[Effect of Hemoglobin on Efficacy of CAR-T Therapy in Patients with Multiple Myeloma].

OBJECTIVE: To investigate the effect of hemoglobin (Hb) on the efficacy of chimeric antigen receptor T cell therapy (CAR-T) in patients with multiple myeloma (MM). METHODS: From June 2017 to December 2020, 76 MM patients who received CAR-T therapy in the Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, with complete clinical data and evaluable efficacy, were selected as the research objects. According to the receiver operating characteristic (ROC) curve, the best cut-off value was obtained. The patients were divided into groups on the basis of Hb 105.5 g/L as the cut-off value. The age, sex, serum calcium, ß2-microglobulin, serum creatinine, lactate dehydrogenase (LDH), and the influencing factors of CAR-T treatment efficacy in MM patients were analyzed. RESULTS: Hb was an influencing factor of efficacy. Univariate analysis showed that Hb, LDH, and albumin affected the efficacy of CAR-T therapy. Multivariate analysis showed that Hb ( OR=1.039, 95% CI: 1.002-1.078) and LDH ( OR=1.014, 95% CI: 1.000-1.027) were the influencing factors for the efficacy of CAR-T therapy. CONCLUSION: The efficacy of CAR-T therapy in MM patients with low Hb is poor, and Hb is a factor affecting the efficacy of CAR-T therapy.

Clinical efficacy of recombinant human thrombopoietin combined with glucocorticoids in the treatment of immune thrombocytopenia.

OBJECTIVE: Herein, we aimed to determine the clinical efficacy of recombinant human thrombopoietin (rhTPO) combined with glucocorticoids for treating immune thrombocytopenia (ITP). METHODS: Clinical data of 87 patients with ITP admitted to our hospital were retrospectively analyzed, and patients were divided into two groups according to the treatment employed: 42 patients in the control group (CG) were prescribed glucocorticoids, and 45 patients in the study group (SG) received rhTPO combined with glucocorticoids. RESULTS: The total effective treatment rate in the SG (95.56%) was higher than that in the CG (76.19%) (P < 0.05). The SG achieved a platelet (PLT) count > 50 × 109/L faster and required fewer PLT transfusions than the CG (P < 0.05). At 1, 7, and 14 days after treatment, the PLT count increased in both groups and was higher in the SG than in the CG (P < 0.05). After treatment, CD3+, CD4+, and CD4+/CD8+ T cells increased, whereas CD8 + decreased in both groups, with the SG exhibiting a superior improvement to the CG (P < 0.05). Considering prothrombin time, activated partial thromboplastin time, and fibrinogen, differences between the two groups were not statistically significant, both before and after treatment (P > 0.05). CONCLUSION: rhTPO combined with glucocorticoids for treating ITP can effectively enhance the therapeutic effect, regulate the T lymphocyte subpopulation, rapidly increase the PLT level, and induce no significant effect on the coagulation function of patients, with good safety and high clinical promotion value.

Cellular Immunotherapies for Multiple Myeloma: Current Status, Challenges, and Future Directions.

Multiple myeloma (MM) remains incurable due to relapse, although the use of proteasome inhibitors, immunomodulatory drugs, CD38-targeting antibodies, and autologous stem cell transplantation (auto-SCT) significantly improve the clinical outcomes of patients with newly diagnosed MM. In recent years, the introduction of chimeric antigen receptor T-cell (CAR T-cell) therapy has brought hope to patients with refractory and relapsed MM. The graft-versus-myeloma effect of allogeneic SCT provides the possibility for curing a subset of MM patients. In this review, we summarize the recent advances and challenges of cellular immunotherapies for MM, focusing on auto-SCT, allogeneic SCT, and CAR T-cell approaches. We also discuss future directions, and propose a specific algorithm for cellular therapies for MM and probability of minimal residual disease-directed therapy.

Donor-derived CD19 CAR-T cell therapy of relapse of CD19-positive B-ALL post allotransplant.

