Characteristics of acute myeloid leukemia with myelodysplasia-related changes: A retrospective analysis in a cohort of Chinese patients.

We retrospectively analyzed 449 patients with AML under the WHO classification of AML 2008 and probed implications of this classification in diagnosis and treatment of acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) among them. The clinical presentations, biological features, treatments, and prognosis of patients diagnosed with AML-MRC were analyzed and compared with those of AML not otherwise specified (AML-NOS). In all patients, 115 (25.6%) were diagnosed as AML-MRC including 64 males and 51 females with median onset age of 48 years (range from 17 to 78). Their complete remission (CR) rate was 60.9% and relapse rate was 57.1%. The observed median overall survival (OS) and disease-free survival (DFS) were 10 and 5 months, respectively, which was significantly shorter than those of AML-NOS patients (P < 0.05). The prognosis of AML-MRC patients with myelodysplastic syndrome (MDS)-related cytogenetics sole was similar to those with history of MDS or myelodysplastic/myeloproliferative neoplasm (MDS/MPN). Patients with MDS-related cytogenetic abnormalities and/or history of MDS or MDS/MPN predisposed significantly shortened CR, OS, and DFS than AML-MRC patients with only multilineage dysplasia (MLD) and AML-NOS patients (P < 0.05). Multivariate analysis showed that age, cytogenetics, and history of MDS or MDS/MPN were independent prognostic factors. Patient diagnosed as AML-MRC presented distinctive clinical and biological features. Presence of MLD does not change the prognosis.

[Comparison of protein expression profiles between bortezomib-resistant JurkatB cells with PSMB5 mutation and their parent cells].

This study was purposed to investigate the differences of cyto biological characteristics and protein expression levels between bortezomib-resistant T-lymphoblastic lymphoma/leukemia cell lines JurkatB containing PSMB5 G322A mutation and their parent cell line Jurkat, The cytotoxicities of bortezomib and chemotherapeutic drugs to JurkatB5 cells (end selection concentration of bortezomib was 500 nmol/L), JurkatB8 (end selection concentration 800 nmol/L) and Jurkat cells were analyzed. The cell growth curves were drawn with viable cell counts by trypan blue assay, the colony formation rate were assayed by soft-agar colony culture, and the cell distributions in cell cycle were analyzed by flow cytometry, mRNA expression levels of multidrug resistance (MDR) genes MDR1, LRP and MRP were measured by real-time fluorescence quantitative RT-PCR, the differences of protein expression levels were detected by SpringBio antibody microarray containing 720 proteins. The results showed that the drug resistance multiples for 48 hours of JurkatB5 and JurkatB8 cells (relative to Jurkat) to bortezomib were increased by 33.52 and 39.04 times, respectively. JurkatB5 and JurkatB8 cells did not display significant cross-resistance to daunorubicin, adriamycin, vindesine, and etoposide after exposure for 48 hours. There were no significant differences in the cell growth curve, colony formation rate and cell distributions in cell cycle between JurkatB5, JurkatB8 and Jurkat cells (p > 0.05). There were no significant differences of mRNA expression levels of MDR1, LRP, MRP between JurkatB5 and Jurkat cells (p > 0.05). There were 264 analyzable expression points detected by antibody microarray. Among them, 252 protein expression levels were not significantly different between JurkatB5, JurkatB8 and Jurkat cells (< 2-fold), including 15 drug resistance-related proteins. 12 proteins were detected at higher or lower expression levels in JurkatB5 or JurkatB8 cells then that in Jurkat cells (cell division cycle protein 34, cell division cycle protein 37, CD34 Type II, matrix metalloproteinase-2, tenascin, Golgi complex, involucrin, histone deacetylase 1, perforin, prolactin, retinoic acid receptor ß, integrin ß-1), but no proteins were detected in JurkatB5 and JurkatB8 cells with higher or lower expression levels than that in Jurkat cells. It is concluded that there are no significant differences in the characteristics of cellular biology between Jurkat and JurkatB with bortezomib-resistant and PSMB5 G322A mutation. There are no significant phenotype change of MDR and overexpression of genes related to MDR in PSMB5 mutated cells. There are no significantly differential expressions of a majority of known proteins related to drug resistance, tumor cells growth, proliferation, apoptosis, malignancy degree, aggressiveness.

