Study Of Experiment And Clinical Review Of Childhood Acute Leukemia

PartⅠStudy of Molecular Mechanism of Berberine Inducing Proliferation and Apoptosis on Jurkat cellObjectiveRecent studies have shown the significant effect of berberine on cancer cells with anti-proliferation and induction of apoptosis. Our previous studies have shown berberine can induce apoptosis of Molt-4 cells combined with TRAIL, involved with the suppression of NF-κ/P65 expression and activation with caspase 3 and caspase 8 expressions. However, P53 and MDM2 are not known whether to be involved in the apoptosis of leukemia cells induced by berberine.Survivin is the member of IAP (Inhibitors of Apoptosis Protein, LAP) family, with the bi-direction effect of inhibition of apoptosis and regulation of proliferation. However, the isomers of survivin, survivin-2B and survivin-△Ex3 would have the different biological roles in the oncogenic development. Also our studies in pediatric acute leukemia patients have shown the expression of survivin would not be the same trend as its isomers, but we have not identified the role of survivin and its isomers in the apoptosis of leukemia cells induced by berberine, nor the relationships with the roles of P53 and MDM2.This study would be focus on:1,The effect of berberine inducing proliferation and apoptosis of Jurkat cells, and the molecular mechanism;2,The different mechanisms of berberine and daunorubicin on proliferation and apoptosis of Jurkat cells.Methods 1,Jurkat cell was divided to three groups and cultured for 12h and 24h as:1) blank control group; 2)berberine group:the concentration of berberine as 25μM,50μM,100μM, seperately; 3)daunorubicin group:the concentration of daunorubicin as 0.1mg/L, 1mg/L,10 mg/L, seperately.2,The inhibition of cell proliferation of every group was detected by CCK-8 at the time points as 12h and 24h; and the apoptosis of cells was detected by flow cytometry.3,The mRNA expression of survivin, survivin-2B, survivin-△Ex3, P53 and MDM2 was detected by Real-time PCR.4,The proteins expression of survivin, P53 and MDM2 was detected by western-blot.Results1,Berberine inhibited the proliferation of Jurkat cell and induced its apoptosis, and the effect was concentration-dependent and time-dependent, showing the positive trend of inhibition rate would be attained by the increased concentration and action time of berberine, which was the same performance as daunorubicin.2,Berberine inhibited the expression of survivin and survivin-△Ex3, and increased the expression of survivin-2B with concentration-dependent and time-dependent, as daunorubicin did, but the role of berberine and daunorubicin would be inconsistent.3,Berberine increased the expression of P53 with time-dependent as daunorubicin did, but with the different role in concentration-dependent.4,Berberine inhibited the expression of MDM2 with time-dependent, without concentration-dependent; while daunorubcin inhibited the expression of MDM2 with not fully time-dependent and concentration-dependent.ConclusionBerberine could inhibit the proliferation and induce the apoptosis of Jurkat cell via down-expression of survivin, survivin-△Ex3 and MDM2, and up-expression of survivin-2B and P53. The inconsistent roles of berberine and daunorubicin suggest berberine be clinical value in the treatment of daunorubicin-resistant leukemia cells, or used with daunorubicin togher for the therapy of leukemia in the future. (I) Neonatal Transient Abnormal Myelopoiesis in Chinese Children: A Small Cohort ReviewBackgroundTen percent of neonates with constitutional trisomy 21 are predisposed to developing transient abnormal myelopoiesis (TAM), which is characterized by a rapid accumulation of blast cells during the first few days of life that spontaneously resolves in several weeks or months; acute megakaryoblastic leukemia (AMKL) subsequently develops in 20% to 30% of these infants in several years later.ObjectiveTo explore the natural history of neonatal TAM, the clinical characteristics of subsequent AMKL, and the prognosis of TAM and AMKL with constitutional trisomy 21. MethodsThe charts of 4 neonates identified as having trisomy 21 and TAM was retrospectively reviewed and compared their treatment and long-term outcomes with those of similar patients in the literatures.ResultsTrisomy 21 was the only cytogenetic abnormality identified in the blast cells of the 4 patients. In all patients, peripheral blast cells cleared spontaneously, blood counts normalized and complete remission (CR) ensued without chemotherapy. AMKL developed in 3 patients at a mean age of 15 months; they were treated with chemotherapy. All experienced CR for at least 6 years. The remaining patient without AMKL was found to have trisomy 21 only in the blast cells and he has normal phenotype without any Down's stigmata.ConclusionsAll 4 patients'outcomes exceed those of similar patients reported in the literature. Further research should be focused on the role of the cytogenetic interaction locating in trisomy 21 on leukemogenesis and identification of special therapeutic targets. Multicenter collaborations are needed to determine the risk stratification and appropriate treatment of China neonatal TAM and secondary AMKL to minimize the therapy-related toxicity.