Safety and efficacy of allogeneic anti-CD19 chimeric antigen receptor T cells (CAR-T cells) in persons with CD19-positive B-cell acute lymphoblastic leukemia (B-ALL) relapsing after an allotransplant remain unclear. Forty-three subjects with B-ALL relapsing post allotransplant received CAR-T cells were analyzed. 34 (79%; 95% confidence interval [CI]: 66, 92%) achieved complete histological remission (CR). Cytokine release syndrome (CRS) occurred in 38 (88%; 78, 98%) and was ≥grade-3 in 7. Two subjects died from multiorgan failure and CRS. Nine subjects (21%; 8, 34%) developed ≤grade-2 immune effector cell-associated neurotoxicity syndrome (ICANS). Two subjects developed ≤grade-2 acute graft-versus-host disease (GvHD). 1-year event-free survival (EFS) and survival was 43% (25, 62%). In 32 subjects with a complete histological remission without a second transplant, 1-year cumulative incidence of relapse was 41% (25, 62%) and 1-year EFS and survival, 59% (37, 81%). Therapy of B-ALL subjects relapsing post transplant with donor-derived CAR-T cells is safe and effective but associated with a high rate of CRS. Outcomes seem comparable to those achieved with alternative therapies but data from a randomized trial are lacking.

[Expression and Significance of PD-1, TIM-3 and VISTA on T Cell of Acute Myeloid Leukemia Patients].

OBJECTIVE: To study the expression of multiple negative costimulatory molecules on peripheral blood T cells in patients with acute myeloid leukemia (AML) and its affection on prognosis. METHODS: The peripheral blood samples from patients with newly diagnosed AML, complete remission (CR), and no-remission (NR) were collected, the expression levels PD-1、VISTA and TIM-3 in CD4+ and CD8+ T cells were detected by flow cytometry , and the clinical data of patients were analyzed. RESULTS: The expression levels of PD-1、VISTA and TIM-3 of CD4+ and CD8+ T cells in the newly diagnosed AML patients were significantly higher than those in control group (P＜0.05). The expression levels of PD-1、TIM-3 and VISTA of CD4+ and CD8+ T cells in the CR group were significantly lower than those in newly diagnosed and the NR group (P＜0.05). The TIM-3 expression level positively correlated with VISTA expression level of CD4+ and CD8+ T cells in newly diagnosed AML patients (r=0.85 and 0.73). The VISTA and PD-1 expression level of CD4+ T cells in newly diagnosed AML, NR after first induction chemotherapy and high risk patients significantly increased (P＜0.05), the TIM-3 expression level of CD8+ T cells in high risk group significantly increased (P＜0.05), and the VISTA expression level of CD8+ T cells in CBFß-MYH11 mutation-positive group significantly decreased (P＜0.05). CONCLUSION: The expression of PD-1、TIM-3 and VISTA in AML peripheral blood T cells may be involved in the immune escape of AML and can be the targets of treatment for acute myeloid leukemia patients.

Roles of T875N somatic mutation in the activity, structural stability of JAK2 and the transformation of OCI-AML3 cells.

Activating mutations in JAK2 have been described in patients with various hematologic malignancies including acute myeloid leukemia (AML) and myeloproliferative neoplasms. However, mechanism of these mutations in JAK2's activity, structural stability and pathology of AML remains poorly understood. The JAK2 T875N somatic mutation has been detected in about 5.2% of AML patients. But the structural basis and mechanism of JAK2 T875N mutation in the pathology of AML is still unclear. Our results suggested that JAK2 T875N mutation disrupted the T875 and D873 interaction which destroyed the compact structure of JH1 domain, forced it into the active conformation, facilitated the entrance of substrate and thus led to JAK2 hyperactivation. Mutations (T875N, T875A, D873A and D873G) disrupted the T875 and D873 interaction enhanced JAK2's activity, decreased its structural stability and JH2 domain's activity which further enhanced JAK2's activity, while mutations (T875R, D873E, T875R/D873E) repaired this interaction displayed opposite results. Moreover, JAK2 T875N mutation enhanced the activity of JAK2-STAT5 pathway, promoted the proliferation and transformation of OCI-AML3 cells. This study provides clues in understanding structural basis of T875N mutation caused JAK2 hyperactivation and its roles in the pathology of AML.