Clinical and biological characteristics of adult biphenotypic acute leukemia in comparison with that of acute myeloid leukemia and acute lymphoblastic leukemia: a case series of a Chinese population.

BACKGROUND: Biphenotypic acute leukemia is a rare disorder that is difficult to diagnose. It displays features of both myeloid and lymphoid lineage. There is still a lack of studies in biphenotypic acute leukemia in a Chinese population. We present here a comprehensive investigation of the clinical and biological characteristics, and outcome of biphenotypic acute leukemia in our hospital in over a seven year period. DESIGN AND METHODS: We retrospectively analyzed 452 adult acute leukemia patients diagnosed according to French-American-British (FAB) classification and biphenotypic acute leukemia diagnosed according to European Group for the Immunological Characterization of Leukemias (EGIL) classification, respectively. Biological characteristics, response to treatment, and outcome were examined in biphenotypic acute leukemia patients and compared with that in acute myeloid leukemia and acute lymphoblastic leukemia patients with complete follow-up profiles diagnosed in the same period. RESULTS: Of 452 acute leukemia patients, 21 cases (4.6%) were diagnosed as biphenotypic acute leukemia. Among them, 14 (66.7%) were B lymphoid and myeloid, 5 (23.8%) were T lymphoid and myeloid, one (4.8%) was T/B lymphoid and one (4.8%) was trilineage differentiation. When compared with acute myeloid leukemia and acute lymphoblastic leukemia, patients with biphenotypic acute leukemia showed significantly higher incidence of CD34 antigen expression, unfavorable karyotypes, and extramedullary infiltration (p<0.05). In this cohort of patients with biphenotypic acute leukemia, t(9;22) was the most common abnormality in chromosome structure. The median disease-free survival and overall survival in biphenotypic acute leukemia patients was five months and ten months, respectively, significantly shorter than those in acute myeloid leukemia and acute lymphoblastic leukemia patients (p<0.05). CONCLUSIONS: The prognosis of biphenotypic acute leukemia patients is poor when compared with de novo acute myeloid leukemia or acute lymphoblastic leukemia. Biphenotypic acute leukemia patients showed a much higher incidence of CD34 antigen expression, complex abnormal karyotype, extramedullary infiltration, relapse, and resistance to therapy after relapse.

[Synergistic effects of arsenic trioxide and proteasome inhibitor bortezomib on apoptosis induction in Raji cell line].

The aim of this study was to explore the synergistic effect of arsenic trioxide and bortezomib on apoptosis of Raji cell line. The cells were treated with arsenic trioxide, bortezomib, low-dose arsenic trioxide combined with bortezomib, respectively. The cell viability and proliferative curve were estimated by trypan blue dye exclusion. The cell apoptosis and cell cycle status were analyzed by flow cytometry. The apoptosis related elements such as caspase-3, BCL-2, BAX, JNK2 and IkappaB-alpha, were measured with Western blot. The results showed that compared with cells treated with mentioned above drugs alone, the proliferative potential of cells in combination group was significantly inhibited (p < 0.01), and apoptosis rate markedly increased (p = 0.001), while obvious cell cycle arrest was not observed. On the protein level, the expression of Caspase-3, BAX and IkappaB-alpha increased, while the expression of BCL-2, and JNK2 decreased. It is concluded that low-dose arsenic trioxide combined with bortezomib synergistically induced apoptosis in Raji cell line which may be mediated by inhibiting NK-kappaB and JNK2 signaling.

[An analysis of prognostic factors in 80 myelodysplastic syndrome].

