[CLAG Regimen Composed of Continuous Intravenous Infusion of Cladribine in the Treatment of Refractory/Relapsed Acute Myeloid Leukemia].

OBJECTIVE: To study the efficacy and safety of continuous intravenous infusion of 2-Chlorodeoxyadenosine (2-CdA) combined with high-dose cytarabine (Ara-C) and granulocyte colony-stimulating factor (G-CSF) (CLAG regiem) in the treatment of relapsed/refractory acute myeloid leukemia (AML). METHODS: Fifteen patients with refractory/relapsed AML hospitalized in 5 medical units such as Department of Hematology, the Affiliated Tumor Hospital of Zhengzhou University and received one course of CLAG regimen from June 2014 to August 2019 were analyzed retrospectively (specifically: cladribine 5 mg/M2, day 1 to day 5, continuous 24-hour intravenous infusion; Ara-C 2 g/M2, 1 time/day, day 1 to day 5, intravenous infusion; G-CSF 300 mg, 1 time/day, day 0 to day 5, subcutaneous injection). RESULTS: Among the 15 patients with refractory/relapsed AML, 9 males and 6 females, the median age was 35 (13-63) years old. FAB classification: 1 case of M1, 3 cases of M2a, 4 cases of M2b (including 1 case with extramedullary invasion), 1 case of M4 with extramedullary invasion, 5 cases of M5, 1 case of HAL; NCCN classification: 6 cases in intermediate risk group, 9 cases in high risk group; 8 cases refractory, 7 cases relapsed. The median time of pre-chemotherapy was 4 (2-8) (of which NO.15 had received 8 cycles of chemotherapy and received CLL1-CAR-T), and the median white blood cell count before chemotherapy was 12.27 (from 0.78 to 5.29)×109/L. After 1 course of treatment with CLAG regimen, 12 patients achieved complete remission (12/15, 80%), and the median duration of CR was 65 days (0-528) days. IV grade leukopenia and thrombocytopenia was found in all the patients after chemotherapy. The median duration of granulocytosis was 20 (14 to 33) days, and 1 patient died. Seven patients received allogeneic hematopoietic stem cell transplantation. The median EFS and OS time of 15 patients was 85 (19-558) days and 117 (19-558) days, respectively. CONCLUSION: The CLAG regimen consisting of continuous intravenous infusion of cladribine shows high CR in the treatment of AML patients, but the duration of CR is short, myelosuppression is sever, so that infection control is the key. Allogeneic hematopoietic stem cells transplantation should be performed as soon as possible after CR.

[Synergistic Mechanism of Interferon alpha-1b, Interleukin-2 and Thalidomide for Immune Regulation in Patients with Acute Myeloid Leukemia].

OBJECTIVE: To explore the synergistic immunomodulatory mechanism of interferon alpha-1b, interleukin-2 and thalidomide (ITI) regimen on patients with acute myeloid leukemia (AML). METHODS: Sixty eight untreated de novo or relapsed or refractory or maintenance therapy patients with AML admitted in the Affiliated Cancer Hospital of Zhengzhou University and the other 11 medical units from March 2016 to May 2019 were treated with ITI regimen. Peripheral blood specimen per patient was collected into EDTA-K3 anticoagulation vacuum tube before the administration of ITI and 3 months after the treatment; peripheral blood lymphocyte subsets and perforin and Granzyme B expression were analyzed by using flow cytometry; the levels of VEGF, IFN-γ, TNF-α and IL-6 in the plasma were detected by using a cytometric bead array. Thirty-five healthy subjects from the hospital physical examination centre were selected as normal controls. RESULTS: The ratio of CD4+/CD8+ T cells, the percentage of NK cells, the expression of perforin and Granzyme B of NK cells in the peripheral blood of patients with hematological malignancies were lower than those of healthy controls. The level of VEGF, IL-6 and TNF-α in the peripheral plasma were higher than those of the healthy control group, and the difference was statistically significant. The level of IFN-γ was lower, and the difference was not statistically significant. The ratio of CD4+/CD8+ T cells, the percentage of NK cells, the expression of Granzyme B and Perforin of NK cells in peripheral blood were higher after the therapy of thalidomide combined with rhIFNα-1b for 3 months as compared with those before treatment of ITI, the level of the IFN-γ in peripheral plasma was higher while that of VEGF was lower, the difference was statistically significant; after treatment, the ratio of CD3+ CD4+ and CD3+ CD8+ lymphocytes and the level of TNF-α in peripheral blood were higher those that before treatment, IL-6 was lower, while the difference was not statistically significant. CONCLUSION: The ITI regimen can raise the ratio of CD4+/CD8+ T cells and the percentage of natural killer cells, also, can enhance the generation of perforin and granzyme B and the concentration of IFN-γ as well as inhibit the generation of VEGF, suggesting that these activities may enhance the antitumour capacity of patients with AML.

