Minimal residual disease monitoring in chronic myeloid leukemia patients after allogeneic hematopoietic stem cell transplantation using interphase fluorescence in situ hybridization and real-time quantitative reverse transcription PCR.

BACKGROUND AND OBJECTIVE: Interphase fluorescence in situ hybridization (FISH) and real-time quantitative reverse transcription PCR (RQ-PCR) are the common methods for monitoring minimal residual disease (MRD) in chronic myeloid leukemia (CML) patients. This study was to assess the value of monitoring BCR-ABL fusion gene level in CML patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) using FISH and RQ-PCR. METHODS: BCR-ABL fusion gene levels were detected in the bone marrow of 31 patients with CML before and 3-48 months after allo-HSCT using FISH and RQ-PCR. RESULTS: BCR-ABL positive cells detected by FISH were decreased 3-30 months after allo-HSCT and BCR-ABL/ABL mRNA was reduced by 2 logarithmic units in RQ-PCR (P < 0.05). While no BCR-ABL positive cell was detected by FISH 30 months after allo-HSCT, BCR-ABL/ABL mRNA was detected by RQ-PCR and declined by more than 3 logarithmic units, (P < 0.05). CONCLUSIONS: Dynamic monitoring of BCR-ABL gene on molecular level in CML patients after allo-HSCT is useful in the early prediction of susceptibility to recurrence in the patients and in designing intervention, and is thus helpful in improving the overall survival rate after transplantation.

[A prospective multicenter study of rituximab combined with high-dose chemotherapy and autologous peripheral blood stem cell transplantation for aggressive B-cell lymphoma].

OBJECTIVE: To investigate the feasibility and efficacy of rituximab combined with high-dose chemotherapy supported by autologous peripheral blood stem cell transplantation (ASCT) in patients with aggressive B-cell non-Hodgkin lymphoma (NHL). METHODS: Twenty-eight patients with aggressive B-cell NHL (22 newly diagnosed, 6 relapsed) were enrolled in this study. The high-dose chemotherapy included CHOP regimen (CTX + ADM + VCR + PDN) for the newly diagnosed patients and DICE (DEX + IFO + DDP + VP-16) or EPOCH (VP-16 + PDN + VCR + CTX + ADM) for the relapsed patients. Each patient received infusion of rituximab at a dose of 375 mg/m(2) for four times, on D1 before and on D7 of peripheral blood stem cell mobilization, and on D1 before and D8 after stem cell reinfusion. RESULTS: Complete remission was achieved in all patients after high dose chemotherapy and ASCT. At a median follow-up of 37 months, the estimated overall 4-year survival and progression-free survival rate for all patients were 75.0% and 70.3%, respectively, while both were 72.7% for the previously untreated patients. The therapy was generally well tolerated with few side-effects attributable to rituximab. CONCLUSION: These results suggest that adding rituximab to high-dose chemotherapy with peripheral blood stem cell transplantation is feasible and may be beneficial for patients with aggressive B-cell non-Hodgkin lymphoma.

[CD56 and CD11b antigen expressions in patients with acute monocytic leukemia and the clinical implications].