[Prognostic Significance of CD45dimCD117+ Cells in Patients with Acute Myeloid Leukemia after Complete Remission].

OBJECTIVE: To investigate the predictive value of CD45dimCD117+ phenotype-abnormal cells (hereinafter referred to as "abnormal cells") for relapse and prognosis in adult patients with acute myeloid leukemia (AML) within 2 weeks after the first complete remission (CR1). METHODS: The clinical data of patients with newly diagnosed AML (non-acute promyelocytic leukemia) admitted in our department from July 1, 2014 to June 30, 2017 were analyzed retrospectively, and the relationship between clinical features at the initial diagnosis and the abnormal phenotype cells of CD45dimCD117+ within 2 weeks after CR1 with the prognosis were analyzed. RESULTS: A total of 91 patients with CD45dimCD117+ abnormal cells were detected. The median age was 51 years old, the median WBC count was 11.60×109/L, and the median ratio of bone marrow blast cells was 0.35 at initial diagnosis. According to the FAB classification, 1 (1.1%), 7 (7.7%), 38 (41.7%), 20 (22.0%), 21 (23.1%) and 4 (4.4%) patients were classifice as M0, M1, M2, M4, M5, and M6, respectively. According to the NCCN risk stratification, 30 (33.0%), 51 (56.0%), and 10 (11.0%) patients were determined as good, moderate, and poor prognosis, respectively. The median ratio of abnormal cells within 2 weeks after CR1 was 1.8500 (0.0236-8.0000)%. The median time from initiation of induction therapy to the acquisition of CR was 46 days, median recurrence-free survival time was 319 days, and median overall survival time was 352 days. A total of 45 patients relapsed, of which 14 died; 46 patients did not relapse, of which 3 died. The cutoff of abnormal cells by receiver operating characteristic curve (ROC) analysis was 2.055% (Se=0.733，Sp=0.761). The abnormal cell ratio was＞2.055% in 44 patients, the median ratio of abnormal cells was 3.075%, among which 33 patients relapsed and 12 patients died; the abnormal cell ratio was ＜2.055% in 47 patients, the median ratio of abnormal cells was 1.150%, 12 patients relapsed and 5 patients died. Regression analysis showed that WBC count＞50×109/L and abnormal cell ratio＞2.055% were independent risk factors for recurrence. The abnormal cell ratio＞2.055% group had a 2-year RFS rate of 54.3% and a 2-year OS rate of 52.8%. The abnormal cell ratio＜2.055% group had a 2-year RFS rate of 86.6% (P=0.018), and a 2-year OS rate of 85.3% (P＜0.05). CONCLUSION: For adult AML patients, CD45dimCD117+ phenotypical abnormal cells ratio＞2.055% within 2 weeks after CR1 is an independent risk factor for recurrence, which also is an dverse factor for RFS and OS.

[Effect of Stably Down-regulating FMI Expression of K562 Cells on Sensitivity of K562 cells to Imatinib Mesylate].

