Objective:To analyze the immune reconstitution after BTKi treatment in patients with chronic lymphocytic leukemia(CLL).Methods:The clinical and laboratorial data of 59 CLL patients admitted from January 2017 to March 2022 in Fujian Medical University Union Hospital were collected and analyzed retrospectively.Results:The median age of 59 CLL patients was 60.5(36-78).After one year of BTKi treatment,the CLL clones(CD5+/CD19+)of 51 cases(86.4％)were significantly reduced,in which the number of cloned-B cells decreased significantly from(46±6.1)× 109/L to(2.3±0.4)× 109/L(P=0.0013).But there was no significant change in the number of non-cloned B cells(CD19+minus CD5+/CD19+).After BTKi treatment,IgA increased significantly from(0.75±0.09)g/L to(1.31±0.1)g/L(P＜0.001),while IgG and IgM decreased from(8.1±0.2)g/L and(0.52±0.6)g/L to(7.1±0.1)g/L and(0.47±0.1)g/L,respectively(P＜0.001,P=0.002).BTKi treatment resulted in a significant change in T cell subpopulation of CLL patients,which manifested as both a decrease in total number of T cells from(2.1±0.1)× 109/L to(1.6±0.4)× 109/L and NK/T cells from(0.11±0.1)× 109/L to(0.07±0.01)× 109/L(P=0.042,P=0.038),both an increase in number of CD4+cells from(0.15±6.1)× 109/L to(0.19±0.4)× 109/L and CD8+cells from(0.27±0.01)× 109/L to(0.41±0.08)× 109/L(both P＜0.001).BTKi treatment also up-regulated the expression of interleukin(IL)-2 while down-regulated IL-4 and interferon(IFN)-γ.However,the expression of IL-6,IL-10,and tumor necrosis factor(TNF)-α did not change significantly.BTKi treatment could also restored the diversity of TCR and BCR in CLL patients,especially obviously in those patients with complete remission(CR)than those with partial remission(PR).Before and after BTKi treatment,Shannon index of TCR in patients with CR was 0.02±0.008 and 0.14±0.001(P＜0.001),while in patients with PR was 0.01±0.03 and 0.05±0.02(P＞0.05),respectively.Shannon index of BCR in patients with CR was 0.19±0.003 and 0.33±0.15(P＜0.001),while in patients with PR was 0.15±0.009 and 0.23±0.18(P＜0.05),respectively.Conclusions:BTKi treatment can shrink the clone size in CLL patients,promote the expression of IgA,increase the number of functional T cells,and regulate the secretion of cytokines such as IL-2,IL-4,and IFN-γ.BTKi also promote the recovery of diversity of TCR and BCR.BTKi treatment contributes to the reconstitution of immune function in CLL patients.

Objective:To evaluate the efficacy of acute T-cell lymphoblastic leukemia(T-ALL)in children and explore the prognostic risk factors.Methods:The clinical data of 127 newly diagnosed children with T-ALL admitted to five hospitals in Fujian province from April 2011 to December 2020 were retrospectively analyzed,and compared with children with newly diagnosed acute precursor B-cell lymphoblastic leukemia(B-ALL)in the same period.Kaplan-Meier analysis was used to evaluate the overall survival(OS)and event-free survival(EFS),and COX proportional hazard regression model was used to evaluate the prognostic factors.Among 116 children with T-ALL who received standard treatment,78 cases received the Chinese Childhood Leukemia Collaborative Group(CCLG)-ALL 2008 protocol(CCLG-ALL 2008 group),and 38 cases received the China Childhood Cancer Collaborative Group(CCCG)-ALL 2015 protocol(CCCG-ALL 2015 group).The efficacy and serious adverse event(SAE)incidence of the two groups were compared.Results:Proportion of male,age ≥ 10 years old,white blood cell count(WBC)≥ 50 × 109/L,central nervous system leukemia,minimal residual disease(MRD)≥ 1％during induction therapy,and MRD ≥ 0.01％at the end of induction in T-ALL children were significantly higher than those in B-ALL children(P＜0.05).The expected 10-year EFS and OS of T-ALL were 59.7％and 66.0％,respectively,which were significantly lower than those of B-ALL(P＜0.001).COX analysis showed that WBC ≥ 100 x 109/L at initial diagnosis and failure to achieve complete remission(CR)after induction were independent risk factors for poor prognosis.Compared with CCLG-ALL 2008 group,CCCG-ALL 2015 group had lower incidence of infection-related SAE(15.8％vs 34.6％,P=0.042),but higher EFS and OS(73.9％vs 57.2％,PEFS=0.090;86.5％vs 62.3％,PoS=0.023).Conclusions:The prognosis of children with T-ALL is worse than children with B-ALL.WBC ≥ 100 × 109/L at initial diagnosis and non-CR after induction(especially mediastinal mass has not disappeared)are the risk factors for poor prognosis.CCCG-ALL 2015 regimen may reduce infection-related SAE and improve efficacy.