OBJECTIVE: To investigate the expressions of cell surface differentiation antigen CD56 and CD11b antigen in acute monocytic leukemic (AML-M(5)) cells and their clinical significance. METHODS: A total of 113 cases of de nove adult AML-M(5) were examined genetically and immunologically using G-banding technique, interphase fluorescence in situ hybridization (I-FISH) and flow cytometry immunophenotyping, and the results were analyzed in relation to their clinical data. RESULTS: Of the 113 cases, the expression rates of CD56 and CD11b was 28.32% and 73.45%, respectively. The CD56(+) patients had high CD11b expression, and the expression levels of CD11b and CD56 were positively correlated (P<0.05). The incidence of karyotypic abnormalities was 48.57% (55 cases) in these patients, including 25 (22.12%) with 11q23 aberrations. Twenty-five cases were positive for MLL gene abnormalities as found by I-FISH analysis. Compared with the patients positive for both CD56 and CD11b, those negative for both CD56 and CD11b showed increased peripheral blood white blood cell (WBC) count and also increased blast and progenitor cells in the bone marrow (P<0.05); the former patients often had karyotypic abnormalities, commonly involving 11q23 aberrations (P<0.05), whereas the latter patients presented more likely with extramedullary infiltration and refractory leukemia (P<0.01) with lowered complete remission rate and shortened median survival time (P<0.01). CD56-positive patients were more likely to have karyotypic abnormalities and refractory leukemia than CD11b-postive patients (P<0.05), but the peripheral blood WBC counts, bone marrow blast and progenitor cells, extramedullary infiltration, complete remission rate or median survival time showed no significant differences between them (P>0.05). CONCLUSION: AML-M(5) patients with CD56 positivity and high expression of CD11b often have aberrant karyotypes, commonly involving 11q23/MLL gene abnormality. These patients frequently develop extramedullary infiltration and refractory leukemia often with poor prognosis.

[Correlation of Fms-like tyrosine kinase 3 (FLT3) gene expression to FLT3/internal tandem duplication mutation in peripheral blood of acute myeloid leukemia.].

BACKGROUND AND OBJECTIVE: Fms-like tyrosine kinase 3 internal tandem duplication (FLT3/ITD) is associated with an unfavorable prognosis in acute myeloid leukemia (AML). However, the role of FLT3 expression as well as its correlation to FLT3/ITD has not sufficiently studied. This study was to evaluate the relationship between FLT3 gene expression and FLT3/ITD mutation in patients with de novo AML. METHODS: FLT3 gene expression was determined by real-time quantitative polymerase chain reaction (RQ-PCR). FLT3/ITD mutation was detected by PCR in 79 de novo AML patients. RESULTS: FLT3/ITD mutations were found in 22.8% (18/79) patients. FLT3 gene expression (range: 0-7320, median: 312) was detected in 92.4% (73/79) patients, but not in normal controls. Compared to AML patients with low FLT3 expressers and without FLT3/ITD mutation, patients with high FLT3 expressers and FLT3/ITD mutation had a significantly higher white blood count as well as a higher ratio of bone marrow blasts. The positive rate of FLT3/ITD mutation was not correlated to the level of FLT3 expression, and no statistical difference of FLT3 expression was found between AML patients with and without FLT3/ITD mutation. The complete remission (CR) rate of AML patients with FLT3/ITD mutation (58.8%) was significance lower than that of those without FLT3/ITD mutation (82.1%). In AML patients without FLT3/ITD mutation, the CR rate was significantly lower in patients with high FLT3 expressers (69.2%) than in those with low FLT3 expressers (93.3%). CONCLUSION: The FLT3 expression is not associated with FLT3/ITD mutation. High-FLT3 expression may be a poor prognostic factor for AML patients without FLT3/ITD mutation.

[Clinical characteristics and outcomes of 59 patients with acute lymphoblastic leukemia positive for BCR/ABL].