OBJECTIVE: To investigate the effect of stably down-regulating the FMI expression of K562 cells on the sensitivity of K562 cells to Imatinib (IM) and its possible mechanism. METHODS: Western-blot was used to detect the expression of FMI protein in K562 cells and peripheral blood mononuclear cells from the patients with chronic myelogenous leukemia, chronic myeloid blast crisis and healthy volunteers. The specific interference sequences targeting at the human FMI gene were designed and ligated into the lentiviral vector LV3; the three plasmid system-packaged lentivirus particles were used to transfect K562 cells to screen K562 cells that stably down-regulated FMI. CCK-8 assay and flow cytometry were used to determine effect of IM on cell proliferation and apoptosis. The transcription level of FMI and Fz8 in leukemia cells was detected by fluorescent quantitative PCR. The protein expression levels of FMI, Fz8, NFAT1, BCR-ABL and ß-catenin in leukemia cells were detected by Western-blot. RESULTS: The expression of FMI protein could be detected in peripheral blood mononuclear cells of the patients with CML-BC and K562 cells, the FMI expression could not be detected in all the patients with CML-CP and healthy volunteers. The recombinant lentiviral vector LV3/FMI had been successfully constructed the lentivirus was packaged, and the K562 cells stably down-regulating the FMI protein were screened. After stable down-regulation of FMI expression in K562 cells, the proliferation rate of leukemia cells decreased and the apoptosis rate was increased under the same drug concentration. Both the transcription and protein expression levels of Fz8 decreased. The NFAT1 total protein level increased, as well as the nuclear translocation of protein was enhanced. There was no significant change in the expression level of BCR-ABL fusion protein. The expression level of ß-catenin protein decreased. CONCLUSION: After the stable down-regulation of FMI expression, the sensitivity of K562 cells to IM and apoptosis of cells increase, which are performed possibly by inhibiting the FMI-Fz8 signaling pathway and activating the Ca2+-NFAT and Wnt/ß-catenin signaling pathway.

[Role of Ca2+-NFAT Signaling Pathway in Ph+ ALL Drug-resistance Mediated by Bone Marrow Stromal Cells].

OBJECTIVE: To explore the role of Ca2+-NFAT signaling pathway in Ph+-ALL drug resistance mediated by bone marrow stromal cells. METHODS: The transcription level of NFAT mRNA in Sup-B15 cells and Ph+ ALL primary cells was detected by polymerase chain reaction. The expression of P-glycoprotein in Sup-B15 cells was detected by flow cytometry. The change of NFAT protein in Sup-B15 cells was detected by Western blot. AnnexinV/7-AAD was used to label cells. Flow cytometry was used to detect cell apoptosis; Fluo 3-AM dye was used to label cells, and flow cytometry used to detect changes of Ca2+ concentration in leukemia cells. RESULTS: NFAT expression could be detected in both Sup-B15 and Ph+ ALL primary cells; P-glycoprotein could not be detected by flow cytometry; CAS could significantly inhibit NFAT protein expression in clinically applied drug concentrations (2.5, 5 µmol/L); Clinically applied concentration of CAS (2.5, 5 µmol / L) has no significant effect on the apoptosis of Sup-B15 cells, while higher concentration of CAS (10 µmol / L) could induce apoptosis of Sup-B15 cells. Bone marrow stromal cells OP9 could, decrease the sensitivity of Sup-B15 cells and Ph+ ALL primary cells to imatinib (IM); After co-culture with bone were marrow stromal cells, the Ca2+ concentration in Sup-B15 cells was enhanced, the levels of NFAT protein and nullear protein in sup-B15 cells also were enhanced. The addition of CAS in co-culture system could inlibit the Ca2+-NFAT signaling pathway, reduce the protective effect of OP9 on Sup-B15 cells.Conclution:The Ca2+-NFAT sigualing pathway, contributes to the survival of Ph+ ALL cells. Bone marrow stromal cells can mediate the resistance of Ph+ ALL cells to IM by activating Ca2+-NFAT signaling pathway.

[Multivariate Analysis of Factors Influencing Recovery from Hemorrhagic Cystitis after Allo-HSCT].

