Objective: To investigate the effect of feeder layer cells expressing interleukin (IL)-21 on the amplification of NK cells In Vitro .
Methods: The K562 cell line with IL-21 expression on its membrane was constructed by electroporation, and co-cultured with NK cells after inactivation. The proliferation of NK cells was observed. The killing function of the amplified NK cells In Vitro was evaluated by the lactate dehydrogenase (LDH) and interferon-γ (IFN-γ) release assay. A colorectal cancer xenograft model in NOD/SCID mice was established, and a blank control group, a NK cell group and an amplified NK cell group were set up to detect the tumor killing effect of amplified NK cells in vivo.
Results: K562 cells expressing IL-21 on the membrane were successfully constructed by electroporation. After co-culturing with K562 cells expressing IL-21 on the membrane for 17 days, the NK cells increased to 700 times, which showed an enhanced amplification ability compared with control group (P < 0.001). In the tumor cell killing experiment In Vitro , there was no significant difference in the killing activity on tumor cells between NK cells and amplified NK cells, and there was also no significant difference in mice in vivo.
Conclusion: K562 cells expressing IL-21 on the membrane can significantly increase the amplification ability of NK cells In Vitro , but do not affect the killing function of NK cells In Vitro and in vivo. It can be used for the subsequent large-scale production of NK cells In Vitro .
Objective: To investigate the mechanism of DNA damage and repair in MLL -rearranged acute myeloid leukemia（ MLL-r AML）cells by the combination of Chidamide and the BRD4 inhibitor(+)-JQ-1.
Methods: MLL-r AML cell lines Molm-13, MV4-11 and non- MLL-r AML cell line Kasumi were divided into control group（contr）, Chidamide group（chida）, (+)-JQ-1 group and Combination group（combi）, respectively. Cell viability of Molm-13 was measured by CCK-8 to determine optimal the concentrations of Chidamide and(+)-JQ-1. The cell cycle was detected by flow cytometry, and apoptosis-related factors Bcl-2, Bax and caspase-3 were detected by Western blot. DNA damage marker γH2AX was detected by immunofluorescence. The protein expressions of DNA damage factor γH2AX, DNA damage checkpoint kinases p-ATR, p-CHK1, p-ATM, p-CHK2 and DNA damage repair factors Rad51 and 53BP1 were detected by Western blot. The expression of DNA damage repair factors Rad51 and 53BP1 mRNA was detected by qRT-PCR.
Results: Under the treatment of Chidamide (300 nmol/L) and (+)-JQ-1 (400 nmol/L), the proportion of G1 phase cells in MLL-r AML cell lines Molm-13 and MV4-11 was increased in combination group compared with control group. In non- MLL-r AML cell line Kasumi, compared with control group, the proportion of G1 phase cells in combination group was increased (P < 0.05). In Molm-13 and MV4-11 cell lines, compared with control group, the expression level of DNA damage marker γH2AX in combination group was increased (P < 0.05). The expression levels of DNA damage checkpoint and damage repair factors p-ATR, p-CHK1, p-ATM, p-CHK2, Rad51, 53BP1 were decreased (P < 0.05). In Kasumi cell line, compared with control group, there was no significant change in the expression of some of the above factors in combination group (P >0.05), but the expression trend of some factors was opposite. In MLL-r AML cell lines Molm-13 and MV4-11, compared with control group, the expression levels of Bax and caspase-3 protein were increased in combination group, while the expression levels of Bcl-2 protein were decreased (P < 0.05). In non- MLL-r AML cell line Kasumi, there was no significant change in apoptotic factor protein expression in combination group compared with control group (P >0.05).
Conclusion: Chidamide combined with (+)-JQ-1 can inhibit the proliferation of MLL-r AML cells, inhibit the initiation of protective self-repair of these leukemia cells by inhibiting the DNA damage response pathway, and ultimately increase the apoptosis of these cells, but non- MLL-r AML cells have no similar results.
Objective: To discover the relationship between the RNF213 gene and acute myeloid leukemia (AML), and explore the effect of RNF213 on the proliferation and apoptosis of THP-1 cells.
