T-cell-replete haploidentical HSCT with low-dose anti-T-lymphocyte globulin compared with matched sibling HSCT and unrelated HSCT.

We developed an approach of T-cell-replete haploidentical hematopoietic stem cell transplantation (HSCT) with low-dose anti-T-lymphocyte globulin and prospectively compared outcomes of all contemporaneous T-cell-replete HSCT performed at our center using matched sibling donors (MSDs), unrelated donors (URDs), and haploidentical related donors (HRDs). From 2008 to 2013, 90 patients underwent MSD-HSCT, 116 underwent URD-HSCT, and 99 underwent HRD-HSCT. HRDs were associated with higher incidences of grades 2 to 4 (42.4%) and severe acute graft-versus-host disease (17.2%) and nonrelapse mortality (30.5%), compared with MSDs (15.6%, 5.6%, and 4.7%, respectively; P < .05), but were similar to URDs, even fully 10/10 HLA-matched URDs. For high-risk patients, a superior graft-versus-leukemia effect was observed in HRD-HSCT, with 5-year relapse rates of 15.4% in HRD-HSCT, 28.2% in URD-HSCT (P = .07), and 49.9% in MSD-HSCT (P = .002). Furthermore, 5-year disease-free survival rates were not significantly different for patients undergoing transplantation using 3 types of donors, with 63.6%, 58.4%, and 58.3% for MSD, URD, and HRD transplantation, respectively (P = .574). Our data indicate that outcomes after HSCT from suitably matched URDs and HRDs with low-dose anti-T-lymphocyte globulin are similar and that HRD improves outcomes of patients with high-risk leukemia. This trial was registered at www.chictr.org (Chinese Clinical Trial Registry) as #ChiCTR-OCH-12002490.

Inhibition of p38 MAPK Phosphorylation Is Critical for Bestatin to Enhance ATRA-Induced Cell Differentiation in Acute Promyelocytic Leukemia NB4 Cells.

Bestatin has been known as an immunomodulating agent in anti-leukemia treatment. The mechanism by which Bestatin enhances all-trans retinoic acid (ATRA)-induced cell differentiation of acute promyelocytic leukemia (APL) cells is generally attributed to inhibition of cell surface CD13/aminopeptidase N activity. Bestatin also exerts its biological activities besides its ability to inhibit aminopeptidase N enzymatic activity. This article provides data to support an alternative mechanism regarding an important role of inhibition of p38 mitogen-activated protein kinase (MAPK) signal pathway in Bestatin's anti-leukemia effect. Bestatin enhanced ATRA-induced differentiation and inhibited ATRA-driven phosphorylation of p38 MAPK in ATRA-sensitive APL NB4 cells. In contrast, Bestatin could not reverse the differentiation block in ATRA-resistant APL MR2 cells, in which ATRA was unable to induce phosphorylation of p38 MAPK. Moreover, CD13 ligation with anti-CD13 antibody WM-15 resulted in phosphorylation of p38 MAPK, reduced the inhibition of Bestatin on the phosphorylation of p38 MAPK, and completely abolished the enhancement of Bestatin on ATRA-inducing differentiation in NB4 cells. This study shows that inhibition of p38 MAPK phosphorylation is critical for Bestatin to enhance ATRA-induced cell differentiation in ATRA-sensitive APL NB4 cells. Results suggested that pharmacological inhibition of the p38 MAPK pathway might enhance ATRA-dependent differentiation.

[A clinical observation of treatment of peripheral T-cell lymphoma with L-asparaginase-containing combination chemotherapy].