Objective:To explore the clinical efficacy and safety of flumatinib mesylate produced in China in patients with newly diagnosed chronic myeloid leukemia in chronic phase(CML-CP).Methods:32 newly diagnosed CML-CP patients admitted to the Hematology Department of the Affiliated Hospital of Southwest Medical University from March 1,2020 to March 31,2022,who had never received any tyrosine kinase inhibitor(TKI)were included in the study.The patients were treated by flumatinib mesylate 600mg once daily.The hematologic,cytogenetic and molecular responses were assessed at 3-,6-and 12-month,and adverse effects of the drug were evaluated.Results:31 patients were treated with flumatinib for≥3 months,of which 24 patients were treated for ≥ 6 months and 14 patients were treated for ≥ 12 months.At 3rd month of treatment,30 out of 31 patients achieved complete hematologic response(CHR);24 patients underwent cytogenetic testing and 22 cases achieved major cytogenetic response(MCyR),of which 21 cases achieved complete cytogenetic response(CCyR);Among 25 patients who underwent molecular testing,22 patients had BCR-ABLIS ≤ 10％,including 10 patients with BCR-ABLIS ≤ 0.1％,and 6 patients with BCR-ABLIS≤0.01％.At 6th month of treatment,23 out of 24 patients achieved CHR;17 patients underwent cytogenetic testing and all achieved CCyR;Among 23 patients who underwent molecular testing,20 patients had BCR-ABLIS ≤1％,including 16 patients with BCR-ABL1S≤0.1％and 12 patients with BCR-ABLIS ≤ 0.01％.At 12nd month of treatment,all 14 patients achieved CHR and CCyR;Among them,10 patients had BCR-ABLIS ≤ 0.1％,including 9 patients with BCR-ABLIS ≤ 0.01％.The grade Ⅲ/Ⅳ leukopenia,thrombocytopenia and anemia rates in the patients were 13.3％,20.0％and 3.3％,respectively.One patient stopped flumatinib therapy due to severe and persistent hematologic toxicity.The major non-hematologic adverse events were abnormal liver function(20％),diarrhea(10％),bone/joint pain(10％),muscle spasm(10％),rash(6.7％),acute kidney injury(6.7％)and nausea(3.3％),most of which were grade Ⅰ-Ⅱ.No patient experienced grade Ⅳnon-hematologic adverse events.No drug toxicity-related death occurred.Conclusion:Flumatinib mesglate,as the first-line treatment for newly diagnosed CML-CP,can enable the patients to achieve early and deep molecular and cytogenetic responses,and shows good safety.

