Efficacy and safety analysis of the zanubrutinib-based bridging regimen in chimeric antigen receptor T-cell therapy for relapsed/refractory diffuse large B-cell lymphoma / 中华血液学杂志

Objective: To further elucidate the clinical efficacy and safety of a combination regimen based on the BTK inhibitor zebutanil bridging CD19 Chimeric antigen receptor T cells (CAR-T cells) in the treatment of relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) . Methods: Twenty-one patients with high-risk r/r DLBCL were treated with a zanubrutinib-based regimen bridging CAR-T between June 2020 and June 2023 at the Department of Hematology, Tongji Hospital, Tongji University and the Second Affiliated Hospital of Zhejiang University, and the efficacy and safety were retrospectively analyzed. Results: All 21 patients were enrolled, and the median age was 57 years (range: 38-76). Fourteen patients (66.7%) had an eastern cooperative oncology group performance status score (ECOG score) of ≥2. Eighteen patients (85.7%) had an international prognostic index (IPI) score of ≥3. Three patients (14.3%) had an IPI score of 2 but had extranodal infiltration. Fourteen patients (66.7%) had double-expression of DLBCL and seven (33.3%) had TP53 mutations. With a median follow-up of 24.8 (95% CI 17.0-31.6) months, the objective response rate was 81.0%, and 11 patients (52.4%) achieved complete remission. The median progression-free survival (PFS) was 12.8 months, and the median overall survival (OS) was not reached. The 1-year PFS rate was 52.4% (95% CI 29.8% -74.3%), and the 1-year OS rate was 80.1% (95% CI 58.1% -94.6%). Moreover, 18 patients (85.7%) had grade 1-2 cytokine-release syndrome, and two patients (9.5%) had grade 1 immune effector cell-associated neurotoxicity syndrome. Conclusion: Zanubrutinib-based combination bridging regimen of CAR-T therapy for r/r DLBCL has high efficacy and demonstrated a good safety profile.

Detection of Genetic Mutations in Primary Hypereosinophilia Patients / 中国实验血液学杂志

OBJECTIVE@#To explore the potential pathogenetic mutations of primary hypereosinophilia（HEN）by sequencing FGFR1 FLT3, MPL and JAK2 genes, and to clarify their effect on clinical manifestation and prognosis of HEN patients.@*METHODS@#The direct DNA sequencing was employed to detect the gene mutations of FGFR1, FLT3, MPL and JAK2 in HEN patients.@*RESULTS@#One deletion mutation (2654_2753del) within tyrosine kinase domain of FLT3 gene was found in a patient suffered from severe symptoms and ended with dismal outcome, which induced a premature stop codon (G885fsX888). For FGFR1, a new variation described as 1014_1019del AACAGT for nucleotide change was found in 19 cases, resulting in T339_V340del at the protein level.@*CONCLUSION@#The deletion of 6 bases in the FGFR1 gene (1014_1019del AACAGT) is first reported as non-synonymous SNP (nsSNP) site in the patients with primary hypereosinophilia. Deletion mutations in the FLT3 gene may be related with malignant clinical features and poor prognosis.

Clinical Efficacy Comparison of Ultralow Dose of Decitabine and Cyclosporine on Low-risk and Intermediate-risk Type 1 of Myelodysplastic Syndrome / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To evaluate the clinical efficacy and safety of decitabine and cyclosporine for treatment of low-risk and intermediate-risk-1 myelodysplastic syndrome (MDS) patients.</p><p><b>METHODS</b>The clinical data of 27 cases of low risk and intermediate-risk-1 MDS during the past 3 years in Tongji hospital were analyzed retrospectively. These MDS patients were divided into 2 groups: decitabine group (11 cases) and cyclosporine group (16 cases). The MDS patients in the 2 groups were treated with ultra low dose of decitabine and cyclosporine A; the curetive efficacy and adverse reactions were evaluated.</p><p><b>RESULTS</b>In the 11 patients with low-risk and intermediate-risk-1 MDS treated with 2 courses of ultra-low-dose decitabine, 4 cases (36.4%) achieved a hematological improvement, 7 cases (63.6%) showed ineffective, including non-remission in 6 cases (54.5 %) and death in 1 patient (9.1%), total effective rate were 36.4%; 3 cases died within the first year and the overall survival (OS) rate was 72.7%. The causes of death mainly were severe myelosuppression and the associated infection and bleeding. In the 16 patients with low-risk and intermediate-risk-1 MDS treated with cyclosporine (CsA), 10 cases (62.5%) achieved a hematological improvement, 6 cases (37.5%) showed ineffective, the total efficiency of 62.5%; no patients died within 1 year, the 1-year OS was 100%. Changes in neutrophils, hemoglobin and platelet were not significantly different between the two group.</p><p><b>CONCLUSION</b>The clinical efficacy of decitabine on low-risk and intermediate-risk-1 MDS has not confirmed to be superior to cyclosporine (P = 0.252). However, the side effects of serious infection and myelosuppression were more severe in decitabine group than that in the cyclosporine group. Moreover, the 1-year overall survival rate in decitabine group is much lower than that in the cyclosporine group (P = 0.027). In regard to the small number of cases and short follow-up time in our this study, the more patients and longer follow-up time are needed to study.</p>