Methods: Analyze the expression of RNF213 gene in AML and its relationship with prognosis through the GEPIA database. Collecting 30 AML patients and non-tumor hematological patients who went to the Affiliated Hospital of Zunyi Medical University from January 2017 to January 2022. RT-qPCR and Western blot were used to detect the expression levels of RNF213 mRNA and protein. Perform survival of patients was analysed by Kaplan-Meier. Meanwhile, the expression levels of RNF213 mRNA and protein were detected in AML cell lines (THP-1, OCI-AML2 ). CRISPR-Cas9 was used to knockdown the RNF213 gene in THP-1 cells; flow cytometry was used to detect apoptosis rate of cell. CCK-8 and colony formation assay were used to detect cell proliferation. Western blot was used to detect the expression level of Cleaved-Caspase 3 protein.
Results: Compared with the control group, the expression level of RNF213 in AML patients was significantly increased, and patients with high expression of RNF213 have a worse prgnosis. Higher expression level of RNF213 protein in THP-1 cells. After knocking down the RNF213 gene of THP-1 cells, cell proliferation was significantly reduced, and the apoptosis rate and expression of apoptosis related protein Cleared-Caspase3 were significantly increased.
Conclusion: AML patients have high expression of RNF213 , and the prognosis of high expression patients is poor. The RNF213 gene affects AML cell proliferation and apoptosis, and may be a prognostic marker and potential therapeutic target for AML.
Objective: To investigate the value of serum ferritin, folic acid and vitamin B12 in the treatment of multiple myeloma (MM) with bortezomib combined with chemotherapy.
Methods: Clinical data of 40 MM patients admitted to our hospital from January 2020 to August 2022 and 40 hematology outpatients during the same period were reviewed. All MM patients were treated with bortezomib combined with chemotherapy. The diagnostic efficacy of serum ferritin, folic acid and vitamin B12 on MM was analyzed by ROC curve. The changes of serum ferritin, folic acid and vitamin B12 in MM patients before and after treatment were compared. According to the mean values of serum ferritin, folic acid and vitamin B12, patients were divided into high and low expression groups, and the survival of patients between the groups was compared.
Results: Before treatment, the levels of serum ferritin and vitamin B12 in MM patients were significantly higher than those in control group, while folic acid was lower (all P <0.001). The area under the curve (AUC) of MM patients diagnosed with ferritin, folic acid and vitamin B12 were 0.928, 0.843 and 0.867, the specificity was 100%, 67.50% and 72.50%, and the sensitivity was 80.00%, 95.00% and 87.50%, respectively. After 4 cycles of chemotherapy, there were 9 cases of complete remission (CR), 19 cases of very good partial remission (VGPR), 6 cases of PR, 4 cases of stable disease (SD) and 2 cases of progression disease (PD) in 40 MM patients. In CR group, ferritin and vitamin B12 decreased but folic acid increased after treatment compared with before treatment (all P < 0.05). The overall survival (OS) rates of patients in low expression group of ferritin and vitamin B12 were significantly higher than those in high expression group (both P < 0.01). The OS rate of patients in high expression group of folic acid was significantly higher than that in low expression group (P < 0.01). Cox regression analysis showed that ferritin was an independent prognostic factor for MM patients (HR=8.850, 95%CI : 2.267-34.553, P =0.002).
Conclusion: Serum ferritin, folic acid and vitamin B12 have some auxiliary value in the diagnosis and efficacy evaluation of MM, and ferritin is an independent prognostic factor for MM.
Objective: To analyze the gene mutation types and composition characteristics of patients with thalassemia in Chengdu Region, Sichuan Province.
Methods: 6 649 suspected thalassemia patients with positive screening results who visited Chengdu Women's and Children's Center Hospital from January 2017 to December 2020 were selected as the study subjects. Among them, there were 2 273 males and 4 376 females. The frequency and distribution of α and β genotypes of thalassemia in this cohort was analyzed by Luminex liquid-phase microarray method.