(Ⅱ) Clinical Analysis of 30 Infants with Acute LeukemiaObjectiveTo investigate the clinical, cytogenetic and biological characteristics of infant acute leukemia (IAL).MethodsRetrospective analysis of clinical manifestations and laboratory results was done on 30 cases of IAL.Results Fourteen cases with fever,5 cases with subcutaneous hemorrhage,4 cases with sallow complexion,4 cases with pale complexion,4 cases with hepatosplenomegaly,8 cases with superficial lymphadenopathy,5 cases with bone lesion,2 cases with bilateral renomegaly,1 cases with central nervous system (CNS) infiltration of leukemia. The length of time between attack and reception of treatment was from 2-75 days. WBC 114.78 (1.06-681.37)×109/L; Hb 67 (8～101) g/L; PLT 61 (1～355)×109/L. Peripheral blasts were identified in all 30 cases. Of the 30 cases,15 (50%) were ALL,14 (46.7%) were ANLL,1 (3.3%) was unknown. 12 cases were acquired immunology and cytogenetics.5 cases of ALL were diagnosed as B-ALL, with 3 cases of CD 10 positive,1 case of CD7 and CD 15 co-expression.2 of 7 ANLL cases were found with CD 19 expression.3 of 12 cases showed chromosome abnormalities.13 cases were acquired with fusion gene results, and 1 case was E2A/PBX1 positive, one case was BCR/ABL positive; and other cases'results were negative.4 of 30 cases accepted chemotherapy, but eventually gave up. Another 26 untreated cases were confirmed death after four weeks.ConclusionsPatients with IAL have no unique features in clinical cheracterictics and laboratory examinations. The prognosis is poor due to most of the patients gave up chemotherapy after diagnosis. Multicenter should co-operate togher to design the appropriate chemotherapy protocols for this special group patients.(Ⅲ) Long-term Prognosis of Chinese Older Children and Adolescents with Acute Lymphoblastic Leukemia Treated on a Pediatric Protocol: A Small Cohort Review BackgroundOlder children and adolescents with acute lymphoblastic leukemia (ALL) treated on pediatric protocols have significantly better outcomes than those on adult protocols, reflecting the importance of more intensive use of nonmyelosuppressive agents and intrathecal therapy.ObjectiveTo examine the biological and clinical characteristics of Chinese older children and adolescents with ALL, and their outcomes treated on the pediatric ALL protocol.MethodsThe clinical characteristics, effect of treatment and long-term outcomes of 10- to 18-year-old patients with ALL on the Hong Kong pediatric ALL protocol were retrospectively reviewed. ResultsA total of 10 male and 9 female patients were treated; 4 patients' peripheral white cell counts were more than 100×109/L at diagnosis.4 cases were identified as T cell lineage and the remaining had B-cell lineage. Ten had abnormal cytogenetic results. Seventh-day post-oral prednisone treatment responses were favorable for 15 patients. On day 33 of treatment, bone marrow results indicated that 2 patients did not achieve remission. Although the rate of complete remission (CR) was 84%, nine patients experienced significant treatment-related side effects and 2 patients died.4 patients experienced relapse, and the mean time between CR and relapse was 35 months (range,10-63 months). The mean follow-up time was 88 months (5-150months). The 7-year overall survival (OS) and event-free survival (EFS) were 89.5±7.0% and 72.9±10.4%, respectively. ConclusionAlthough apparently with more frequent serious therapy related complications, older children and adolescents with ALL had satisfactory prognosis on pediatric oriented protocol without routine transplantation.(IV) The Role of Hematopoietic Stem Cell Transplantation in Refractory and/or Relapsed Infant Leukemia in China:A small cohort reviewBackgroundAlthough multi-drug chemotherapy combined with hematopoietic stem cell transplantation (HSCT) has improved the long-time outcome of pediatric refractory or relapsed leukemia, the prognosis of infant refractory or relapsed leukemia patients would be still poor on traditional chemotherapy, and HSCT has not been identified to improve the prognosis of these patients.ObjectiveTo explore the role of HSCT performed in infant refractory or relapsed leukemia patients.MethodsWe present the outcomes of 8 infants with refractory or relapsed leukemia who were treated with HSCT.ResultsFour of eight patients had acute myeloid leukemia (AML), and 2 had acute lymphoblastic leukemia (ALL), while 1, juvenile myelomonocytic leukemia (JMML), and 1, mixed lineage leukemia. Four patients had an initial white blood-cell count greater than 50x109/L, and 2 patients had determinable abnormal cytogenetics of MLL rearrangement. Three patients received cord blood from an unrelated donor,1 received HSCT from an unrelated donor, and 4 received HSCT from a related donor. One patient experienced engraftment failure, and 7 patients achieved neutrophil engraftment at a median of 17 days. Four patients relapsed after HSCT. Although 2 of them received a second transplantation, all 4 patients died. Another patient died of transplantation-related causes. HSCT was not well tolerated in these patients; 4 patients experienced Grade 4 acute graft-versus-host disease (GVHD), and 5 patients with hepatic venous-occlusive disease (VOD).Three survival patients experienced some long-term sequelae.Conclusion Treatment of infant refractory or relapsed leukemia with HSCT is still facing with serious challenges.