The effect of apoptosis induced by IL-21 in SUDHL-4 cell line and its mechanism / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the apoptosis effect of diffuse large B-cell lymphoma cell line (DLBCL) SUDHL-4 induced by IL-21 and its related mechanism.</p><p><b>METHODS</b>SUDHL-4 cells were treated with IL-21 at different concentration (1000 ng/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml) for 24 h, 48 h, 72 h, respectively. The inhibitory rate of cell proliferation was detected by CCK-8 assay. The cell growth curves were drawn and half inhibitory concentration (IC(50)) values were calculated. The cell apoptosis were detected by flow cytometry (FCM), the expression of the caspase-9, caspase-3, cleaved caspase-3, Bcl-2, Bcl-XL, Bid, Bax and c-myc protein in SUDHL-4 cells treated with IL-21 by western blot, the mRNA expression of Bcl-2, Bcl-XL, Bid, Bax, c-myc by Survivin gene with RT-PCR.</p><p><b>RESULTS</b>IL-21 markedly inhibited SUDHL-4 cell growth in a time- and dose-dependent manner. The 48 hIC(50) was 140.9ng/ml; The FCM showed that the apoptosis proportion of SUDHL-4 cells treated with 100 ng/ml of IL-21 apoptosis (AnnexinV-FITC(+) positive cells) gradually increased (48 h: 19.7 ± 2.3%). The protein expression of caspase-9, caspase-3, Bcl-2 and Bcl-XL decreased in a time-dependent manner. The Bax and c-myc protein markedly increased, but the Bid protein level did not change. IL-21 up regulated c-myc and Bax gene expression, however down regulated Bcl-2 and BCL-XL gene expression, but the gene expression of Bid and Survivin hadn't been changed significantly.</p><p><b>CONCLUSIONS</b>IL-21 can inhibit proliferation and induce apoptosis of SUDHL-4 cell. The mechanism may involve in endogenous mitochondrial pathway mediated by the c-myc and the Bcl-2 genes.</p>

Silencing of survivin gene in Jeko-1 cell line with small interfering RNA / 中国实验血液学杂志

This study was aimed to investigate the impact of specific siRNA on survivin gene in transfected lymphoma cell line and provide experimental evidences for future treatment of mantle cell lymphoma. The small interfering RNA (siRNA) targeted survivin mRNA was synthesized in vitro and was transfected into Jeko-1 that showed high survivin expression in mRNA level. The levels of survivin mRNA and protein expression were detected by quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot respectively. The apoptosis effect was examined by calculating the ratio of Annexin V-FITC/PI positive cells using flow cytometry. The inhibition of cell proliferation was assayed with CCK-8 reagent after transfection. The results showed that expression of survivin mRNA was markedly suppressed by the siRNA. The relative expression levels were 0.49 ± 0.03, 0.38 ± 0.02 and 0.17 ± 0.02 at time points of 24, 48 and 72 h respectively, compared with the control group; the inhibitive rates of cell proliferation were (31.2 ± 2.1)%, (43.3 ± 3.4)% and (52.6 ± 2.5)%; the apoptotic rates of cells were (6.3 ± 0.5)%, (13.5 ± 1.1)% and (23.6 ± 1.6)% respectively; survivin protein expression levels were gradually reduced. It is concluded that the siRNA targeting survivin down-regulates the expressions of survivin mRNA and protein evidently. The siRNA of survivin displays the potent ability to inhibit the proliferation of lymphoma cell line Jeko-1; survivin may become a potential molecular target for the therapy of lymphoma in the future.

Effect of hypoxia on HIF-1α and its sumoylation in Jurkat cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of hypoxia on sumoylation of HIF-1α, as well as transcriptional activity and protein stability of HIF-1α in Jurkat cells, and explore its effect and significance on modulating the transcriptional activity of down stream target gene.</p><p><b>METHODS</b>CoCl(2) was used as a chemical inducer to simulate the hypoxia environment. Real-time fluorescence quantitative PCR and western blot were used to evaluate transcriptional activity and protein stability of HIF-1α, levels of SUMO-1 and SENP1 protein, and gene transcripts of VEGF, mdr1, mdr3, Mcl-1 and survivin respectively.</p><p><b>RESULTS</b>After 4 h, 8 h, 16 h, 24 h and 72 h after hypoxia induction, the gene transcripts of HIF-1α were 0.79 ± 0.19, 2.65 ± 2.05, 4.19 ± 4.72, 2.77 ± 3.37, 0.09 ± 0.05 and 0.69 ± 0.55-fold (P > 0.01) of that of 0h in Jurkat cells, respectively, while the protein stability increased first, then decreased (P < 0.01). SENP-1 protein up-regulated first, then down-regulated, and the SUMO-1 protein changed in an opposite trend. Excepting for survivin gene, transcriptional activities of VEGF, mdr1, mdr3, and Mcl-1 were affected by the stability of HIF-1α protein.</p><p><b>CONCLUSION</b>Hypoxia induces changes in SENP-1 expression, which increases the stability of HIF-1α by decreasing the sumoylation of HIF-1α and affects biological process by regulating the transcriptional activities of VEGF, mdr1, mdr3 and Mcl-1 gene.</p>