Results: Among the 6 649 samples, 3 787 were genetically diagnosed as thalassemia, with a total positive rate of 56.96%; in which, 2 063 (31.03%) cases were β-thalassemia, 1 629 (24.50%) cases were α-thalassemia, and 95 (1.43%) cases were α combined with β thalassemia. The types of β-thalassemia gene mutation were mainly CD17/N (36.45%, 752/2 063), CD41-42/N (25.30%, 522/2 063), and IVS-II-654/N (24.72%, 510/2 063); and 2 037 cases of simple heterozygous mutations were identified, accounting for 98.74% of β-thalassemia patients. The types of α-thalassemia gene mutation were mainly -- SEA/αα (79.01%, 1 287/1 629), -α3.7/αα (10.62%, 173/1 629), -α3.7/-- SEA (2.95%, 48/1 629), and -α4.2/αα (2.15%, 35/1 629). The α combined with β thalassemia was dominated by -α3.7/αα; CD17/N and -α3.7/αα; IVS-II-654/N, both accounting for 14.74% (14/95) of patients with α combined with β thalassemia.
Conclusion: In Chengdu region, Sichuan province, β thalassemia is more common than α thalassemia, the main type of β thalassemia mutation is CD17/N, and the main type of α thalassemia mutation is -- SEA/αα, with regional characteristics.
Objective: To investigate the clinical efficacy of ibrutinib combined with venetoclax in the treatment of relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL), and to analyze the factors affecting efficacy and prognosis.
Methods: Clinical data of 62 R/R DLBCL patients admitted to our hospital from August 2017 to July 2022 were retrospectively analyzed. All patients were treated with ibrutinib combined with venetoclax. The clinical efficacy and drug safety were evaluated. The effects of clinical features on short-term efficacy and overall survival (OS) were analyzed.
Results: The objective response rate (ORR) of 62 patients was 48.39%. The extranodal lesions, intermediate-high/high risk of NCCN-IPI, intermediate-high/high risk of IPI, progression or recurrence time <12 months were the risk factors affecting the short-term efficacy of chemotherapy in R/R DLBCL patients (all P < 0.05). The most common adverse effect was neutropenia (75.19%), and the incidence of grade Ⅲ-Ⅳ neutropenia was 52.71%. The 1-year and 2-year OS rates of 62 patients were 48.51% and 31.56%, respectively, and the median OS time was 12 months. Multivariate analysis showed that objective remission after chemotherapy [HR =0.080 (95%CI: 0.028-0.235)] was a protective factor for OS in R/R DLBCL patients, and intermediate-high/high risk of NCCN-IPI [HR =4.828 (95%CI : 1.546-15.080)] was an independent risk factor affecting the prognosis of R/R DLBCL patients.
Conclusion: Ibrutinib combined with venetoclax can be used as an effective treatment regimen for R/R DLBCL, and NCCN-IPI can be used as a prognostic indicator. Objective remission after chemotherapy is beneficial for R/R DLBCL patients to achieve better OS.
Objective: To investigate the correlation between bone marrow microvascular density, angiogenesis factors and bortezomib resistance in multiple myeloma (MM).
Methods: The data of 200 patients with MM treated in our hospital from January 2020 to August 2023 were retrospectively analyzed, and the patients with MM were divided into drug-resistant group(n=68) and non-drug-resistant group(n=132) according to their drug resistance during bortezomib treatment. The univariate and multivariate logistic analysis were used to screen the independent influencing factors of bortezomib resistance in MM patients during treatment. The receiver operating characteristic (ROC) curve and clinical decision curve (DCA) were used to evaluate the predictive performance and clinical application value of the risk prediction model, the consistency between the actual incidence rate and the predicted incidence rate was judged by validating the calibration chart, and the goodness-of-fit of the model was judged by H-L test.