OBJECTIVE: To investigate the WHO classification, clinical and hematological features and risk group of International Prognostic Scoring System (IPSS) in patients with myelodysplastic syndrome (MDS). METHODS: The diagnosis and classification of MDS patients were defined according to the WHO classification. The clinical manifestations, hemogram, bone marrow biopsy and prognosis were retrospectively analyzed. RESULTS: The median age at diagnosis of MDS was 47 yrs being younger than that in some foreign reports. The frequency of abnormal karyotype was 35.14% and +8 was the most frequent abnormal karyotype in our study. Eleven of 74 patients transformed into leukemia. Univariate analysis showed that age, chromosome abnormality, percentage of bone marrow blast cells and number of cytopenias were significantly related to prognosis. There was a statistical difference in cum survival rate between IPSS subcategories (P < 0.05) except that between low- and intermediate I-risk subcategory (P > 0.05). There were statistical differences for refractory anemia (RA) vs RA with excess blast (RAEB), refractory cytopenias with multilineage dysplasia (RCMD) vs RAEB and RAEB-I vs RAEB-II (P < 0.05). CONCLUSIONS: There were differences in age of disease onset, distribution of WHO, sub-classification and abnormal karyotype in this cohort of MDS patients as compared with those in Europe and Japan. It is helpful in diagnosis, treatment and prognosis to divide RAEB into RAEB-I and RAEB-II. IPSS was well applicable in Chinese MDS patients.

[Effects of proteasome inhibitors on leukemias].

The proteasome is primarily responsible for intracellular protein degradation. The abnormality of its activity is sign of tumorigenesis. It was confirmed that proteasome inhibitors have activities against a variety of malignancies. Bortezomib, the first proteasome inhibitor, obtained permission of clinical trial and on sale. Multiple myeloma patients treated with bortezomib have gained a high overall response rate and complete remission rate. A lot of studies on effects of proteasome inhibitors on leukemias, including plasma cell leukemia; chronic lymphocytic leukemia, adult T cell lymphoma/leukemia, chronic myeloid leukemia and acute myeloid leukemia, were reviewed in this article.

[A clinical observation of fludarabine-containing regimens in the treatment of low grade non-Hodgkin's lymphoma].

OBJECTIVE: To evaluate the therapeutic efficiency and adverse effect of the fludarabine-containing regimens in the treatment of low grade non-Hodgkin's lymphoma. METHODS: Thirty-two patients with low grade non-Hodgkin's lymphoma consisting of 19 primary one and 13 relapsed or refractory were treated with fludarabine-containing regimens, which included FMD (fludarabine, mitoxantrone and dexamethasone); FMC (fludarabine, cyclophosphamide and mitoxantrone) and FC ( fludarabine and cyclophosphamide). RESULTS: The average course completed in these 32 patients was 4.1 with a complete response rate (CR), partial response rate (PR) and overall response rate (OR) of 65.6%, 18.8% and 84.4% , respectively. There were no significant difference in CR, PR and OR between primary and relapsed or refractory group (71.4%, 21.0%, 92.4% vs. 46.2%, 13.1%, 59.3%, respectively). Myelotoxicity and immunotoxicity was the dominating adverse effects. Ill to IV grade granulocytopenia and thrombocytopenia were observed in 31.3% (10/32) and 9.4% (3/32) of these patients respectively. Infection developed in 7 patients, and two of them died of pulmonary infection. The median follow-up period was 16 months (1-30 months) with 2-year overall-survival rate (OS) and progression-free survival rate (PFS) of 93.8% and 84.4%, respectively. No significant difference was observed between primary and relapsed or refractory group in OS (100% vs. 76.9%) and PFS (94.7% vs. 69.2%). CONCLUSION: Fludarabine-containing regimens is well tolerated and effective in the treatment of low grade non-Hodgkin's lymphoma.

[Spectrum of gene expression of a multi-drug resistant leukemia cell line with high tumorigenicity in nude mice].

OBJECTIVE: To investigate the mechanism of multi-drug resistance of K562-n/VCR cell line with both bcr-abl and mdr-1 expressions by clustering analysis of differential gene expression profiles. METHODS: By DNA microarray technique, genes differentially expressed by K562-n/VCR and K562-n cell lines were identified and analyzed. RESULTS: DNA microarray analysis of K562-n/VCR and K562-n cells was repeated three times and revealed 58 genes significantly differentially expressed among 12,800 genes arrayed. All but one was up-regulated in K562-n/VCR cells. The only gene down-regulated was a-myb. The up-regulated genes were MDR-associated genes, oncogenes, cytoskeleton, protein kinases and phosphatases, apoptotic and antiapoptotic factors, metabolism, transcriptional regulators associated with stress response, cell cycle checkpoint control, and genes for signal transduction proteins. CONCLUSION: These results indicate that, besides MDR-associated genes, other known and unknown genes may also be involved in the mechanism of multi-drug resistance.