[Effect of RNA Interference-silenced TAK1 on Kasumi-1 cell Proliferation Inhibition Induced by As2O3 and Its Mechanism].

OBJECTIVE: To explore the effect of transforming growth factor-ß activated kinase-1(TAK1) gene silenced by RNA interference on proliferation inhibition of Kasumi-1 cells induced by As2O3 and its mechanism. METHODS: The experiments were divided into 4 groups, including control group(Kasumi-1 cells treated with non-specific siRNA), TAK1 specific siRNA treated group (Kasumi 1 treated with TAK specific siRNA), As2O3 treated group (Kasumi 1 cells treated with As2O3) and combined treated group (Kasumi 1 cells treated with TAK1 specific siRNA plus As2O3). The proliferation inhibition rate of Kasami 1 cells was detected by CCK-8 method, the apoptotic rate of cells was detected by flow eytometry, the expressions of TAK1, phosphorylated c-Jun N-terminal kinase(p-JNK) and apoptosis-related proteins were detected by Western blot. RESULTS: As2O3 could inhibit Kasumi-1 cell proliferation in a dose-dependent manner between 0.5 to 20 µmol/L with IC50 of (3.79±0.36) µmol/L at 24 h, and also inhibit Kasumi-1 cell proliferation in a dose-dependent manner between 0.5 to 10 µmol/L with IC50 of (2.38±0.17) µmol/L at 48 h, but then the inhibitory effect reached plateau. After treating Kasumi-1 cells with TAK1 siRNA and 3.5 µmol/L As2O3 for 24 h, the proliferation inhibition rate was (10.86±1.64)% and (49.80±2.19)%, meanwhile the apoptosis rate was (8.47±0.75)% and (24.78±2.14)%, all significantly higher than those in control group (P<0.05, P<0.01). The proliferation inhibition rate and apoptosis rate of the combined treated group were significantly higher than that in control and single treated groups (P<0.05, P<0.01), TAK1 silencing and 3.5 µmol/L of As2O3 could decrease the expression of TAK1, p-JNK, c-Fos, c-Jun and BCL-2 in different degrees, and increase the expression levels of BAX and the activated (cleaved) caspase-3, 9 with statistically significant differences as compared with control group (P< 0.05). When Kasumi-1 cells were treated with TAK1 specific siRNA plus As2O3 for 24 h, protein expression levels were all significantly greater than that in the single-treated groups (P< 0.05). CONCLUSION: TAK1 silencing and As2O3 can separately and synergistically inhibit Kasumi-1 cell proliferation which probably relates with the inducing apoptosis via the JNK and mitochondrial pathway. Meanwhile, TAK1 silencing enhances the inhibitory effect of As2O3 on Kasumi-1 cell proliferation.

Efficacy and Safety of Danshen Compound Tablets in Preventing Thalidomide-Associated Thromboembolism in Patients with Multiple Myeloma: A Multicenter Retrospective Study.