OBJECTIVE: To analyze the incidence of hemorrhagic cystitis (HC) after allogeneic hematopoietic stem cell transplantation and the factors affecting HC, so as to provide clinical evidence for further treatment of HC. METHODS: The HC of 113 patients after allogeneic hematopoietic stem cell transplantation in Affiliated Hospital of Xuzhou Medical University between the years 2014-2016 was analyzed respectively. All cases of HC were divided into HC group and non-HC(control) group. The follow-up time: from preeonditionig day to 180 d after transplantation. The 10 clinical parameters were selected for univariate analysis with COX regression analysis: sex, age (＜25 years and 25 years), primary disease, conditioning regimen with anti-thymoglobulin(ATG), sex-mismatch in recipients, haploidential HSCT, cytomegalovirus (CMV) viremia, EB viremia, graft-versus-host disease (GVHD), and primary disease relapse, the factors significant at the 0.1 level in univariate analysis should be further evaluated by multivariate analysis using a COX regression analysis. The difference was significant at P＜0.05 in multivariate analysis. RESULTS: The HC occured in 31 of 113 patients (27.4%), with 5 cases of grade I (5.5%), 19 of grade II (16.8%), 5 of grade III (4.4%), and 2 of grade IV (1.8%). The median time of HC onset was 37 days (26-70 d) after transplantation. The median duration of HC was 14 days (5-55d). Univariate analysis showed that conditioning with anti-thymoglobulin (ATG) (RR=6.170, 95%CI: 1.875-20.306, P＜0.01), CMV viremia (RR=7.633, 95%CI：2.318-25.133) (P＜0.01), haploidentical HSCT (RR=0.307, 95%CI：0.137-0.686, P＜0.01), GVHD (RR=1.891, 95%CI：0.918-3.898, P＞0.05) were the risk factors for recovery from HC. The multivatiate analysis of above-mentioned risk factors with statistical significance showed that only CMV viremia (RR=4.770, 95%CI: 1.394-16.326, P＜0.05) was the indentified risk factor affecting the recovery from HC. CONCLUSION: Monitoring CMV viremia and antivirotic treatment are effective measurs to prevent the occurrence of HC and promote the recovery from HC.

Disruption of R867 and Y613 interaction plays key roles in JAK2 R867Q mutation caused acute leukemia.

Janus tyrosine kinase 2 (JAK2) mediates downstream signaling of cytokine receptors in all hematological lineages, constitutively active somatic JAK2 mutations were important for the leukemogenesis of acute leukemia (AL). The JAK2 R867Q somatic mutation is detected in a subset of AL patients. However, roles of JAK2 R867Q mutation in the pathogenesis of AL remain unclear. In this study, homology modeling analysis showed that loss of interaction between R867 and Y613 disrupted the JAK2 JH1/JH2 domain's interactions was responsible for its activation. JAK2 R867Q and mutations (R867A and R867G) abolished this interaction caused JAK2 constitutive activation. While, mutations (R867K, Y613E, R867K/Y613E) repairing this interaction reduced JAK2 R867Q mutation's activity. Furthermore, our studies showed that abolished R867 and Y613 interaction disrupted JH1/JH2 domains' interactions and led to JAK2 constitutive activation. More importantly, mutations (R867Q, R867A and R867G) disrupted this interaction enhanced the activity of JAK2-STAT5 pathway and the proliferation of Ba/F3 and MV4-11 cells. Further study showed that JAK2 R867Q mutation promoted the expression of proliferation marker and inhibited the differentiation marker of Ba/F3 and MV4-11 cells. Thus our studies provide clues in understanding the pathogenesis of JAK2 R867Q mutation in AL.

Loss of K607 and E877 interaction is a key reason for JAK2 K607N mutation caused acute myeloid leukemia.

Oncogenic activation of tyrosine kinase signaling pathway is recurrent in human leukemia. The acquired Janus kinase 2 (JAK2) K607N somatic mutation was detected in about 6.8% of acute myeloid leukemia (AML) patients. However, roles of JAK2 K607N mutation in the leukemogenesis of AML remain unclear. In this study, loss of interaction between K607 and E877 was identified as key reasons for JAK2 K607N mutation constitutive activation. JAK2 K607N and mutations (K607A, K607G and E877A) abolished the K607 and E877 interaction caused JAK2 constitutive activation. While, mutations (K607R, E877D) repairing this interaction reduced K607N mutation's activity. Furthermore, our studies showed that disruption of K607 and E877 interaction abolished JH1/JH2 domains' interactions and led to JAK2 constitutive activation. More importantly, JAK2 K607N and mutations disrupted this interaction enhanced JAK2-STAT5 pathway activation and the proliferation of Ba/F3 cells. Thus our studies provide clues in understanding the leukemogenesis of JAK2 K607N mutation in AML.