Objective:To evaluate the efficacy and safty of China-made flumatinib mesylate in the treatment of chronic myeloid leukemia in chronic phase(CML-CP).Methods:42 CML-CP patients treated with Chinese produced flumatinib(oral,600 mg,1/d)were included in the study,including 14 newly diagnosed patients and 28 patients underwent conversion therapy.The hematological,cytogenetic and molecular response and safety were observed and evaluated after 3,6 and 12 months of treatment.Results:35 patients were treated for more than 3 months,among which 31 patients were treated for more than 6 months and 17 patients were treated for more than 12 months.After 3 months of treatment,33 patients underwent hematological,cytogenetic and molecular examination.Of these,32 patients achieved complete hematological response(CHR),13 patients achieved complete cytogenetic response(CCyR),20 patients showed BCR-ABLIS≤10％and 7 patients reached major molecular response(MMR).After 6 months of treatment,all 30 patients who could evaluate efficacy achieved CHR,of which 17 patients achieved CCyR,18 patients showed BCR-ABLIS ≤ 1％and 16 patients reached MMR.After 12 months of treatment,all 17 patients were evaluated for efficacy,all achieved CHR,10 patients obtained CCyR,7 patients reached MMR.Grade Ⅲ or Ⅳ thrombocytopenia,leukopenia and anemia occurred in 7,2 and 1 patients,respectively.The non-hematological adverse reactions were diarrhea in 6 cases,renal function damage in 4 cases,rash and pruritus in 3 cases,liver function damage in 3 cases,nausea in 1 case,fever in 1 case,bone/joint or muscle pain in 1 case.Conclusion:In the real world,China-made flumatinib mesylate has a positive short-term efficacy and reliable safety in the treatment of CML-CP patients,whether as first-line treatment or second-and third-line conversion therapy.

Objective:To investigate the clinical significance of genetic and molecular changes in primary myeloid sarcoma(MS).Methods:Fourteen patients with primary MS were selected in Jiading District Central Hospital Affiliated Shanghai University of Medicine & Health Sciences,The First People's Hospital of Lianyungang from September 2010 to December 2021.AML1-ETO fusion,PML-RARα fusion and CBFβ breakage were detected by fluorescence in situ hybridization(FISH),and the mutations of NPM1,CEBPA,FLT3,RUNX1,ASXL1,KIT and TP53 genes were detected by new generation sequencing(NGS).Results:Among 14 patients,the MS occurred in bone,breast,epididymis,lung,chest wall,cervix,small intestine,ovary,lymph nodes and central nervous system.The tumor cells expressed MPO(13 cases),CD34(7 cases),CD43(8 cases),CD68(7 cases),CD99(8 cases)and CD117(6 cases).Cytogenetic abnormalities were observed in 4 cases,including 3 cases of AML1-ETO fusion and 1 case of CBF β breakage,while no PML-RAR α fusion was detected.There were no significant differences in overall survival(OS)and leukemia-free survival(LFS)between patients with and without AML1-ETO fusion/CBFβ breakage(both P＞0.05).Among the 14 patients,the number of NPM1,CEBPA,FLT3-ITD,RUNX1,ASXL1,KIT and TP53 gene mutations was 5,3,5,3,2,2,1.respectively,of which 7 cases had at least one mutation in FLT3-ITD,RUNX1,ASXL1 and TP53 gene.The OS and LFS of patients with FLT3-ITD,RUNX1,ASXL1 or TP53 mutation were shorter than those without mutations(both P＜0.01).Conclusion:The genetic and molecular abnormalities of primary MS can be detected by FISH and NGS techniques.FLT3-ITD,RUNX1,ASXL1 or TP53 mutation indicates a worse prognosis,but further clinical studies are needed to confirm it.