BACKGROUND Currently available antithrombotic prophylaxis is not perfectly reliable in elderly patients. The aim of this retrospective study was to evaluate the efficacy and safety of Compound Danshen Tablet (CDT) in preventing thromboembolism in multiple myeloma (MM) patients treated with thalidomide-based regimens. MATERIAL AND METHODS MM patients treated with thalidomide-based regimens were retrospectively reviewed between January 2008 and March 2015. Patients were categorized into 3 cohorts based on thromboembolic prophylaxis used: CDT, Warfarin Tablet, and no prophylaxis. Venous thromboembolism (VTE), other adverse effects (AEs), and the changes of D-dimer and fibrinogen levels were monitored. RESULTS Seven out of 313 MM patients (2.24%) developed venous thrombosis events (VTE) in this retrospective study, all clustering in the no prophylaxis cohort. Three patients of the Warfarin cohort (3.19%) experienced hemorrhage. Neither VTE events nor serious AEs were observed in the CDT cohort. Following Compound Danshen or Warfarin treatment for 3 months, the D-dimer and fibrinogen levels (in particular the D-dimer level) (all P<0.05), were obviously decreased relative to their respective baselines and the no prophylaxis cohort. In contrast, the 2 blotting parameters were significantly increased in the no prophylaxis cohort relative to the baseline level (All P<0.05), and were even higher in the patients experiencing VTE compared to the no VTE patients (P<0.0001 and P=0.016, respectively). CONCLUSIONS Our findings indicate CDT is an effective therapy for preventing VTE in MM patients treated with thalidomide-based regimens, and is well tolerated in long-term use.

[Effects of As2O3 in combination with TPA on K562 cells].

This study was aimed to investigate the effects of arsenic trioxide (As2O3) combined with TPA on cell cycle, cell differentiation and apoptosis of K562 cell line, and their possible mechanisms. K562 cells were treated with 200 nmol/L TPA, 2 µmol/L As2O3 alone and 200 nmol/L TPA combined with 2 µmol/L As2O3. The proliferative inhibition rates were determined with CCK-8. Annexin V and agarose gel electrophoresis were adopted to detect apoptosis. Colony formation test was used to determine the colony-formation efficiency. Flow cytometry was used to detect the cell differentiation and cell cycle changes. Western blot was employed to detect the expression of P38 and p-P38 proteins. The results showed that combination treatment had synergistic effects on the proliferative inhibition and apoptosis, which were much higher than those treated alone. As2O3 could decrease the colony formation ability of K562 cells. The cells treated with both TPA and As2O3 expressed far more CD11b antigens compared with cells exposed to As2O3 alone. K562 cells treated with TPA were arrested in G1 phase compared with the control group, As2O3 increased the percentage of K562 cells in the G2 phase. The combination treatment increased the expression of p-P38 of K562 cells compared with the cells exposed As2O3 alone. It is concluded that TPA can enhance the effect of As2O3 on inducing apoptosis and adjusting cell cycle , which will expect to provide a new therapeutic program.

[Clinical study of COAD-B regimen in treatment of patients with relapsed/refractory non-Hodgkin lymphoma].

OBJECTIVE: To investigate the efficacy, adverse events and long-term survival of cyclophosphamide, vindesine, cytarabine, dexamethasone and bleomycin (COAD-B) regimen for relapsed and refractory non-Hodgkin lymphoma (NHL). METHODS: Eighty six patients diagnosed with relapsed or refractory NHL were included in our study from January 2007 to January 2013. The chemotherapy regimen was COAD-B, the therapeutic efficacy was evaluated every 2 courses. Once the stable disease (SD) or progress of the disease (PD) achieved, the patients would switch to other second-line regimens. RESULTS: The overall response rate (ORR) was 67.4%, median remission duration was 13 months (3-51 months); 1-,2- and 4-year overall survival (OS) rates were 75.4%, 56.8% and 40.0%, respectively; 1-, 2- and 4-year progression-free survival (PFS) rates were 50.3%, 39.4% and 27.5%, respectively. The main adverse reaction of patients was myelosuppression. The response to chemotherapy and long- term survival of the relapsed patients were significantly better than that of the refractory ones, and the difference had statistical significance. CONCLUSION: COAD-B could be the salvage regimen for relapsed and refractory NHL.