[Relationship between BCR-ABLIS and Prognosis and Affecting Factors for Prognosis in CML Patients Treated with TKI for 12 Months].

OBJECTIVE: To evaluate the long-term prognosis of CML patients whose BCR-ABL transcript level was warning and best response at 12 months of treatment with tyrosine kinase inhititor (TKI), and to investigate the factors affecting therapeutic efficacy and prognosis. METHODS: The clinical data of patients with newly diagnosed CML were analyzed retrospectively. According to BCR-ABL transcript level, the 80 patients were divided into group A and group B, the patients with BCR-ABLIS >0.1% and ≤ 1% (warning response) were entolled in group A, and the patients with BCR-ABLIS ≤ 0.1% (best response) were enrolled in group B as control. The ratio of patients with main molecular response (MMR) and deep molecular response (DMR), as well as aquistation rate and cummulative rate of MR4 (DMR) at specified fine points in 2 groups were compared, the independent risk factors affecting the therapeutic efficacy and prognosis were analyzed. RESULTS: The MMR and MR4 of the B group at 15, 18 and 24 months after TKI treatment were significantly higher than those of the A group, and the patients in the B group reached MR4 faster. In the 3 months, 6 months and 12 months after the demarcation point (TKI 12 months), the A group was much less easy to obtain MR4 (P<0.05) than the B group. Through survival analysis, there were more patients in the B group than the A group at different time points to reach MR4, and the difference was statistically significant (P<0.01). The single factor analysis showed that the splenomegaly (below rib edge)> 10cm (P<0.01) and lactate dehydrogenase > 400 U/L (P<0.05) were the long-term warning factors for patients. Multivariate analysis showed that the size of the spleen was an independent factor (P<0.01) to affect the prognosis of the patients who had been warned for 12 months. CONCLUSION: Patients at 12 months warning effect are slower and less easier to get DMR, which has a poor long-term prognosis. The size of the spleen in the patient at warning for 12 months of treatment effect can predict the relatively poor long-term prognosis. For a patient with a 12 months response to the warning, an early replacement therapy is available on the basis of combining other factors..

CT of ovarian cancer: 3D cinematic rendering for preoperative evaluation.

BACKGROUND: Ovarian cancer is the second most common gynecologic malignancy. As the primary imaging modality, computed tomography (CT) can provide staging information for preoperative planning and determination of surgical resectability. As a new three-dimensional postprocessing tool for CT images, cinematic rendering (CR) has the potential to depict anatomic details accurately. CASE PRESENTATION: (Case 1) A 44-year-old married woman was diagnosed with recurrent ovarian cancer. CT images indicated the recurrent nodules and masses in the pelvic cavity and the upper middle abdominal peritoneum. The CR image showed that the multiple metastatic lesions and lymph nodes could not be completely removed by reoperation. The patient agreed to receive continued chemotherapy. (Case 2) A 51-year-old woman was admitted to our hospital due to abdominal distension and defecation that had increased for 6 months, with aggravation over the past 3 days. CT examination found cystic and solid masses in the bilateral ovarian area. The CR image demonstrated that the ovarian mass violated the posterior wall of the bladder and the anterior rectal wall. The preoperational imaging evaluation ensured the safety of the operation. CONCLUSION: CR could improve the visualization of ovarian cancer masses, metastatic lymph nodes, and peritoneal metastases. CR has a good clinical value and will be more helpful in the preoperational evaluation of ovarian cancer.

[Risk Factors and Prognosis of Hepatic cGVHD after Allogeneic Hematopoietic Stem Cell Transplantation].