OBJECTIVE: To investigate the efficacy and adverse effects of L-asparaginase (L-ASP) containing regimens in patients with newly diagnosed peripheral T-cell lymphoma. METHODS: A total of 102 newly diagnosed patients with peripheral T-cell lymphoma who received combination chemotherapy with or without L-ASP were enrolled in the study between January 2011 and December 2013 in our hospital. Therapeutic and adverse effects were retrospectively analyzed, including the short-term efficacy such as complete remission (CR) rate, partial remission (PR) rate, overall remission (OR) rate, and long-term efficacy such as overall survival (OS) rate, progressive free survival (PFS) rate. RESULTS: The OR rate in patients treated with L-ASP containing regimens (L-ASP group) was apparently higher than the patients treated without L-ASP (non L-ASP group) [83.3% (35/42) vs 61.7% (37/60), P = 0.016]. Furthermore, the difference was especially significant in patients with stage III/IV [82.4% (28/34) vs 54.0% (27/50), P = 0.007] or IPI score ≥ 2 [82.1% (23/28) vs 50.0% (21/42), P = 0.006]. The 3-year OS rate of L-ASP group and non L-ASP group were 48.9% and 65.0% respectively (P = 0.974). Three-year PFS rate of L-ASP group and non L-ASP group were 40.8% and 61.0% respectively (P = 0.479). Neither had statistical significance. Although the incidence of adverse effects was higher in L-ASP group, most of them were mild and controllable after supportive treatment. There was no significant difference in serious infections caused by III-IV degree neutropenia between the two groups (P = 0.777). Other severe side-effects in L-ASP group such as hematencephalon and acute pancreatitis were only seen in one case respectively. CONCLUSIONS: Combination chemotherapy with L-ASP showed better short-term efficacy in newly diagnosed peripheral T-cell lymphoma patients and the adverse effects were controllable. Large scale prospective clinical trial of using L-ASP in peripheral T-cell lymphoma is worthy of developing and further studying.

First report of multiple CEBPA mutations contributing to donor origin of leukemia relapse after allogeneic hematopoietic stem cell transplantation.

Donor cell leukemia after allogeneic hematopoietic stem cell transplantation might provide a unique human model for our understanding of leukemogenesis in vivo. We hypothesized that the "2-genetic-hits model" may contribute to the "leukemization" of donor cells and first evaluated these genetic mutations that are implicated in the development of acute myeloid leukemia in a donor cell leukemia patient and donor. The patient and his donor-sister both harbored a germline mutation in CEBPA (584_589dup). Susceptible donor hematopoietic cells evolved to overt acute myeloid leukemia by developing 2 somatic CEBPA mutations (247dupC and 914_916dup) in the patient's microenvironment. These were identical to the acquired mutations identified in leukemic cells that originated from the patient during de novo acute myeloid leukemia. Our results provide the first report of multiple mutations of CEBPA contributing to the transformation of donor cells to the leukemic phenotype and provide clues to support the multiple-genetic-hits mechanism of donor cell leukemia.

Clinical analysis and prognostic significance of lymphoma-associated hemophagocytosis in peripheral T cell lymphoma.

This study aims to retrospectively analyze the clinical characteristics, treatments, and prognosis of aggressive peripheral T cell lymphoma (PTCL) patients with a lymphoma-associated hemophagocytosis syndrome (LAHS). We compared the clinical features and the overall survival (OS) rates of 159 PTCL patients with and without LAHS as well as the treatment outcomes of these patients with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or intensive chemotherapy regimens. We observed that in 23 % (36/159) patients PTCL was associated with LAHS. Different subtypes of PTCL in LAHS patients were diagnosed and peripheral T cell lymphoma, not otherwise specified (PTCL-NOS) was the main subtype (78 %). The median survival rates of the LAHS and non-LAHS groups were 3 and 16 months, respectively. The elevated rates of serum ß2-microglobulin, ferritin, fasting triglycerides, and hypofibrinogen levels were higher in the LAHS group, so were bone marrow involvement, liver dysfunction, hepatosplenomegaly, and B symptoms. Three patients who were treated with a plasma exchange had a longer survival time. There was no statistically significant difference in the OS rates between the intensive chemotherapy and CHOP regimen groups (P > 0.05). PTCL patients with LAHS had a poorer prognosis. Awareness of the clinical symptoms and laboratory findings are crucial in order to diagnose LAHS in an early stage and repeated biopsies of multiple bone marrows from different locations in those patients without enlargement of superficial lymph nodes are necessary to improve the diagnosis. Intensive chemotherapy due to its severe toxicity was not obviously advantageous for the OS rate compared to the CHOP regimen.