Objective:To explore the role of bone marrow mesenchymal stem cells(BMSC),an essential element of the bone marrow microenvironment,in multidrug resistance(MDR)of K562 cells,as well as the reversal effect of tetrandrine(TET)on BMSC-mediated MDR and its potential mechanism.Methods:A mixed co-culture system and a transwell co-culture system for BMSC and K562 cells were established,and the cells were divided into different groups and treated with daunorubicin(DNR)alone or combined with TET and DNR.The CCK-8 assay was used to detect the proliferation of K562 cells in each group,and the cell inhibition rate was calculated.Cytometric bead array(CBA)was used to detect the expression levels of IFN,IL-2,IL-6 and IL-10 in the supernatant of different groups.RT-qPCR and Western blot were used to detected the expression of STAT3 at mRNA and protein levels,respectively.Results:Compared with K562+DNR group,the inhibition rate of DNR on K562 cell proliferation in K562+BMSC+DNR group was significantly decreased(P＜0.05),while the levels of IL-6 in the culture supernatant and phosphorylated STAT3 in K562 cells were significantly increased(P＜0.05).Compared with K562+BMSC+DNR group,the inhibition rate of DNR on K562 cell proliferation in K562+BMSC+DNR+TET group was significantly increased(P＜0.05),while the level of IL-6 and phosphorylated STAT3 was significantly decreased(P＜0.05).Conclusion:BMSC can promote the drug resistance of leukemia cells,and TET may reverse the BMSC-mediated drug resistance via inhibiting IL-6/STAT3 signaling pathway.

Objective:To investigate the effects of miR-217 on proliferation and adriamycin sensitivity of acute myeloid leukemia(AML)cells.Methods:The mimic NC and miR-217 mimic vectors were constructed and transfected into HL-60 cells,and transfection efficiency was detected by qPCR.The cells were treated with different concentrations of adriamycin for 24 h and 48 h.CCK-8 assay was used to detect the chemical sensitivity of adriamycin and screen the optimal concentration and time of adriamycin treatment.Cells were divided into control group,mimic NC group,miR-217 mimic group,adriamycin group and miR-217 mimic+adriamycin group.Apoptosis was detected by flow cytometry,and the expressions of miR-217,PI3K and Akt3 were detected by qPCR.Western blot was used to detect the expression of PI3K/Akt pathway proteins PI3K,Akt3 and apoptosis proteins Bcl-2,Bax,and double luciferase was used to verify the relationship between miR-217 and Akt3.Results:MiR-217 mimic could enhance the sensitivity of HL-60 cells to adriamycin.The optimal concentration and treatment time of adriamycin were 160 ng/ml and 48 h,respectively.Compared with control group,apoptosis rate,miR-217 and Bax protein levels were significantly increased in miR-217 mimic and adriamycin groups(P＜0.01),while Bcl-2 protein,PI3K,Akt3 mRNA and protein levels were significantly decreased(P＜0.01).Compared with adriamycin group,apoptosis rate,miR-217 and Bax protein levels were significantly increased in miR-217 mimic+adriamycin group(P＜0.01),while Bcl-2 protein,PI3K,Akt3 mRNA and protein levels were significantly decreased(P＜0.0 1).Dual luciferase assay showed that there was a targeted regulatory relationship between miR-217 and Akt3.Conclusion:MiR-217 regulates the PI3K/Akt pathway targeting Akt3,inhibits cell proliferation,promotes cell apoptosis and enhances the sensitivity of adriamycin to AML cells.

