Influence of initiation time and white blood cell count on the efficacy of cytotoxic agents in acute promyelocytic leukemia during induction treatment.

The present study retrospectively analyzed 96 newly diagnosed acute promyelocytic leukemia (APL) patients with low-intermediate mortality risk to identify the optimum timing to initiate cytotoxic chemotherapy following all-trans retinoic acid (ATRA) administration. Based on white blood cell (WBC) at chemotherapy initiation, the patients were divided into three groups: low WBC (WBC count ≤4×109/l), intermediate WBC (WBC count >4×109/l and <15×109/l) and high WBC group (WBC count ≥15×109/l). According to the period from ATRA commencement to chemotherapy, 96 patients were further divided into two groups: ≤3 days group (chemotherapy within 3 days of ATRA) and >3 days group (chemotherapy >3 days after ATRA). Clinical effects were compared by univariate analysis and multivariate analyses. The incidence rate of differentiation syndrome (DS; also termed retinoic acid syndrome) was 0.0, 11.1 and 40.0% in the low, intermediate and high WBC groups, respectively (P<0.001); complete remission (CR) rate was 90.5, 100.0 and 73.3%, respectively (P<0.001); and the rate of early mortality (defined as fatality during induction treatment) was 4.8, 0.0 and 26.7%, respectively (P<0.001). No differences were identified in clinicolaboratory parameters between the ≤3 days and >3 days groups, except in time to achieve CR (P=0.004) and rate of bleeding related to chemotherapy (P=0.009), both being higher in the >3 days group. Multivariate analyses indicated WBC count at chemotherapy was the only independent risk factor for the occurrence of DS [P=0.002; odds ratio (OR) =1.058, 95% confidence interval (CI) =1.021-1.095] and early mortality (P=0.036; OR =1.036, 95% CI =1.002-1.070). For newly diagnosed APL patients with low-intermediate risk, chemotherapy initiation should be recommended until WBC count rises to between 4×109/l and 15×109/l during induction treatment.

[Influence of FLT3-ITD Mutation and ITD Length on the Outcome on Patients with Acute Myeloid Leukemia].

OBJECTIVE: To explore the influence of FLT3-ITD mutation and ITD length on the overall survival (OS) and relapse free survival(RFS) in patients with non-M3 acute myeloid leukemia. METHODS: Clinical features and therapeutic effect were retrospectively analyzed in 75 AML patients with FLT3-ITD mutation and 76 FLT3-ITD- AML patients with a normal karotype from June 2011 to April 2016. Genomic DNA was amplified by PCR, and FLT3-ITD mutation length was analyzed by DNA sequencing in 40 patients. RESULTS: AML patients with FLT3-ITD mutation had higher WBC count and the ratio of BM blast cells at initial diagnosis was also higher than those in AML patients without FLT3-ITD mutation (95.13 vs 10.85)(P<0.01); 72% vs 59%(P<0.01). The CR rates in AML patients with FLT3-ITD mutation less than those in AML patients without FLT3-ITD mutation(70.42% vs 94.7%)(P<0.01). OS (P<0.01) and RFS (P<0.01) were significantly increased in patients with AML who received allo-HSCT as compared with the patients who received consolidation chemotherapy and similar to AML patients without FLT3-ITD mutation who received HSCT. Patients with maintenance sorafenib after HSCT had longer OS (P<0.05) and RFS (P<0.05) than controls. ITDs exceeding 60 bp in length were associated with decreasing OS as compared with shorter ITD in AML patients with FLT3-ITD mutation (P<0.05). OS and RFS were similar among the 2 groups receiving consolidation chemotherapy. Besides, the patients with allo-HSCT had shorter ITDs and longer OS than ITDs exceeding 60 bp (P<0.05) and similar to AML patients without FLT3-ITD mutation. CONCLUSION: AML patients with FLT3-ITD mutation has poorer outcome, among which the prognosis was worse in patients with ITD exceeding 60 bp, and the chemotherapy alone can not improve the prognosis of FLT3-ITD+. Allo-HSCT is an effective treatment for AML patients with FLT3-ITD mutation; Sorafenib appears to be an effective maintenance therapy after allo-HSCT in FLT3-ITD AML.

Association between the concentration of imatinib in bone marrow mononuclear cells, mutation status of ABCB1 and therapeutic response in patients with chronic myelogenous leukemia.