OBJECTIVE: To study the clinical characteristics and outcomes of BCR/ABL-positive acute lymphoblastic leukemia (BCR/ABL360888725-ALL) and screen the prognostic factors for BCR/ABL360888725-ALL. METHODS: From January 2001 to May 2008, 59 patients (median age of 32 years ranging from 3 to 69 years) with the diagnosis of BCR/ABL360888725-ALL by fluorescence in situ hybridization received induction chemotherapy with VDLP-/+Ara-C regimen. The patients who failed to respond to the chemotherapy received subsequent consolidation chemotherapy with imatinib (400-800 mg/day) (17 cases) or allogeneic hematopoietic stem cell transplantation (allo-HSCT) (16 cases). RESULTS: Of the 59 patients, 32 (58.3%) achieved complete remission (CR) after the first induction cycle. In patients with peripheral white blood cell (WBC) count <30=10(9)/L, 30-99.9(9)/L and > or =100(9)/L, the CR rates were 75.0% (18/24), 56.3% (9/15) and 26.3% (5/19) (P=0.006), and the overall survival probability of 2 years ( OSs of 2-yrs) was 24.7%, 22.5% and 21.1%, respectively (P=0.180). According to the FAB classification, 56 cases were divided into L1, L2 and biphenotypic acute leukemia (BAL) subgroups, and their CR rates were 66.7% (6/9), 63.2% (24/38) and 22.2% (2/9) (P=0.029), with OSs of 2-yrs of 22.2%, 27.0% and 22.0%, respectively (P=0.623). In terms of immunophenotype grouping by EGIL, the patients with ALL, myeloid antigen-positive ALL and BAL had CR rates of 61.1% (11/18), 60.6% (20/33) and 12.5% (1/8) (P=0.039), and the OSs of 2-yrs of 22.7%, 21.0% and 18.8%, respectively (P=0.643). In 55 patients with known karyotype, the CR rates were 71.4%(5/7), 70.8% (17/24) and 37.5% (9/24) in normal, sole t(9;22) abnormality, t(9;22) with additional abnormalities groups (P=0.046), with the OSs of 2-yrs of 42.9%, 34.0% and 7.3%, respectively (P=0.000). The patients complicated by septicemia had significantly lower OSs of 2-yrs than those without septicemia (0% vs 38.8%, P=0.005). The OSs of 2-yrs were significantly higher in patients with consolidation chemotherapy with imatinib than those without (48.0% vs 11.2%, P=0.001), and allo-HSCT was associated with significantly higher OSs of 2-yrs than exclusive chemotherapy (54.2% and 8.5%, P=0.000). CONCLUSION: BCR/ABL360888725-ALL with WBC> or =100 x 10(9)/L, presence of BAL diagnosed by FAB or FACM, t(9;22) with additional chromosome abnormalities all adversely affect the treatment results, and additional chromosome abnormalities and septicemia are associated with lower OSs of 2-yrs. Imatinib treatment and allo-HSCT can both improve the OSs of 2-yrs of the patients with BCR/ABL(+)-ALL.

[A clinical study of chronic disseminated candidiasis in patients with acute leukemia].

OBJECTIVE: To deepen the understanding of chronic disseminated candidiasis (CDC) in patients with acute leukemia (AL). METHODS: CDC was investigated in 119 AL patients who received induction chemotherapy from August 2004 to May 2005. Clinical manifestations, laboratory tests, imaging modalities, diagnosis and treatment were investigated retrospectively. RESULTS: Three patients (2.5%) were identified to be suffering from CDC. All the three patients had an absolute neutrophil count (ANC) <0. 5 x 10(9)/L for more than 15 days. Two patients had normal ANC when they were diagnosed to have CDC. The common manifestations in these three patients were persistent fever, splenohepatomegalia and percussion pain in hepatic region. Meanwhile, 2 of them were accompanied with cough, expectoration and dyspnoea. The abnormal laboratory test observed during the course of infection in two of them was increase of alkaline phosphatase. Computed tomography scan showed multiple hypodense lesions in the liver and spleen in all the three patients; two of them showed multiple nodular patchy shadows in lungs. Nuclear magnetic resonance imaging showed multiple abnormal signal in liver, spleen and kidneys in one of the patients. Two patients had positive bleed fungal cultures and histologic examination in one of the patients were positive for Candida tropicalis. Two patients received amphotericin B therapy empirically, but it was replaced by amphotericin B colloid dispersion (ABCD) later in one and combined with voriconazole in another because of unresponsiveness to the drug. One patient took a favorable turn after receiving ABCD therapy for 45 d, which was replaced by voriconazole because of the emergence of fever after discontinuation of ABCD. All the three patients received further chemotherapy smoothly after the diagnosis of CDC. CONCLUSION: The diagnosis of CDC remains difficult. Fungal blood cultures and histologic examination have been considered in many studies as the golden standard for the diagnosis of CDC. Amphotericin B is the cornerstone of treatment in patients with CDC and lipid formulations of amphotericin B can be used in CDC patients who are intolerant of or refractory to conventional amphotericin B. Voriconazole has a favorable response for refractory/relapse patients and could be used for second line treatment. The development of CDC in patients with acute leukemia does not preclude further chemotherapy.