Genetic variations in the mycophenolate mofetil target enzyme are associated with acute GVHD risk after related and unrelated hematopoietic cell transplantation.

Inosine monophosphate dehydrogenase (IMPDH) is the target enzyme of mycophenolate mofetil (MMF). Single nucleotide polymorphisms (SNPs) in the IMPDH1 gene are reportedly relevant to acute rejection in renal transplant patients receiving MMF. The objective of this study was to identify the impact of IMPDH1 gene polymorphisms on the outcomes of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Four IMPDH1 gene SNPs (IVS7 +125 G>A, IVS8-106 G>A, exon15 1572 G>A, and 5' flanking intron-exon region C>T) were analyzed in 240 consecutive pairs of transplant recipients and their donors. The presence of the IMPDH1 IVS8-106 G/G genotype in recipients was associated with a significantly higher incidence of acute graft-versus-host disease (aGVHD) than other genotypes, in both unrelated and sibling transplantation cohorts (unrelated cohort: 83.3% vs 63.9%, P = .048; sibling cohort: 47.6% vs 17.3%, P = .008). Multivariate analysis confirmed that recipients with the IVS8-106 G/G genotype were at significantly higher risk of developing aGVHD (relative risk [RR] = 2.018, 95% confidence interval [CI]: 1.354-3.009, P = .001) and grades II-IV aGVHD (RR = 2.232, 95% CI: 1.352-3.685, P = .002). There was no association among IVS7 +125, exon15 1572, and 5' flanking intron-exon region genotypes and the risk of aGVHD. These results represent the first report of an association between IMPDH1 gene polymorphisms and the risk of aGVHD in allo-HSCT.

Genetic variations in T-cell activation and effector pathways modulate alloimmune responses after allogeneic hematopoietic stem cell transplantation in patients with hematologic malignancies.

BACKGROUND: Recently, several important polymorphisms have been identified in T-cell activation and effector pathway genes and have been reported to be associated with inter-patient variability in alloimmune responses. The present study was designed to assess the impact of these genetic variations on the outcomes of allogeneic hematopoietic stem cell transplantation. DESIGN AND METHODS: We first investigated ten single nucleotide polymorphisms in six genes, CD28, inducible co-stimulator, cytotoxic T-lymphocyte antigen 4, granzyme B, Fas and Fas ligand, in 138 pairs of patients and their unrelated donors and a second cohort of 102 pairs of patients and their HLA-identical sibling donors. RESULTS: We observed that patients receiving stem cells from a donor with the cytotoxic T-lymphocyte antigen 4 gene CT60 variant allele (AA genotype) had a reduced incidence of grades II-IV acute graft-versus-host disease; however, they experienced early cytomegalovirus infection and relapsed more frequently, which suggested an interaction between the donor cytotoxic T-lymphocyte antigen 4 gene CT60 AA genotype and reduced T-cell alloreactivity. Furthermore, an unrelated donor with the granzyme B +55 variant genotype (AA) was an independent risk factor for development of grades II-IV acute graft-versus-host disease (P=0.024, RR=1.811). Among patients with acute myelogenous leukemia, those with the Fas -670 TT genotype were at higher risk of relapse (P=0.003, RR=3.823). The presence of these susceptible alleles in the donor and/or patient resulted in worse overall survival (54.9% versus 69.5%, P=0.029). CONCLUSIONS: Our data suggest that genotype analysis of T-cell activation and effector pathway genes can be used for risk assessment for patients with hematologic malignancies before hematopoietic stem cell transplantation.