OBJECTIVE: To analyze the risk factors and prognosis of hepatic chronic GVHD after allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: The clinical data of 147 patients undergoing allo-HSCT from January 2013 to December 2016 were analyzed, the correlation between recipient age and sex, disease state, matched degree of HLA, donor sex, stem cell sources, ATG in GVHD prophylaxis, liver dysfunction during conditioning period, pre-transplant HBsAg, prior aGVHD and hepatic cGVHD were studied, and the correlation between hepatic cGVHD and prognosis were analysed. RESULTS: Thirty-two patients had hepatic cGVHD, cumulative incidence of 26.4%. In univariate analysis, pre-transplant HBsAg+and liver dysfunction during conditioning period were not significantly related with hepatic cGVHD (P>0.05). In multivariate analysis, prior acute GVHD (HR=2.087, P=0.045) was the independent risk factor for hepatic cGVHD, ATG (HR=0.231, P=0.000) was significantly related with a lower incidence of hepatic cGVHD. In univariate analysis, patients with hepatic cGVHD had a lower 2 years relapse rate (P=0.038). CONCLUSION: Prior acute GVHD is the independent risk factor for hepatic cGVHD, the ATG can significantly reduce the incidence of hepatic cGVHD. Hepatic cGVHD has been found to relate with a lower 2 years relapse rate.

[Effect of Steadily Down-regulating VE-Cadherin Expression on Susceptibitity of K562 Cells to Chemotherapy].

OBJECTIVE: To investigate the effect of steadily down-regulating the expression of VE-cadherin on the chemotheraputic sensitivity of K562 cells, and explore its possible mechanism. METHODS: Specifically targeting interference sequences carrying human VE-cadherin were designed, the recombinant lentiviral vector containing the IRES-GFP and NEO segment was constructed; recombinant lentivirus was generated by three-plasmids packing system, and transfected into K562 cells, then the cells steadily down-regulated were sorted. CCK-8 assay was performed to evaluate the VE-cadherin of chemotherapeutic (Imatinib) sensitivity of K562 cells. The apoptosis was analyzed by flow cytometry with Annexin V/7-AAD double labeling. The expressions of CD133 and ALDH1 mRNA were determined by real time PCR. The protein expressions of VE-cadherin, BCR-ABL and ß-catenin were analyzed by Western blot. RESULTS: The recombinant lentiviral vector pLB-shVEC-NEO-IRES-GFP was successfully constructed, packed into the lentivirus, then the K562 cells steadily down-regulating VE-cadherin expression was obtained. When VE-cadherin was down-rengulated in K562 cells, the proliferation rate was reduced while the the apoptosis rate was increased; the mRNA levels of CD133 and ALDH1 also were reduced; BCR-ABL fusion protein was not obviously changed; the total ß-catenin protein, as well as the nuclear ß-catenin protein were decreased in the K562/shVEC cells. Conclution: K562 cells are more susceptible to chemotherapy when VE-cadherin is down-regulated, that may be realized via reducing the stability and the nuclear transfer of ß-catenin protein.

[A Modified Protocol for Rapid Establishing Mouse Model with Ph+ Positive Acute Lymphoblastic Leukemia].

OBJECTIVE: To investigate a modified protocol of establishing mouse Ph+ ALL model so as to provide a more convenient and more powerful tool for Ph+ ALL studies. METHODS: Immature B cells from BALB/c mice were transfected with the Mig190 retrovirus and infused into irradiated syngeneic mice. The immunophenotype was identified by flow cytometry, the BCR-ABL was identified by RT-PCR and Western blot. Leukemia cells isolated from sick mice were re-infused into syngeneic mice without irradiation. RESULTS: The acute lymphoblastic leukemia was established in mice after Mig190 retroviral transfection, the lymphoblasts were observed by blood smears, the immunophenotype of these leukemic cells was CD19+, and the BCR-ABL was detected by RT-PCR and Western blot, the immunophenotype was conformed as Ph+ ALL, the isolated leukemia cells infused into mice rapidly developed B-ALL. CONCLUSION: A new protocol is established to generate mice with Ph+ ALL. This mouse model can provide a more simple and effective tool in comparison with the conversional protocols.

[Clinical Significance of Serum Thrombopoietin Levels in Patients with Aplastic Anaemia].