[Polymorphisms in the breakpoint cluster region of bcr gene].

This study was aimed to explore the single nucleotide polymorphism (SNPs) of breakpoint cluster region of bcr gene in Chinese people and the relationship between SNPs and chronic myelogenous leukemia (CML). A 3.12 kb region spanning from exon 13 to exon 15 in the bcr region were screened by DNA pooling and denaturing high performance liquid chromatography (dHPLC), and the results were verified by sequencing. The results indicated that 6 novel SNP sites and 2 bases different from reference sequence were confirmed in the region studied, and the frequency of 6 novel SNP sites in studied population was obtained, one SNP of which was found in exon 13 and caused a nonsynonymous mutation. The gene frequencies of novel SNPs had no significant difference between CML and control people. It is concluded that sequence polymorphisms in the major breakpoint cluster region of bcr gene are found, most of which are SNPs, No relationship can be confirmed between SNPs and CML disease.

[Are leukemic patient bone marrow mesenchymal stem cells malignant?].

The study was aimed to explore whether there are leukemic characteristics in the bone marrow mesenchymal stem cells (BMMSC) from leukemic patients as compared with normal controls. The mesenchymal stem cells from bone marrow of normal volunteers and patients with APL and CML were isolated, then cultured and proliferated in vitro. The morphology, growth curve and cell surface markers of two different sources mesenchymal stem cells were investigated for detecting whether the bone marrow mesenchymal stem cells derived from leukemia patients have the specific abnormal fusion gene of leukemia cells through fluorescent in situ hybridization. The results indicated that there was no significant difference between the mesenchymal stem cells derived from different subjects, the bone marrow mesenchymal stem cells derived from leukemia patients did not have the clonal malignant fusion gene as seen in the leukemia cells. Taken altogether, mesenchymal stem cells derived from leukemia patients had no biological differences as compared with those from normal volunteers, and no malignant clonal abnormality was found. It is concluded that mesenchymal stem cells derived from leukemia patients as an alternative vehicle may be used for assistant of autologous hematopoietic stem cell transplantation or cell therapy and gene therapy.

[Gene expression profile in K562 cells treated by interferon alpha].

To study the gene expression profile in K562 cells treated by IFN-alpha, so as to provide some information about the potential mechanism of IFN-alpha curing CML, the changes of gene expression were examined with the DNA array in K562 cells before and after treatment with IFN-alpha. The results showed that no gene expression difference more than 2.5 times in K562 cells was found on the first day after treatment with IFN-alpha (200 U/ml), then the genes significant expression difference increased step by step, and reached the peak on the forth day. In all examined genes, 97 genes significant expression difference were detected, 86.60% (84/97) gene of interest out of those gene were up-regulated, 13.40% (13/97) were down-regulated. In these 97 genes with significant expression difference, cell regulator protein genes accounted to 23.71% (23/97), surface receptor genes 14.43% (14/97), oncogenes and tumor suppressors 11.34% (11/97), extracellular communication proteins 9.28% (9/97), cell adhesion molecular genes 8.25% (8/97) and the other genes accounted to 32.99% (32/97). JAK1 was up-regulated to 3.78 times, JAK2 to 15.43, STAT1 and STAT2 were up-regulated to 11.98 and 8.11 times respectively, and these genes are components of JAK-STAT pathway. The number of different genes began to decrease on the fifth day. There were still 9 genes that had expression difference more than 3 times on the twenty-first day. It is concluded that when concentration of IFN-alpha was 200 U/ml, the forth day should be considered as the best time to examine change of gene expression in K562 cells treated by IFN-alpha. IFN-alpha realizes its biological functions through the JAK-STAT pathways and it may be one of the mechanisms for curing CML with IFN-alpha.