Expression and clinical significance of antiapoptotic gene (survivin) in NB4 and acute promyelocytic leukemia cells.

To study survivin gene expression in APL cells and to explore its correlation with clinical manifestations. PML/RARα and survivin mRNA expression were analysed using RT-PCR. By treatment of ATRA, the survivin mRNA expression in NB4 cells gradually decreased with time and was almost undetectable in the 72th hour. Survivin was expressed in 67% of the 36 APL cases (de novo and relapse patients) with PML/RARα fusion gene expression. However, in 22 cases of remission stage patients without PML/RARα fusion gene expression, survivin was expressed in 36%. The survivin mRNA expression positive rate in de novo and relapse groups, and PML/RARα fusion gene L-type positive groups, was obviously higher than those in remission period groups and was significantly lower than those in acute leukemia groups. In 36 cases of de novo and relapse APL patients, all cases could obtain complete remission, irrespective of the survivin expression. APL patients expressed with survivin mRNA had DIC and serious infection (one patient died). The clinical symptom included slight skin or mucosa bleeding, fever, and asthenic for patients without the survivin mRNA expression. Later, two cases of APL patients with the survivin mRNA expression were treated by ATRA, induction differentiation sign in their peripheral blood and bone marrow figure was not obvious. It was concluded that the survive gene expression was lower in APL than those in any other types of leukemia, thus closely associated with clinical manifestation.

Immunosuppressive cytokine gene polymorphisms and outcome after related and unrelated hematopoietic cell transplantation in a chinese population.

Cytokine gene polymorphisms can affect the outcome of allogeneic hematopoietic stem cell transplantation. We analyzed 6 single nucleotide polymorphisms in 3 immunosuppressive cytokine genes, TGFß1-509(C>T), +869(T>C), TGFß1 receptor II (TGFß1RII) +1167(C>T, codon389 AAC/AAT), and IL-10-1082(A>G), -819(T>C), -592(A>C), in a cohort of 138 pairs of recipients and their unrelated donors and a second cohort of 102 pairs of recipients and their HLA-identical sibling donors. TGFß1-509 T/T genotype in the donors or T allele-positivity in the recipients was associated with a significant protective effect against acute graft-versus-host disease (aGVHD) and grades II-IV aGVHD in the unrelated transplantation cohort. In the combined cohort, multivariate analysis confirmed that donors with the TGFß1-509 T/T genotype also conferred protection against the risk of aGVHD and grades II-IV aGVHD. In both the unrelated transplantation cohort and the sibling transplantation cohort, the IL-10-819 C/C and -592 C/C genotypes in either recipients or donors were significantly associated with a higher incidence of aGVHD. In the combined cohort, the IL-10 promoter haplotype polymorphisms at positions -1082, -819, and -592 influenced the occurrence of aGVHD and death in remission. Recipients without the A-T-A haplotype or those transplanted from donors without the A-T-A haplotype had a higher incidence of aGVHD than those who were A-T-A homozygotes or heterozygotes. Estimates for death in remission showed a clear advantage for recipients transplanted from donors with the A-T-A haplotype. In multivariate analysis, recipients without the A-T-A IL-10 haplotype had a higher risk of aGVHD (relative risk [RR] = 0.764; 95% confidence interval [CI]: 0.460-1.269; P = .096) and grades II-IV aGVHD (RR = 0.413; 95% CI: 0.245-0.697; P = .001). These results provide the first report of an association between TGFß1, TGFß1RII, and IL-10 polymorphic features and outcome of allo-HSCT in a Chinese population, and suggest an interaction between TGFß1-509 genotypes and IL-10 promoter haplotype polymorphisms at positions -1082, -819, and -592 and the risk of aGVHD.

Homoharringtonine-induced apoptosis of human leukemia HL-60 cells is associated with down-regulation of telomerase.