Low concentrations of imatinib (IM) in bone marrow cells have been linked with poor prognosis in patients with chronic myeloid leukemia (CML), which may be caused by the emergence of ATP-binding cassette transporter B1 (ABCB1) mutations. The aim of present study was to investigate how clinical outcomes vary among patients with different single nucleotide polymorphisms (SNPs) of ABCB1. A total of 48 adult patients with CML and higher than median ABCB1 mRNA levels were selected for testing of ABCB1 SNPs. In 28 of the 48 patients, the IM concentration and expression levels of human organic cation transporter 1 (hOCT1) and ABCB1 in bone marrow mononuclear cells (BMMCs) were also tested. Correlations between treatment outcomes and IM concentration or the SNP status of ABCB1 were analyzed. Patients were classified by therapeutic response as major molecular response (MMR) (n=11), complete cytogenetic response (CCyR) (n=19) and non-CCyR (n=18) groups. It was found that the concentration of IM in BMMCs of the CCyR group was significant higher than that of the resistant groups (P=0.013). In addition, the IM concentration was positively correlated with the expression of hOCT1 mRNA (R=0.456, P=0.033), but negatively correlated with the expression of ABCB1 mRNA (R=-0.491, P=0.015). Furthermore, the mRNA expression level of ABCB1 was not associated with therapeutic response, but SNPs of the ABCB1 gene were associated with the response to IM. In conclusion, the concentration of IM in BMMCs may be regulated by the ABCB1 gene, and SNPs of the ABCB1 gene predict the therapeutic response to IM in patients with CML.

Immunophenotypes and immune markers associated with acute promyelocytic leukemia prognosis.

CD2+, CD34+, and CD56+ immunophenotypes are associated with poor prognoses of acute promyelocytic leukemia (APL). The present study aimed to explore the role of APL immunophenotypes and immune markers as prognostic predictors on clinical outcomes. A total of 132 patients with de novo APL were retrospectively analyzed. Immunophenotypes were determined by flow cytometry. Clinical features, complete remission (CR), relapse, and five-year overall survival (OS) rate were assessed and subjected to multivariate analyses. The CD13+CD33+HLA-DR-CD34- immunophenotype was commonly observed in patients with APL. Positive rates for other APL immune markers including cMPO, CD117, CD64, and CD9 were 68.7%, 26%, 78.4%, and 96.6%, respectively. When compared with patients with CD2- APL, patients with CD2+ APL had a significantly higher incidence of early death (50% versus 15.7%; P = 0.016), lower CR rate (50% versus 91.1%; P = 0.042), and lower five-year OS rate (41.7% versus 74.2%; P = 0.018). White blood cell (WBC) count before treatment was found to be the only independent risk factor of early death, CR failure, and five-year mortality rate. Flow cytometric immunophenotype analysis can facilitate prompt APL diagnosis. Multivariate analysis has demonstrated that WBC count before treatment is the only known independent risk factor that predicts prognosis for APL in this study population.

Synergistic effect of panobinostat and bortezomib on chemoresistant acute myelogenous leukemia cells via AKT and NF-κB pathways.

In this study, we investigated the synergistic effects of panobinostat and bortezomib on adriamycin-resistant HL60/ADR cells and refractory acute myelogenous leukemia (AML) primary cells. Combination of both agents had synergistic cytotoxicity on these cells, and increased the sensitivity of HL60/ADR cells to adriamycin. Panobinostat plus bortezomib was shown to modulate multiple apoptotic and drug metabolic related molecules, including activation of caspases, down-regulation of XIAP, Bcl-2 and MRP1. These effects were likely to be mediated via inhibition of AKT and NF-κB pathways. These findings provide evidence for clinic protocols using panobinostat and borezomib to overcome drug resistance in refractory AML patients.

Imatinib and bortezomib induce the expression and distribution of anaphase-promoting complex adaptor protein Cdh1 in blast crisis of chronic myeloid leukemia.

Anaphase promoting complex cofactor Cdh1 plays a critical role in tumor suppression and genomic stability in cancer. However, its role in chronic myeloid leukemia (CML) remains unclear. We treated both wild-type and imatinib-resistant K562 cells with imatinib or nilotinib and bortezomib, respectively. The siRNAs of Cdh1 and Skp2 were designed and transiently transfected with HiPerFect transfection reagent into CML cells. Expression of Cdh1-Skp2-p27 pathway proteins were detected by Western blotting. Cell cycle, cell apoptosis and cellular morphology were detected by flow cytometry and Wright staining. Our study revealed that Cdh1 was expressed at lower levels in imatinib-resistant CML blast crisis (BC) patients than imatinib-sensitive ones. Moreover, imatinib and bortezomib induced cell cycle quiescence or arrest, upregulation and nuclear relocation of Cdh1 in CML cells. Furthermore, nilotinib and bortezomib resulted in upregulation of Cdh1 in imatinib-resistant CML cells. Conversely, Cdh1 silencing resulted in stabilization of Skp2 and Cdc20, subsequently promoting G1-S transition and formation of multinucleated cells. Our study shows that TKIs and bortezomib can regulate the cell cycle and cell apoptosis via regulation of the expression and redistribution of Cdh1 in CML-BC, which sheds light on the orchestration of crosstalk between TKIs and bortezomib in imatinib-resistant CML-BC. Additionally, Cdh1 tends to play an important role in maintenance of genomic stability, the detailed mechanisms deserve further study.

[Effect of RAD001 or plus LBH589 on the proliferation, apoptosis and drug resistance in chemoresistant acute myeloid leukemic cells].