Detection of FLT3 gene and FLT3/ITD mutation by polymerase chain reaction-single-strand conformation polymorphism in patients with acute lymphoblastic leukemia.

OBJECTIVE: To analyze Fms-like tyrosine kinase 3 (FLT3) gene and FLT3 internal tandem duplication (ITD) mutation in acute lymphoblastic leukemia (ALL) patients of different immunological subtypes. METHODS: Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) was used to detect FLT3 gene and FLT3/ITD mutation in 63 ALL cases. RESULTS: Among the 63 ALL cases, FLT3 gene was detected in 41 (61.5%) cases. The positivity rate of FLT3 gene in pre-pre B-lineage ALL, pre-B-ALL, B-lineage ALL and T-lineage ALL cases were 93.3% (14/15), 77.8% (14/18), 41.7% (5/12) and 28.6% (4/14), respectively. The positivity rate of FLT3 gene was significantly higher in pre-pre B-ALL/pre B-ALL subtypes (84.8%) than in B-ALL subtypes (41.7%, P<0.005), and the rate was significantly higher in B-ALL subtypes (73.3%) than in T-ALL subtypes (28.6%, P<0.001). Two cases (3.2%) were found to have FLT3/ITD mutation, which were also positive for myeloid antigen expression and diagnosed as acute mixed-lineage leukemia, showing leukocytosis and high percentage of bone marrow blast cells with poor prognosis. CONCLUSIONS: FLT3 gene can be detected in both B-and T-lineage ALL patients, but more frequently in the former. In B-lineage ALL patients, FLT3 gene is more frequent in cases with undifferentiated than those with differentiated blast cells. FLT3/ITD is rarely detected in ALL patients and FLT3/ITD mutation detection might be helpful to identify the genotypes and evaluate the prognosis of acute leukemia.

[Analysis of sequence-tagged site in bcr and abl genes by DNA pooling and dHPLC].

To investigate the relationship between the single nucleotide polymorphism (SNPs) of the bcr and abl gene and chronic myelogeous leukemia (CML), the 9 sequence-tagged sites (STS) in bcr and abl gene were screened by DNA pooling and denaturing high performance liquid chromatography (dHPLC), and the results were varified by sequencing. The results showed that the polymorphism sites were detected in 4 out of the 9 STS fragments and there were 3 bases different from the reference sequence found in 3 fragments. In conclusion, the novel SNP in U07000 fragment shows significantly different frequencies between CML and controled people.

[Recombinant adeno-associated virus 2-mediated green fluorescent protein expression in bone marrow mesenchymal stem cells derived from acute myelogenous leukemia patients].

OBJECTIVE: To explore the possibility of using autologous bone marrow mesenchymal stem cells (BMSC) as a vehicle to deliver recombinant adeno-associated virus 2-mediated enhanced green fluorescent protein (rAAV-2-eGFP) in vitro, therefore to find an alternative solution for gene therapy of hematological malignancy. METHODS: BMSCs isolated from the bone marrow of patients with acute myelogenous leukemia (AML) at the onset of disease were infected by rAAV-2-eGFP at different multiplicity of infection (MOI=10(2), 10(3), 10(4), 10(5), 10(6), and 10(7), respectively). Phase-contrast fluorescent microscope and flow cytometry were employed to evaluate the expression of enhanced green fluorescent protein (eGFP). RESULTS: Ten to fourteen days after the transfection, eGFP expression began to be detected and the transfection efficiency ranged between 0.3% to 2%, which failed to be increased with the increase of MOI. The transduced eGFP could maintain a long-term stable expression in vitro in the 61 days of observation, and from 12 to 33 days after transfection, eGFP percentage underwent a decrease from the initial 1.16% to 0.5%-0.6% and maintained this expression level till 61 days after transfection. CONCLUSION: rAAV can be used with BMSCs for in vitro gene therapy, but the poor transfection efficiency of these cells remains a significant obstacle for its further application.