Homoharringtonine (HHT), first isolated from the Chinese evergreen Cephalotaxus Harringtonia, has been shown inhibiting activity in leukemia in initial studies in China and in later studies in the US, but the detailed mechanism of action is still unclear. The goal of the experiments shown here is to explore the effect of HHT on the telomerase activity and apoptosis of human leukemia HL-60 cells. The telomerase activity of HL-60 cells was examined by the telomeric repeat amplification protocol (TRAP)--an enzyme-linked immunosorbent assay (ELISA). Apoptosis was analyzed by morphological observation, DNA agarose gel electrophoresis, flow cytometry (FCM), and TdT-mediated dUTP-biotin nick end labeling (TUNEL). After treatment with HHT at 5-500 microg/l for 48 hours, the level of telomerase activity in HL-60 cells decreased in a dose-and time-dependent manner. Simultaneously, HL-60 cells underwent apoptosis. In conclusion, our data suggest that HHT can inhibit the telomerase content of HL-60 cells effectively and induce apoptosis.

A study of the relationship between expression level of TRF1 protein and telomerase activity in human acute leukemia.

OBJECTIVE: To study the expression level of TRF1 (telomeric repeat binding factor 1) protein in human acute leukemia and relationship between expression level of TRF1 protein and telomerase. METHODS: A quantitative Western-Blot technique was developed using anti-TRF1(33-277) monoclonal antibody and GST-TRF1 purity protein as a standard to further determine the expression level of TRF1 protein in total proteins extracted from clinical specimens. RESULTS: Bone marrow tissues of 20 acute leukemia patients were studied, 11 healthy donors' bone marrows were taken as a control. The expression level of TRF1 protein was significantly higher (P<0.01) in normal bone marrow ((2.217+/-0.462) microg/microl) than that of acute leukemia patients ((0.754+/-0.343) microg/microl). But there was no remarkable difference between ALL and ANLL patients ((0.618+/-0.285) microg/microl vs (0.845+/-0.359) microg/microl, P>0.05). After chemotherapy, TRF1 expression level of patients with complete remission elevated ((0.772+/-0.307) microg/microl vs (1.683+/-0.344) microg/microl, P<0.01), but lower than that of normal ((2.217+/-0.462) microg/microl, P<0.01). There was no significantly difference after chemotherapy ((0.726+/-0.411) microg/microl vs (0.895+/-0.339) microg/microl, P>0.05). TRF1 expression level of patients with complete remission is higher than that of patients without complete remission ((1.683+/-0.344) microg/microl vs (0.895+/-0.339) microg/microl, P<0.01). All samples were determined for telomerase activity. It was confirmed that the activity of telomerase in normal bone marrow was lower than that of acute leukemia patients ((0.125+/-0.078) microg/microl vs (0.765+/-0.284) microg/microl, P<0.01). There was no significant difference of expression level of TRF1 protein between ALL and ANLL patients ((0.897+/-0.290) microg/microl vs (0.677+/-0.268) microg/microl, P>0.05). After chemotherapy, telomerase activity of patients with complete remission decreased ((0.393+/-0.125) microg/microl), but was still higher than that of normal ((0.125+/-0.078) microg/microl, P<0.01). CONCLUSION: The expression level of TRF1 protein has correlativity to the activity of telomerase (P<0.001).

[Impaired microfilament cytoskeleton rearrangement in cytomegalovirus infected cells].