Objective:To investigate the effect of tripipartite motif 59(TRIM59)expression interference on the chemosensitivity of daunorubicin(DNR)in chronic myeloid leukemia(CML)K562 cells and the related molecular mechanism.Methods:The expressions of TRIM59 mRNA in bone marrow tissues of patients with CML and K562 cells were detected by RT-qPCR.Liposome-based transfection technology was used to transfect TRIM59-specific siRNA(si-TRIM59)into K562 cells which then were treated with DNR.The proliferation and apoptosis of cells were detected by CCK-8 assay and flow cytometry,respectively,and the expressions of apoptosis-related protein and Wnt/β-catenin signaling pathway-related protein were detected by Western blot.Results:Compared with the bone marrow tissue of CML patients at the time of initial treatment,the expression of TRIM59 mRNA in bone marrow tissue of CML patients at the time of chemotherapy resistance was significantly increased(P＜0.05).Compared with control group,the cell proliferation inhibition rate and apoptosis rate in si-TRIM59 group and DNR group were significantly increased(P＜0.05),the expression of Bax,Caspase3 and Cleaved-Caspase3 protein were significantly increased(P＜0.05),while the expressions of Bcl-2,Wnt3 α,GSK-3 β protein and the ratio of p-β-catenin/β-catenin were significantly decreased(P＜0.05).Compared with si-TRIM59 group and DNR group,the proliferation inhibition rate and apoptosis rate of si-TRIM59+DNR group were significantly increased(P＜0.05),the expression of Bax,Caspase3 and Cleaved-Caspase3 protein were significantly increased,while the expression of Bcl-2,Wnt3 α,GSK-3 β protein and the ratio of p-β-catenin/β-catenin were significantly decreased(P＜0.05).Conclusion:TRIM59 expression interference may enhance the chemosensitivity of K562 cells to DNR,and its mechanism may be related to the regulation of Wnt/β-catenin signaling pathway.

Objective:To construct a acute myeloid leukemia(AML)cell line in which HOXA5 gene is stably knocked out by CRISPR-Cas9-mediated gene editing technique,so as to clarify the effect of HOXA5 gene knockout on the proliferation of AML cells,and preliminarily explore the role of HOXA5 gene in the pathogenesis of AML.Methods:The expression of HOXA5 in bone marrow mononuclear cells(BMMC)of non-tumor hematological patients and newly diagnosed AML patients was detected by quantitative real-time PCR(qRT-PCR)and Western blot,respectively.The AML cell line KO-HOXA5-THP-1 was constructed in which HOXA5 gene was knocked out by CRISPR-Cas9-Mediated gene editing technique,and the knockout of HOXA5 gene was verified by qRT-PCR and Western blot,and the cell proliferation was detected by CCK-8 assay.Results:Compared with non-tumor hematological patients,the levels of HOXA5 gene and protein in BMMC of newly diagnosed AML patients were significantly increased(P＜0.05).The stable HOXA5 knockout cell line can be obtained by CRISPR-Cas9-Mediated gene editing technique,and the proliferation ability of THP-1 cells with HOXA5 gene knockout was significantly decreased(P＜0.05).Conclusion:HOXA5 is highly expressed in AML cells,and knocking out HOXA5 can significantly affect the proliferation ability of AML cells,which provides a new potential therapeutic target for the precise treatment of AML.

Objective:To observe the effect of resveratrol(Res)on T-acute lymphoblastic leukemia(T-ALL)mice,and further explore its mechanism on Notch1 signaling pathway.Methods:Twenty-five 6-8 weeks old female C57BL/6 mice were randomly divided into control group,T-ALL group and Res group.Res group was further divided into low-Res.middle-Res and high-Res group.The percentage of leukemia cells in peripheral blood and spleen cell suspension were detected by flow cytometry and Wright-Giemsa staining,pathological morphology of spleen and bone marrow tissues were observed by HE staining,the expression levels of Notch1,Hes-1,c-Myc,miR-19b and PTEN mRNA in spleen tissue were detected by RT-qPCR,and the protein levels of Notch1,Hes-1,c-Myc,p-PTEN and PTEN were detected by Western blot.Results:Compared with control group,the leukemia cells in peripheral blood of mice in T-ALL group were markedly increased,accompanied by diffuse infiltration of leukemia cells in spleen and bone marrow tissues,the mRNA levels of Notch1,Hes-1,c-Myc,miR-19b and the protein levels of Notch1.Hes-1,c-Myc were increased(P＜0.01),while the expression of PTEN mRNA and protein were significantly decreased in the spleen tissue of T-ALL mice(P＜0.01).The above indicators in the H-Res group were reversed compared with T-ALL group after administration of resveratrol.Conclusion:Resveratrol may play a role in anti T-ALL by inhibiting Notch1 signaling pathway in mice.