[Transfection of human 293 and CD34+ cells with recombinant green fluorescent protein adeno- associated virus].

OBJECTIVE: To investigate the expressions of recombinant green fluorescent protein (GFP) in 293 cells (human embryonic kidney cells) and CD34(+) cells transfected with adeno-associated virus carrying the gene encoding the recombinant GFP. METHOD: Mononuclear cells from the bone marrow were collected on cell separator CS-3000, and CD34(+) cells were separated on immunomagnetic MiniMACS columns. CD34(+) cells and 293 cells were transfected with recombined adeno-associated virus respectively, and the expressions of the GFP were detected by flow cytometry and fluorescence microscopy. RESULT: GFP expression was observed in the two cells under fluorescent microscope. The highest transfection efficiency of the recombined adeno-associated virus was about 32.8% in 293 cells and 25% in CD34(+) cells, and decreased with the prolongation of transfection time. CONCLUSION: Adeno-associated viral vector encoding recombinant GFP can be transfected into human 293 cells and CD34(+) cells, which provides a basis for future gene therapy.

[Therapeutic efficacy of hematopoietic stem cell transplantation in patients with chronic myelogenous leukemia].

OBJECTIVE: To evaluate the clineffects of autologous and allogeneic hematopoietic stem cell transplantation (HSCT) for chronic myelogenous leukemia (CML). METHODS: Fifty-seven patients with CML were treated by HSCT, including 8 patients treated with autologous transplantation in vivo and vitro purging minimal residual disease (MRD), 39 with related donor allogeneic HSCT (allo-HSCT), and 10 with unrelated donor allo-HSCT. For the conditioning regimen, total-body irradiation with cyclophosphamide (CTX) was given in 32 patients, modified BuCY protocol (hydroxyurea, busulfan, Ara-C, CTX) in 24 patients, and MACC protocol (melphalan, Ara-C, CTX and lomustine) in one patient. Cyclosporine (CsA) and methotrexate (MTX) were used in patients with related donor allo-HSCT, and the combination of CsA, MTX , mycophenolate mofetil (MMF), and antithymocyte globulin (ATG) in unrelated donor all-HSCT to prevent graft versus host disease (GVHD). Kaplan-Meier survival analysis model was used to estimate the overall survival and the disease-free survival (DFS) at 5 years after transplantation. RESULTS: In 8 patients with autologous transplantation, 7 obtained partial or complete cytogenetic remission (CR) within 3 months after transplantation and one died of transplantation-related complication. In 49 patients with allo-HSCT transplantation, all patients obtained CR except for two patients, one of whom failed to obtain CR and the other died of hepatic veno-occlusive disease. The incidence of infection and veno-occlusive disease during transplantation was 33.3% and 7.0%, respectively. The incidence of hemorrhagic cystitis and cytomegalovirus interstitial pneumonia after transplantation was 22.8% and 8.8%, respectively. Veno-occlusive disease, hemorrhagic cystitis or cytomegalovirus interstitial pneumonia did not occur in patients with autologous transplantation. The incidence of acute GVHD was 41.0% and 48.6%, and that of chronic GVHD 40.0% and 42.9% in patients with related and unrelated transplantation, respectively. The rate of relapse was 57.1% and 12.8%, with DFS at 5 years of 25.0% and 61.7%, respectively, in patients with autologous and related donor transplantation. The DFS at 5 years was 70.7% and 34.1%, respectively, in patients with chronic/accelerated phases and blast crisis be fore trans plantation. CONCLUSION: allo-HSCT can produce a higher clinical cure rate in CML patients in chronic phase CsA+MTX+MMF+ ATG protocol is more effective for prevention and alleviation of acute GVHD in patients with unrelated donor transplantation. Autologous transplantation with bone marrow purging helps prolong the patients' survival and even obtain clinical cure of CML.