OBJECTIVE: To investigate the effect of cytomegalovirus (CMV) infection on actin and microfilament in human embryo fibroblast cells (HF) and its relationship with CMV replication. METHODS: Cell morphology was observed after the infection of CMV. Western-blot was used to measure the expression levels of beta-actin, G-actin and F-actin proteins. CMV immediately early antigen (CMV IE) in HF cells was analyzed by indirect immunofluorescence assay. Microfilament alteration was determined by cytoskeleton fluorescence probe. RESULT: CMV IE was demonstrated in more than 95% of HF cells after infection, which was primarily located in nucleus. The shape of HF cells changed from thin shuttle like to round and thick ball like, even escaping from wall after infection by CMV. Compared with control group, the expression of G-actin protein increased at 24 h of CMV infection (0.941 +/-0.061 compared with 0.714 +/-0.119, P <0.05), then decreased at 72 h, 96 h respectively(0.218 +/-.035, 0.230 +/-0.055 compared with 0.714 +/-0.119, P <0.05). The levels of F-actin in infected cells gradually decreased at 24 h, 72 h and 96 h compared with control HF cells (0.256 +/-0.021, 0.127 +/-0.032, 0.026 +/-0.008 compared with 0.373 +/-0.050, P<0.05). In infected HF cells, microfilaments were found ruptured, arranged turbulently. Cells fused and fluorescence density of microfilament markedly reduced. CONCLUSION: Cytomegalovirus can induce alteration of actins and microfilament, which may be associated with its infection, replication and reactivity in host cells.

[Detection, enrichment and expansion of T lymphocytes mediating alloresponse based on cytokine].

OBJECTIVE: To detect, enrich and expand the cytokine secreting T lymphocytes after allogeneic PBMNCs stimulation. METHODS: The novel cytokine secretion assay (CKSA) was applied to detect T lymphocytes secreting IFN-gamma at single cell level in human mixed lymphocytes reaction. IFN-gamma secreting T cells were enriched by means of magnetic sorting system and expanded with OKT(3), anti-CD(3)mAb and IL-2 combination. Antigen specificity of the expanded cells was confirmed using enzyme linked immunospot assay. RESULTS: A sizable proportion of IFN-gamma secreting T lymphocytes could be detected [(1.12 +/-0.13)% compared with (0.23 +/-0.07)%] and be further enriched to (67.3 +/-10.5)%, or (93.8 +/-22.1) fold. T lymphocytes could be expanded up to 600-fold within 21-28 days and the specific IFN-gamma response of expanded cells was confirmed with stimulation of the relevant allogeneic PBMNC, which was significantly higher than the irrelevant PBMNC control. CONCLUSION: It is feasible to detect significantly increased IFN-gamma secreting T lymphocytes after allogeneic PBMNCs stimulation based on the CKSA technique at single cell level and these cells can be efficiently enriched and expanded for further research.

[Evaluation of interferon-gamma producing-cells using enzyme linked immunospot assay in mice model of acute graft versus host disease].

OBJECTIVE: To investigate IFN-gamma producing-cells (IFN-gamma PCs) in allogeneic mixed lymphocyte reaction (MLR) and acute graft versus host disease (aGVHD) model of mice. METHODS: Enzyme linked immunospot assay (ELISPOT) was applied to study IFN-gamma PCs in MHC mismatched mice spleen cell MLR and aGVHD model of mice. RESULT: IFN-gamma PCs increased significantly in MLR after allogeneic mice spleen cell stimulation. In the experimental mice aGVHD model, IFN-gamma PCs were significantly higher in the severe aGVHD group than those in the moderate aGVHD. In the moderate aGVHD group, mice with GVHD prophylaxis regimen demonstrated significantly lower level of IFN-gamma PCs, compared with those without prophylaxis. IFN-gamma PCs were significantly correlated with the GVHD clinical scores in the group with moderate aGVHD and prophylaxis regimen. CONCLUSION: ELISPOT is a fast, sensitive and specific approach to evaluate alloresponse in allogeneic mice MLR and IFN-gamma PCs are correlated closely with the severity of aGVHD and prophylaxis regimen in the MHC-mismatched mice model.

[Study on unrelated donor allogeneic bone marrow transplantation with Bu-CY2 conditioning regimen for myelodysplastic syndrome].