OBJECTIVE: To evaluate the clinical significance of thrombopoietin (TPO) level in diagnosis of pateints with aplastic anaemia (AA). METHODS: The TPO levels in sera from 54 AA patients and 119 healthy controls were examined. A total of 92 samples were collected from AA patients including 43 samples harvested at diagnosis, 23 samples in the cytopenic period after treatment, and 26 samples of patients in partial (n=10) or complete remission (n=16) following immunosuppressive treatment. Serum TPO levels were assessed by a sandwich-antibody ELISA that utilized a polyclonal rabbit antiserum for both capture and signal. RESULTS: Serum samples from normal donors revealed a mean TPO level of 95.3±54.0 pg/ml, the mean TPO levels in AA sera collected at diagnosis and before onset of treatment were 2728±1074 pg/ml (P<0.001) compared with normal controls; mean platelet count at that time: 27×109/L). Serum TPO levels of AA patients in partial or complete remission after immunosuppressive treatment were significantly lower than TPO levels at diagnosis (P<0.001). However, despite normal platelet counts (mean 167×109/L), TPO levels remained significantly elevated in complete remission (mean TPO 1009±6590 pg/ml (P<0.001) compared with normal controls. There was a significant negative correlation between serum TPO levels and platelet counts in AA patients (coefficient of correlation r=-0.70, P<0.001). CONCLUSION: TPO levels are highly elevated in sera of patients with AA. Thrombopoietin did not return to normal levels in remission, indicating normal platelet count in remission of AA needs sustained high level of TPO.

[Effect of BRD4 Inhibitor GSK525762A on Proliferation and Apoptosis of SUP-B15 Cells In Vitro and Its Possible Mechanism].

OBJECTIVE: To investigate the effect of BRD4 inhibitor GSK525762A on the proliferation and apoptosis of Philadelphia chromosome-positive acute lymphoblastic leukemia SUP-B15 cells and its mechanism. METHODS: SUP-B15 cells were treated with different concentration of GSK525762A, the proliferation-inhibition curve was assayed and plotted by CCK-8 method, the cell viability and apoptosis were detected by flow cytometry with Annexin V and 7-AAD staining. The transcripts of anti-apoptotic genes C-MYC, CDK6 and BCL-2 were detected by real-time PCR. RESULTS: GSK525762A could inhibit significantly SUP-B15 cell proliteration in dose-and time-dependent manner; GSK525762A treatment could induce apoptosis of SUP-B15 cells. The levels of C-MYC,CDK6 and BCL-2 mRNA transcripts in SUP-B15 cells were reduced in GSK525762A-treated group. CONCLUSION: The GSK525762A can remarkably inhibit proliferation and induce apoptosis of SUP-B15 cells. The down-regulation of apoptosis-related genes C-MYC,CDK6 and BCL-2 may be involved in the process of apoptosis.

[Establishment and Identification of Mouse Model of Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia].

OBJECTIVE: To establish a mouse model of Ph+ acute lymphoblastic leukemia (ALL) for providing a valuable tool to facilitate the researches on Ph+ ALL. METHODS: CML-like mice were generated by transfection to bone marrow cells of BABL/c with Mig190 retrovirus. The Ph+ ALL mouse model was established by infusion of sorted CML like mouse-derived BCR-ABL+ B cells into the mice of same linege. Immonophenotypes, BCR-ABL transcription and expression of these leukemic cells were detected by flow cytometry, RT-PCR and Western blot respectively. RESULTS: CML-like presentation appeared in the mice after Mig190 virus transfection. After infusion with sorted BCR-ABL+ B cells, all the receipted mice eventually presented the clinical signs of acute leukemia. Flow cytometry analysis showed that these leukemic cells were positive for CD19+; both RT-PCR and Western blot indicated that this mouse model is consistent with Ph+ ALL phenotypecally and molecalarlly. CONCLUSION: A novel method to establish Ph+ ALL mouse model has been developed successfully, and this model can provide useful tool for the study of Ph+ ALL.