[Effects of allogeneic hematopoietic stem cell transplantation with very-high-dose conditioning regimen for refractory leukemia].

OBJECTIVE: To explore the regimen-related toxicity (RRT) and therapeutic effects of very-high-dose conditioning regimen combined with induction of graft-versus-leukemia (GVL) effects in allogeneic hematopoietic stem cell transplantation (allo-HSCT) for refractory leukemia with unattainable complete remission (CR) before transplantation. METHODS: Eighteen patients who failed to obtain CR before transplantation received very-high-dose conditioning regimen protocol (experimental group), and 62 patients with acute leukemia with CR or with chronic myeloid leukemia in the chronic phase before transplantation received total body irradiation plus cyclophosphamide (CTX) or modified BuCY (hydroxyurea, busulfan, Ara-C, CTX) protocol (control group). In patients with refractory leukemia who did not develop graft-versus-host disease (GVHD) 30 d after the transplantation, GVL was induced by rapid reduction of the dosage of cyclosporin A or by donor lymphocytic infusion. The incidence and mortality of RRT and the rates of CR, GVHD and leukemia relapse after transplantation were investigated. Kaplan-Meier survival analysis model was used to estimate the disease-free survival (DFS) rate at 3 years post-transplantation. RESULTS: Except for one patient in the experimental group and two in the control group who died of transplantation- related complications, all the other patients obtained hematopoietic reconstitution. The total incidence of RRT was 100% in both groups, involving most frequently the stomach and intestines at the rate as high as 83.3% in the experimental group and 85.5% in the control group. RRT involving the oral cavity occurred in 44.4% and 62.9%, and that involving the bladder in 16.7% and 33.9% of the cases in the experimental group and control group, respectively, all similar between the groups (P=0.823, 0.172 and 0.244, respectively). The RRT mortality was 0 and 5% in the experimental and control groups, respectively (P=0.341). With the exception of one patient who died of infection, all the other patients treated with very-high-dose conditioning regimen obtained CR. The incidences of acute/chronic GVHD were 58.8%/92.6% and 40.0%/55.8%, respectively, in the experimental and control groups. The incidence of leukemia relapse was 11.8% and 18.3%, and DFS at 3 years after transplantation was (61.2+/-12.3)% and (65.0+/-7.4)% (P=0.6311) in the two groups, respectively. CONCLUSION: Consecutive very-high-dose conditioning regimen combined with GVL induction after transplantation can increase the rate of CR and DFS, without increasing RRT incidence and mortality in allo-HSCT for the refractory leukemia with unattainable CR pre-transplantation.

[Two-dimensional polyacrylamide gel electrophoresis analysis of apoptosis in K562 cells induced by harringtonine].

BACKGROUND & OBJECTIVE: Harringtonine (HT),an anti-tumor drug,has been widely used to treat acute or chronic myeloid leukemia,and obtained satisfactory effects. Studies showed that the anti-tumor activity of HT is related with the apoptosis-inducing effect,but the molecular mechanisms remain unclear. This study was to analyze the protein maps contributed to the apoptosis in K562 cells induced by HT,and to screen the apoptosis-associated proteins. METHODS: Flow cytometry was used to distinguish K562 cells of early apoptosis stage from those of late apoptosis stage through Annexin V and PI staining. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used to separate and compare the HT-induced apoptotic K562 cells and control K562 cells. RESULTS: When K562 cells were treated with 10 microg/ml HT for 5,a nd 24 hours,percentages of early apoptotic cells (Annexin V+/PI-) were 28.3%, and 18.1%(P< 0.01), while percentages of late apoptotic cells (Annexin V+/PI+) were 9.1%,and 20.2% (P< 0.01), respectively. Statistical analysis showed (1300+/-50) protein spots were resolved with a match rate of (88.3+/-2.0)% in control K562 cells. Ten protein spots in late apoptotic cells displayed changes in expression after induction of HT for 24 hours (P< 0.01), of which 8 showed higher expression, 1 decreased,and 1 only expressed in control k562 cells. CONCLUSION: These proteins may be apoptosis-associated proteins of K562 cells induced by HT.