[Detection of NPM1, FLT3 and C-KIT mutations in acute myeloid leukemia and their prognostic analysis].

This study was aimed to evaluate the frequencies and prognostic significance of the nucleophosmin 1 (NPM1) mutation, the fms-like tyrosine kinase 3 (FLT3) mutation and c-KIT mutation in acute myeloid leukemia (AML) and to explore their relevance to clinical characteristics, cytogenetics and survival. Genomic DNA from 78 newly diagnosed AML from August 2010 to October 2012 was screened by PCR and sequencing or capillary electrophoresis (CE) for NPM1, FLT3 and c-KIT mutations. The results showed that the incidence of NPM1 mutation was 14.1% in AML patients and 26.7% in normal karyotype AML patients. NPM1 mutant cases were significantly associated with old age (P < 0.05), high peripheral white cell count and platelet counts (P < 0.05) and low expression of CD34 (P < 0.05), but no statistic difference was found in sex, percentage of bone marrow blasts, Hb, expression of CD117 and HLA-DR, complete remission rate, overall survival and relapse rate (P > 0.05). The prevalences of FLT3-ITD and FLT3-TKD mutations were 11.5% (9/78) and 3.8% (3/78) respectively, and no one patient has both of the two mutations. Patients with FLT3-ITD mutation had higher white blood cell counts and percentage of in bone marrow blasts (P < 0.05), and lower overall survival (P < 0.05), more relative to normal karyotype (P < 0.05), while no statistic difference was found in sex, age, platelet count, Hb level, complete remission rate and relapse rate (P > 0.05). No statistic analysis was performed due to the cases of less FLT3-TKD mutation. C-KIT mutation accounts for 7.7% (6/78). Patients with C-KIT mutation had a higher percentage in abnormal karyotype (P < 0.05), and higher relapse rate (P < 0.05), and lower overall survival, whereas no statistic difference was found in sex, age, percentage of bone marrow blasts, peripheral blood cell count, complete remission rate (P > 0.05). It is concluded that the detection of NPM1, FLT3 and C-KIT mutations may contribute to guiding treatment and evaluating prognosis of patients with AML.

[Effect of TAK1 gene silencing on the apoptosis of Kasumi-1 cells induced by arsenic trioxide].

OBJECTIVE: To study the effect of transforming growth factor-ß activated kinase-1 (TAK1) gene silencing on the proliferation and apoptosis of Kasumi-1 cells induced by arsenic trioxide (As2O3). METHODS: Acute myeloid leukemia with t(8;21) cell line Kasumi-1 cells were treated with As2O3 or in combination with TAK1 siRNA interference technology. The experiment was divided into four groups: Kasumi-1 cells without any treatment, TAK1 specific siRNA transfection alone, Kasumi-1 cells treated with different concentration of As2O3, TAK1siRNA transfection combined with As2O3. CCK-8 was used to detect the cell viability. The expression of phosphorylated c-Jun N-terminal kinase (P-JNK) was determined by Western Blot. Cell apoptosis and growth were examined by morphological and colony formation assay. RESULTS: After Kasumi-1 cells were treated with As2O3, the rate of cell inhibition was concentration-dependent, and the 50% inhibitory concentration was 3.5 µmol/L. The highest expression level of P-JNK appeared in 30 minutes after cells were treated with As2O3. The apoptosis rates of Kasumi-1 cells without any treatment, TAK1 siRNA interference alone group, As2O3 alone group and the combined group were (5.02 ± 1.13)%, (6.18 ± 0.28)%, (48.33 ± 2.70)% and (86.07 ± 2.21)%; colony formation rates were (73.83 ± 2.78)%, (76.03 ± 1.46)%, (55.07 ± 1.50)% and (22.20 ± 1.15)%; apoptosis rate of TAK1 siRNA group and the untreated group has no significant difference (P = 0.052); colony formation rate between TAk1 siRNA group and the untreated group has no significant difference (P = 0.179), but the difference in other groups was significant (P = 0.000). CONCLUSION: Silencing the expression of TAK1 can enhance the anti-proliferative and pro-apoptotic effect of As2O3 on Kasumi-1 cells, and its mechanism may be through the TAK1 downstream JNK signal pathway.