OBJECTIVE: To evaluate the efficacy and safety of Bu-CY(2) conditioning regimen on allogeneic bone marrow transplantation (BMT) with unrelated donor for myelodysplastic syndrome. METHODS: Six patients received chemotherapy regimen of busulfan (Bu) and cyclophosphamide (CY) before allogeneic BMT (Bu 4 mg . kg(-1) . d(-1), -7 d - -4 d, CY 60 mg . kg(-1) . d(-1), -3 d - -2 d). Mycophenolate mofetil combined with cyclosporin A and methotrexate was used for prevention of acute graft-versus-host disease after transplantation. Lipo prostaglandin E(1)was used in prophylactic regimen for hepatic veno-occlusive disease. RESULT: Neutrophil count began to be higher than 0.5 x 10(9)/Lat the 18th day after BMT. Platelet count began to be higher than 20 x 10(9)/Lat the 21st day after BMT. Disease-free survival in the six patients was 27 months. CONCLUSION: Bu-CY(2) conditioning regimen on allogeneic bone marrow transplantation with unrelated donor is an effective therapy for patients with myelodysplastic syndrome.

Minimal Residual Disease at First Achievement of Complete Remission Predicts Outcome in Adult Patients with Philadelphia Chromosome-Negative Acute Lymphoblastic Leukemia.

We evaluated the prognostic effect of minimal residual disease at first achievement of complete remission (MRD at CR1) in adult patients with Philadelphia chromosome-negative acute lymphoblastic leukemia (ALL). A total of 97 patients received treatment in our center between 2007 and 2012 were retrospectively reviewed in this study. Patients were divided into two arms according to the post-remission therapy (chemotherapy alone or allogeneic hematopoietic stem cell transplantation (allo-HSCT)) they received. MRD was detected by four-color flow cytometry. We chose 0.02% and 0.2% as the cut-off points of MRD at CR1 for risk stratification using receiver operating characteristic analysis. The 3-year overall survival (OS) and leukemia free survival (LFS) rates for the whole cohort were 46.2% and 40.5%. MRD at CR1 had a significantly negative correlation with survival in both arms. Three-year OS rates in the chemotherapy arm were 70.0%, 25.2%, 0% (P = 0.003) for low, intermediate, and high levels of MRD at CR1, respectively. Three-year OS rates in the transplant arm were 81.8%, 64.3%, 27.3% (P = 0.005) for low, intermediate, and high levels of MRD at CR1, respectively. Multivariate analysis confirmed that higher level of MRD at CR1 was a significant adverse factor for OS and LFS. Compared with chemotherapy alone, allo-HSCT significantly improved LFS rates in patients with intermediate (P = 0.005) and high (P = 0.022) levels of MRD at CR1, but not patients with low level of MRD at CR1 (P = 0.851). These results suggested that MRD at CR1 could strongly predict the outcome of adult ALL. Patients with intermediate and high levels of MRD at CR1 would benefit from allo-HSCT.

Induction of apoptosis by homoharringtonine in G1 phase human chronic myeloid leukemic cells.

BACKGROUND: Homoharringtonine (HHT) is a cephalotaxine ester derived from an evergreen tree found wildely throughout southern China, which has antileukemic activities against a variety of acute myeloid leukemic cells. For the sake of illustrating the mechanisms of HHT in the treatment of leukemia, we assessed the effect of HHT on the apoptosis of human chronic myeloid leukemic cell line K562. METHODS: The apoptosis of K562 cells induced by HHT was analyzed by transmission electron microscopy, agarose gel electrophoresis of DNA, flow cytometry and terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick labeling. RESULTS: Characteristic apoptosis-related features emerged in K562 cells after exposed to HHT at a concentration 0.05-100 microg/ml. Transmission electron microscopy of HHT treated K562 cells displayed chromatin condensation and aggregation under the nuclear membrane, nuclear fragmentation and apoptosis body formation. Typical DNA ladder in agarose gel electrophoresis was observed in the cells exposed to HHT. The cell cycle analysis measured by flow cytometry showed G1 phase cells decreased with the increase of S phase cells while apoptosis was induced by HHT in K562 cells. The percentage of apoptotic cells in K562 cells treated with 50 microg/ml of HHT decreased significantly when pretreated with 1 microg/ml of cycloheximide, 0.05 microg/ml of Actinomycin D respectively. CONCLUSIONS: HHT has apoptotic effects on K562 cells. The HHT induced apoptosis mainly of the cells in G1 phase and this process required RNA transcription and protein synthesis.