[Detection of FLT3 gene and FLT3/ITD gene mutation in chronic myeloid leukemia and its significance].

BACKGROUND & OBJECTIVE: Fms-like tyrosine kinase 3 (FLT3)gene abnormal expression could be detected in most of acute myeloid leukemia (AML)patients, and 20%-30% of them have FLT3/ITD gene mutation which indicate poor prognosis. This study was to detect FLT3 gene expression, and FLT3/ITD gene mutation in chronic myeloid leukemia (CML)patients, and analyze their relationships with prognosis. METHODS: Polymerase chain reaction (PCR)was used to detect FLT3 gene expression, and FLT3/ITD gene mutation in 53 CML patients of chronic phase,and 34 CML patients of accelerated phase or blast crisis. RESULTS: FLT3 gene was detected in 5.7%(3/53)CML patients of chronic phase, and in 55.9% (19/34)CML patients of accelerated phase or blast crisis,the difference of FLT3 gene positive rate between these 2 groups was significant (P< 0.001). Only 2 of 87 CML patients (2.3%)were found with FLT3/ITD mutation. CONCLUSIONS: FTL3 gene expresses mainly in CML patients of accelerated phase or blast crisis. FTL3/ITD gene mutation was seldom involved in CML patients. The CML patients with FLT3 gene expression and FTL3/ITD gene mutation may have poor prognosis.

[Proteome analysis of apoptotic K562 cells induced by harringtonine].

OBJECTIVE: To screen and identify apoptosis related proteins and explore the mechanism of harringtonine (HT)-induced K562 cells apoptosis. METHODS: Flow cytometry was used to distinguish K562 cells in the earlier stage of apoptosis from those in the later stage of apoptosis by annexin V and PI staining. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) coupled with computer image analysis was used to detect the changes in protein expression in the two stages of apoptosis. Proteins were identified by peptide mass fingerprint in combination with database searching. RESULTS: K562 cells treated with HT for 5 and 24 hours were in the early and later stages of apoptosis respectively. Statistical analysis showed 3 spots disappeared, 7 spots with decreased intensity and 10 spots with increased intensity in the 24 h HT induced apoptotic cells as compared with that in 5 h HT induced ones. Ten spots were selected on the basis of the intensity and the significant changes in abundance. Among them, 5 apoptosis related proteins were successfully identified by MALDI-TOF: keratin 9, BTF3, TrpRS, RS and prohibitin. CONCLUSIONS: Up-regulation of TrpRS, RS, prohibitin and down-regulation of BTF3 were involved in inhibition of transcription and protein synthesis in the apoptotic K562 cells induced by HT, whereas up-regulation of keratin 9 was related to apoptosis resistance.

[Polymorphism analysis of 5' promotor region of BCR gene].

BACKGROUND & OBJECTIVE: BCR-ABL fusion gene is regarded as the molecular hallmark of chronic myelogenous leukemia (CML), and its expression is controlled by the BCR gene promoter. This study was designed to investigate the polymorphism of the promoter region of BCR gene, and its possible correlation with the disease. METHODS: A 1.13 kb fragment of BCR gene 5' promotor region was amplified and sequenced from 30 CML patients and 19 controls. Transcription factor binding sites and repeat sequences in this region were analyzed using softwares and online tools. RESULTS: Four novel single nucleotide polymorphisms (SNPs) and 3 bases different from the reference sequence were detected in the region studied. Among these 2 novel SNPs and 1 different base were located in or near several bases of binding sites. The gene frequencies of the novel SNPs had no significant difference between CML and control people. CONCLUSION: Sequence polymorphisms were found in the 5' promotor region of BCR gene, most of them being SNPs. No relativity can be validated between the SNPs and the disease. But it appears that some SNPs might have the probability of bringing influence to the transcription and expression of the gene.