[Clinical significance of dynamic monitoring of thymic recent output function in different stages of treatment in patients with diffuse large B-cell lymphoma].

OBJECTIVE: To detect the changes of naive T cell level of thymic recent output at different stages of treatment in patients with diffuse large B-cell lymphoma (DLBCL), thereby to evaluate the relationship of thymic recent output function with prognosis and the impact of chemotherapy on the potential of immunological recovery. METHODS: The levels of T-cell receptor rearrangement excision circles (TREC) in DNA of peripheral blood mononuclear cells (PBMNC) from 30 DLBCL patients were monitored before, during, until 3 months and 6 months after chemotherapy by real-time PCR (TaqMan), and TREC-level was detected according to the number of CD3 positive(CD3(+)) cells. Twelve normal individuals who matched in age were served as controls. RESULTS: There was a dramatic reduction of TREC values in all DLBCL patients among which TREC values in germinal center B-cell-like-DLBCL (GCB-DLBCL) were higher than those in non-GCB-DLBCL, as compared with TREC values of normal individual in peripheral blood. The mean values of TREC were 0.91 ± 0.15/1000 PBMNCs and (1.22 ± 0.69)/1000 CD3(+) cells in GCB-DLBCL, (0.43 ± 0.29)/1000 PBMNCs and (0.64 ± 0.44)/1000 CD3(+) cells in non-GCB-DLBCL before chemotherapy. TREC values were significantly associated with lower international prognostic index (IPI) grade (r = -0.441, P = 0.015). TREC-level in DLBCL patients was further decreased after chemotherapy, and reached to the lowest level after the 6th cycle of chemotherapy, and during the corresponding period, the mean values of TREC were (0.63 ± 0.34)/1000 PBMNCs and (0.89 ± 0.65)/1000 CD3(+)cells in GCB-DLBCL, (0.19 ± 0.11)/1000 PBMNCs and (0.27 ± 0.25)/1000 CD3(+) cells in non-GCB-DLBCL. TREC-level began to rise obviously 3 months after the last cycle of chemotherapy in most of the DLBCL patients, and came close to normal level in five cases of patients 6 months after the last cycle of chemotherapy. CONCLUSIONS: Thymic recent output function was impaired severely in DLBCL patients. There was an important relationship between thymic recent output function before chemotherapy and prognosis, and chemotherapy had influenced the potential of immunological recovery.

[Clinical significance of IDH1 and IDH2 mutations in patients with acute myeloid leukemia].

OBJECTIVE: To assess the frequencies and prognostic significance of the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) mutations in acute myeloid leukemia (AML) and to explore their relevance to clinical, cytogenetic and molecular feature. METHODS: Genomic DNA from 96 newly diagnosed AML patients from Sep. 2009 to Jan. 2011 was screened by RT-PCR and sequencing for IDH1 and 1DH2 mutation. RESULTS: The prevalence of IDH1 (p. P127 and p. I130) and IDH2 mutations (p. R140) was 14.6% (14/ 96) and 2.17% (2/96) respectively. The IDH1 mutations of p. P127 and p. I130 were not reported so far in literature. Of 14 IDH1 mutation patients, 10 were with normal karyotype and the differences had statistical significance (P=0.021). Two patients with IDH2 mutation were also with normal karyotype. IDH2 mutations were in older patients at diagnosis. Patients with IDH mutation had higher white blood cell counts, lower platelet counts, expression of HLA-DR, CD34, CD33 and CD13, lower remission rate and higher relapse rate. CONCLUSION: IDH mutation is recurring genetic change in AML and indicates poor prognosis.