Correlation between survivin mRNA expression and homoharringtonine induced apoptosis of malignant hematopoietic cells.

BACKGROUND: The inhibitor of apoptosis (IAP) gene family is involved in the suppression of apoptotic cell death as well as an increasing number of seemingly unrelated cellular functions. It is not known, however, whether IAP expression in malignant hematopoietic cells is affected by chemotherapeutic agents such as homoharringtonine (HHT). In this study, we investigated mRNA expression levels of IAPs, especially survivin, in various hematopoietic cell lines in relation with apoptosis induced by HHT. METHODS: Semiquantitative reverse transcriptase polymerase chain reaction was used to determine survivin mRNA levels. Cell apoptosis was examined by flow cytometry. Cell viability and proliferation assay was evaluated by MTT. The experiments were performed on the malignant hematopoietic cell lines MUTZ-1, K562, Jurkat, RMPI and HL60, with or without survivin antisense-oligodeoxynucleotides (AS-ODN) and HHT. RESULTS: The expression levels of survivin mRNA were variable in the cell lines and negatively correlated to HHT induced cell apoptosis. Survivin AS-ODN significantly decreased mRNA level of survivin, but not those of bax and bcl-2. Survivin also inhibited MUTZ-1 cell growth and induced apoptosis in a dose dependent manner. AS-ODN and HHT showed synergistic effect on MUTZ-1 cell growth. CONCLUSION: The apoptotic effect of HHT on the hematopoietic cell lines is associated with decreased level of survivin expression. Survivin could be a new marker for drug sensitivity and a new target for cancer treatment.

[Expression of C II TA gene in five human malignant hematological cell lines and its significance].

OBJECTIVE: To investigate the role of MHC class II Transactivator (C II TA) in expression of HLA molecules in five human malignant hematological cell lines. METHODS: The expressions of HLA molecules and C II TA protein were detected by immunohistochemistry and flow cytometry. The expression of C II TA gene was detected by RT-PCR. The response of peripheral T cells after stimulation by Jurkat cells was detected by mixed lymphocyte reaction. RESULT: The HLA II-positive tumor cells expressed the C II TA and IFN-gamma induced the expression of HLA I, II in tumor cells, which were able to express C II TA constitutively. CONCLUSION: There is a correlation between the inability of the tumor cells in response to IFN-gamma for HLA expression and the deficiency in the inducible expression of C II TA.

Invasive fungal infection in allogeneic hematopoietic stem cell transplant recipients: single center experiences of 12 years.

Invasive fungal infection (IFI) is a growing cause of morbidity and mortality among patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We retrospectively reviewed the records of 408 patients undergoing allo-HSCTs during the period November 1998 to December 2009, analyzed the incidence and risk factors of IFI, and examined the impact of IFI on overall survival. A total of 92 (22.5%) episodes suffered proven or probable IFI (4 patients were proven, 88 patients were probable). Candida was the most common pathogen for early IFI, and mold was the most frequent causative organism for late IFI. A prior history of IFI, human leukocyte antigen (HLA) mismatch, long-time neutropenia, and acute graft-versus-host-disease (GVHD) were risk factors for early IFI. A prior history of IFI, corticosteroid therapy, cytomegalovirus (CMV) disease, and chronic GVHD were risk factors for late IFI. IFI-related mortality was 53.26%. The 12-year overall survival (OS) rate for IFI was significantly lower than that of patients without IFI (41.9% vs. 63.6%, P<0.01).