell lines decreased in a concentration-dependent manner (r =0.860, r =0.970); LCS-1 significantly promoted the apoptosis of DLBCL cell lines OCI-Ly18 and OCI-Ly19 at a concentration of 3 μmol/L (P < 0.001). KEGG enrichment analysis suggested that SOD1 may play an important role through oxidative phosphorylation (P =0.002, FDR=0.003) and ribosome (P =0.004, FDR=0.005) pathways in DLBCL.

Conclusion: The expression levels of SOD1 in tumor tissues of DLBCL patients were significantly increased. As a SOD1 inhibitor, LCS-1 can significantly inhibit the viability and proliferation of DLBCL cell lines OCI-Ly18 and OCI-Ly19, and promote cell apoptosis, which provides a new idea for the treatment of DLBCL.

Objective: To establish a method for preserving viral nucleic acids in plasma using a blood collection card based on the dry spot method, to predict the duration of nucleic acid preservation by establishing the Arrhenius equation, and to demonstrate the feasibility of this preservation method for the re-testing of nucleic acids in blood samples retained by blood banks.

Methods: Plasma samples positive for HBV, HCV, and HIV nucleic acids were prepared into preservation cards in the form of dry plasma spots for storage. The prepared preservation cards were placed under accelerated storage conditions at 37, 45, 50, and 55 ℃. The preservation cards were periodically retrieved from each temperature condition for nucleic acid extraction, and the nucleic acid samples were purified for subsequent PCR testing, with the recorded CT values. An Arrhenius equation model was established between the expiration time and the storage temperature, thereby predicting the validity period of nucleic acid preservation in blood collection cards under specified storage temperature conditions.

Results: For the plasma samples positive for HBV, HCV, and HIV nucleic acids preserved using the dry spot method, the regression equations for the duration with temperature were as follows: y=-11546 x + 31.74 for HBV, y=-12949x + 36.88 for HCV, and y=-12204x + 34.48 for HIV, with the correlation coefficient r greater than 0.98 for all. It was predicted that at a storage temperature of 4 ℃, the preservation periods for HBV, HCV, and HIV viral nucleic acids using the dry spot method would be 20 792 days, 19 289 days, and 14 285 days, respectively. At a storage temperature of 20 ℃, the preservation periods would be 2 135 days 1 502 days, and 1 289 days, respectively.

Conclusion: The nucleic acids of the three common viral pathogens in blood samples, when preserved using the dry spot method, conform to a first-order reaction pattern in the accelerated degradation experiment. The relationship between the rate of nucleic acid degradation and the absolute temperature of storage is consistent with the Arrhenius equation. Based on the calculations using this equation, the stability and validity period of plasma nucleic acid samples preserved using the dry spot method can reach a minimum of 3.5 years under storage conditions not exceeding 20 ℃, which essentially meets the requirements for the preservation period of blood samples retained by blood banks.