Results: 68 of the 200 MM patients developed resistance and poor clinical efficacy during bortezomib treatment, and the clinical resistance rate of bortezomib was 34.0%. The results of multivariate analysis showed that high bone marrow microvessel density (MVD) and high bone marrow supernatant VEGF, HGF, and bFGF expression levels were independent risk factors for bortezomib resistance in MM patients (P < 0.05). The area under the ROC curve (AUC) of the model jointly constructed by bone marrow MVD, serum VEGF, HGF, bFGF and TNF-α levels was 0.924, and its sensitivity and specificity were 92.6% and 78.8%, which were higher than those of the bone marrow MVD model (AUC=0.743) and the vasogenesis factor model (AUC=0.878). The calibration curve of the joint prediction model was close to the standard curve, indicating that the model is more consistent. The results of H-L goodness -of - fit test showed χ2=14.748， P =0.164, the joint prediction model had a good fit. The DCA curve showed that the clinical net benefit of intervention in the range of 0.0~1.0 was greater than that of full intervention and no intervention.
Conclusion: The prediction model based on bone marrow MVD and vasogenesis factors (VEGF, HGF, bFGF) in MM patients has higher clinical evaluation performance and predictive value.
Objective: To analyze the clinical and genetic characteristics of acute myeloid leukemia, myelodysplasia-related (AML-MR) patients and evaluate their prognostic risk stratification, to guide clinical treatment decisions and improve understanding of the biological characteristics and disease progression.
Methods: The study analyzed cellular and molecular genetic information of 307 AML-MR patients, diagnosed based on clinical history, bone marrow morphology, cytogenetics, and molecular genetic abnormalities. The risk stratification followed the 2022 ELN guidelines.
Results: 57 cases (18.6%) met the AML-MR diagnostic criteria based on morphology and clinical history, 110 cases (37.2%) met the AML-MR diagnostic criteria based on cytogenetic results, and 210 cases (74.5%) met the AML-MR diagnostic criteria based on molecular testing results. Among different type of mutations, ASXL1 mutation was the most frequent, followed by SRSF2 and BCOR mutations. Except for 2 cases with incomplete data that could not be classified, 263 (86.2%) of the 305 patients were classified as poor prognosis, 20 (6.6%) were classified as good prognosis group, and 22 (7.2%) were classified as intermediate prognosis group.
Conclusion: Molecular genetic information plays a crucial role in diagnosing AML-MR, highlighting the importance of genetics in diagnosis and prognosis. Most AML-MR patients fall into poor prognosis categories, necessitating early intensive and targeted therapy for better survival outcomes.
Objective: To investigate the clinical characteristics and prognosis of single center adult chronic myeloid leukemia in chronic phase (CML-CP).
Methods: Clinical data of 41 adult CML-CP patients in Department of Hematology, Shanghai Fengxian District Central Hospital from January 2015 to May 2021 were retrospectively analyzed. The clinical characteristics and prognosis of patients between <60 years group and ≥60 years group were compared.
Results: The 41 patients included 27 (65.9%) males and 14 (34.1%) females. The median age of the patients was 56(19-84) years, with 22 cases (53.7%) <60 years and 19 cases (46.3%) ≥60 years. Univariate analysis indicated that the proportions of patients with comorbidities, intermediate/high-risk Sokal score, myelofibrosis, and lactate dehydrogenase ≥1 000 U/L were significantly increased in ≥60 years group compared with <60 years group at initial diagnosis (all P <0.05). There were no statistical differences in the distribution of sex, ELST score, white blood cell count, platelet count, peripheral blood basophil percentage, peripheral blood eosinophil percentage and bone marrow primitive cell percentage between the two groups (P >0.05). The proportion of patients taking reduced-dose imatinib in ≥60 years group significantly increased (P <0.001). Patients <60 years had a higher proportion of molecular biological remission after treatment of tyrosine kinase inhibitors (TKIs) than patients ≥60 years (P <0.001). The incidence of non-hematologic adverse reactions to TKI therapy significantly increased in patients ≥60 years (P <0.001). Multivariate analysis showed that no adverse factors affecting the efficacy and prognosis of TKI.
Conclusion: Compared with adult CML-CP patients <60 years, patients ≥60 years gain fewer benefits from TKI treatment and increased adverse reactions.
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