[Establishment of a mouse model for detecting multi-drug resistant minimal residual leukemia cells expressing green fluorescent protein].

This study aimed to investigate the pathophysiology and therapy of multi-drug resistant model of minimal residual leukemia in mice. The multi-drug resistant model of minimal residual leukemia was established by using P388/VCR-G cell line expressing enhanced green fluorescent protein (EGFP) and DBA mice. The results showed that P388/VCR-G were inoculated in the abdominal cavities of DBA mice, the incidence of leukemia was 100%. Any of these mice with leukemia could not obtain remission spontaneously. The model of leukemia was sensitive to cyclophosphamide (Cy) and the time of survival was related to the dose of Cy received. The logarithm of cells inoculated in mice correlated regressionally with the dose of Cy. So this model was ideal for research on minimal residual leukemia. The distribution of residual leukemia cells in complete remission was not uniform in different organs including liver, spleen, thymus and bone marrow. Minimal residual leukemia cells could be found by fluorescent microscopy in freezing tissue slice. It is concluded that the multi-drug resistant model of minimal residual leukemia expressing EGFP can be established by using P388/VCR-G cell line and DBA mice. The minimal residual leukemia cells can be observed by fluorescence microscopy in complete remission stage.

[Differential gene expression analysis of human leukemic cell line with different tumorigenic potentials in nude mice].

OBJECTIVE: To study the molecular mechanism of tumorigenicity in nude mice of human leukemia cell lines. METHODS: K562-n, is a human leukemic cell line with much higher tumorigenecity in nude mice compared with the parental K562 cell line by repeated in vitro and in vivo passages. Genes differentially expressed between K562 and K562-n cells were analyzed by using DNA microarray technique. RESULTS: Of 12 000 genes screened were differentially expressed significantly, among which 42 genes were up-regulated and 97 genes were down-regulated in K562-n cells. Eighty-five of the 139 genes have been registered in the GeneBank and 54 are unknown genes. The genes accessible from the GenBank include: 1. oncogenes and tumor-supressor genes, 2. genes related to transcription regulation, cell cycle and apoptosis, 3. genes related to cytoskeleton and cytokinetics, 4. genes related to metabolism and transport, 5. genes related to immune function. There were also some differentially expressed genes with mixed functions and some with unknown function differentially expressed. CONCLUSION: There are many genes differentially expressed between K562-n and K562 cells. The high tumorigenicity in nude mice of human leukemia cell line K562-n might be related to its specific gene expression profile.

[Expression of d-amino acid oxidase gene and green fluorescence protein gene transferred into k562e cells by retroviral vector containing internal ribosome entry site sequence].

The internal ribosome entry site (IRES) sequence was derived from encephalomyocarditis virus. It allows to translate two open reading frames at one mRNA, so two genes conjoined by IRES have the same expression rate. K(DfGC) and K(DfGd) cell lines, stably expressing D-amino acid oxidase (DAAO) gene and green fluorescence protein (GFP) genes, were obtained by transfection of K562e cells with retroviral vector pLDfG containing IRES sequence, DAAO cDNA and GFP gene. Fluorescence positive rate and fluorescence intensity of the two cell lines were measured with flow cytometry. H(2)O(2) production by K(DfGC) and K(DfGd) cells treated with D-alanine was measured by the phenol red oxidation assay. The fluorescence positive rate and fluorescence intensity in K(DfGC) and K(DfGd) cell were 94.64% and 96.31% and 202 units and 174 units per 2 x 10(4) cells, respectively. There was exponential correlation between fluorescence intensity and H(2)O(2) level. The above-mentioned results demonstrate that DAAO gene and GFP gene were simultaneously expressed in K562e cell line by the regulation of IRES sequence, and DAAO level was correlated with fluorescence intensity of GFP.