Effect of VEGFR1 gene silencing by shRNA on proliferation, migration and apoptosis of U937 line / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of lentivirus-mediated RNA interference targeting vascular endothelial growth factor receptor 1 (VEGFR1) gene on the proliferation, migration and apoptosis of leukemic cell line U937.</p><p><b>METHODS</b>Short hairpin RNAs (shRNA) targeting VEGFR-1 was synthesized and cloned into pRNAT-U6.2 lentiviral vector. The expression vectors were transfected into 293T cell line to produce packaged lentivirus. After infected with the packaged lentivirus, the expression of VEGFR-1 gene of U937 cells at mRNA and protein level was detected by real-time PCR and Western blot. VEGF production by the cells was determined by ELISA. Cell proliferation and survival under regular culture and in the presence of cytarabine (Ara-C) was determined by CCK-8 assay. Migration assays were performed by 5 µm pore transwell inserts.</p><p><b>RESULTS</b>The lentiviral shRNA vector targeting VEGFR-1 was successfully constructed and transfected into U937 cells. The shRNA vector effectively inhibited the expression of VEGFR-1 gene in U937 cell line at mRNA and protein levels. As compared to that of the control, the proliferation rate of U937-shVEGFR-1 cells reduced; The VEGF production and migrated cell number of U937-shVEGFR-1 cells decreased dramatically. After treated with Ara-C, the inhibition rate and apoptotic rate of U937-shVEGFR-1 cells increased significantly. The number of migrated cells in the KD group under regular culture and in the presence of VEGF was markedly lower than that in the NC group and CON group. Bevacizumab could decrease the number of migrated cells in the NC group and CON group, but could not in the KD group.</p><p><b>CONCLUSIONS</b>Lentivirus-mediated RNA interference targeting VEGFR1 gene reduces the proliferation, migration of U937 cell line and enhances its sensitivity to Ara-C.</p>

Preliminary study of VEGF and its receptor expression on childhood acute lymphoblastic leukemia and its relativity to clinical manifestations / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between differential expression of VEGF and its receptors and clinical characteristics of childhood acute lymphoblastic leukemia (ALL).</p><p><b>METHODS</b>Expressions of VEGF and its receptors (Flt-1, KDR) were assayed by ELISA and RT-PCR in healthy donors(20 cases), ALL patients in remission (20 cases), with low risk (29 cases) and with high risk (10 cases). The clinical data of all the patients and volunteers enrolled in this study were collected and analyzed according to the expression of VEGF and its receptors.</p><p><b>RESULTS</b>The expressions of VEGF were (574.37 +/- 208.45) ng/L, (387.93 +/- 175.86) ng/L, (135.80 +/- 111.28) ng/L and (91.16 +/- 41.34) ng/L in patients with high risk, standard risk, in remission and healthy donors, respectively. Expression levels of VEGF receptors were downwards with risk grades. The clinical manifestations were also in accord with the expression levels of VEGF and its receptors.</p><p><b>CONCLUSION</b>ALL patients with highly expressed VEGF and its receptors are usually with higher tumor burden, and refractory treatment. Detection of VEGF and its receptors might be one of prognostic marker for ALL treatment.</p>

Significance of TPMT activity and TGNs level detection for individualizing 6-mercaptopurine chemotherapy / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the activity and genotype of thiopurine methyltransferase (TPMT) and the concentration of thioguanine nucleotides (TGNs) as parameters for individualizing mercaptopurine (6-MP) therapy.</p><p><b>METHODS</b>Erythrocyte TPMT activity was measured by means of radiochemical assay. Sequence specific primer (SSP) PCR and restriction fragment length polymorphism (RFLP) were used to determine the mutations in TPMT genomic DNAs. Erythrocyte TGNs concentration in acute lymphoblastic leukemia (ALL) patients after 6-MP treatment was detected by high-performance liquid chromatography (HPLC).</p><p><b>RESULTS</b>The erythrocyte TPMT activity in Han population was 16.6 +/- 4.5U/ml pRBCs (man 16.8 +/- 5.0 U/ml pRBCs, woman 16.5 +/- 4.4 U/ml pRBCs), the activity in 8.1% of the samples was lower than 10 U/ml pRBCs. There was no difference for TPMT activity by gender, age, and between healthy donors and ALL patients. For TPMT genotypes, there were 5 cases of TPMT * 2, 4 TPMT * 3A, 6 TPMT * 3B, 10 TPMT * 3C and 5 unknown in 30 subjects who had low TPMT activity. In children with ALL, the TGNs level did show a negative correlation with TPMT activity at diagnosis and 7 approximately 14 days after 6-MP therapy and with WBC count 14 days after the determination of TGNs.</p><p><b>CONCLUSION</b>Detection of TPMT activity before 6-MP therapy and TGNs level during 6-MP therapy may be helpful for preventing side effects from antipurine metabolic drug overdose, and individualizing 6-MP chemotherapy in children with ALL.</p>