OBJECTIVE: To investigate the effects of everolimus (RAD001) or plus panobinostat (LBH589) on the proliferation, apoptosis and drug resistance in chemoresistant acute myeloid leukemic cells. METHODS: HL-60/ADM cells were treated with RAD001 alone or with LBH589. Proliferation and apoptosis were evaluated by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, Hoechst33342 and AnnexinV-FITC/PI stain. The altered expressions of multidrug resistance-associated protein 1 (MRP1) and intercellular adriamycin accumulation were analyzed by flow cytometry. The change in protein level was analyzed by Western blot. RESULTS: Effective proliferative inhibition and apoptotic induction in HL60/ADM cells were observed in the treatment of 10 - 50 µmol/L RAD001. The maximal effect was shown for the concentration of 30 µmol/L RAD001 at 48 and 72 hours. The inhibition ratio remained unchanged with the adjustment of drug doses (P < 0.05). Moreover, there was no synergistic effects in the treatment with different concentration of RAD001 and LBH589 (CI ≥ 1.0). A down-regulation of MRP1 (93.9% ± 4.2% vs 79.10 ± 3.28%) and an up-regulation of adriamycin (8.53 ± 0.68% vs 15.37% ± 1.46%) were induced by the treatment with 10 µmol/L RAD001 (both P < 0.01). RAD001 inhibited the p53-dependent expression of MRP1 via an inhibition of phosphoinositide 3-kinase (PI3K)/Akt/mTOR signaling pathway. CONCLUSION: The combined treatment of RAD001 and LBH589 has no synergistically inhibitory effect on HL60/ADM cells. But the sole treatment of RAD001 may inhibit proliferation, induce apoptosis and accumulate intercellular adriamycin through a down-regulated expression of MRP1 in HL60/ADM cells via an inhibition of PI3K/Akt/mTOR signaling pathway.

[Reversal effect of LBH589 alone or in combination with bortezomib on drug-resistance in myeloid leukemia and its mechanism].

OBJECTIVE: To investigate reversal effect of histone deacetylase inhibitor LBH589 alone or in combination with proteasome inhibitor bortezomib on drug resistance in acute myeloid leukemia (AML) and its mechanism. METHODS: Ex vivo cultures of HL-60/ADM cells and fresh refractory AML cells were treated with LBH589, bortezomib or their combination at varying concentrations. Proliferation capacity, apoptosis rate and reversal of drug resistance were evaluated by MTT assay, dual staining of Hoechst 33342 and Annexin VFITC/PI by flow cytometry, and adriamycin uptake rate with proliferation inhibition, respectively. The change of signal pathway at protein level was analyzed by Western blot. RESULTS: Synergistic cytotoxicity was observed in the combination treatment with LBH589 and bortezomib against HL-60/ADM cells, as well as the fresh AML cells, the most powerful synergy being observed at 21 nmol/L LBH589 plus 12 nmol/L bortezomib, with CI values of 0.531 and 0.498, respectively by Calcusyn software analysis. Moreover, the accumulation of adriamycin in HL-60/ADM cells was increased more in combination treatment [(64.81 +/- 3.69)%] than in either LBH589 [(28.96 +/- 2.52)%] or bortezomib [(37.29 +/- 3.71)%] alone (P < 0.05), and so did the uptake rate of adriamycin being (64.81 +/- 3.69)%, (28.96 +/- 2.52)% and (37.29 +/- 3.71)% respectively (P < 0.05). The combination treatment induced multiple apoptotic molecules co-action and intracellular drug accumulation contributed to the synergistic cytotoxicity, including caspase activation, PARP cleavage, XIAP downregulation, p53-dependent suppression of Bcl-2 and MRP1 expression via the inhibition of phosphoinositide 3-kinase (PI3K)/Akt/nuclear factor-kappaB (NF-kappaB) signaling pathway. CONCLUSIONS: Combination treatment of drug resistant AML cells with LBH589 and bortezomib produces a synergistic effect of in creating sensitivity to chemotherapy. The mechanism may be mainly resulted from inhibition of PI3K/ Akt/NF-kappaB signaling pathway.

[Gene expression of breast cancer resistance protein in adult acute lymphocytic leukemia and its clinical significance].

The objective of this study was to investigate the relationship between the expressions of breast cancer resistance protein (BCRP) gene and drug resistance as well as prognosis in adult patients with acute lymphocytic leukemia (ALL). Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of BCRP gene in 97 adult patients with acute lymphocytic leukemia (ALL) and 30 normal subjects. Beta(2) microglobulin (beta(2)-MG) was used as an internal reference. BCRP/beta(2)-MG ratio >or= 0.48 was defined as BCRP positive. The results showed that the positive ratio of BCRP gene expression in untreated group was 28.7%, in contrast that in refractory and relapsed patients was 51.2%. The first complete remission rates in patients with negative and positive expression were 71.7% and 29.7% respectively. In ALL subtypes BCRP gene expression of L3 type was distinctly higher than that of L(1), L(2) types. In immune types the BCRP gene expression of B-ALL was higher than that of T cell type, especially in mature B cell type with obviously statistical significance (p<0.01). It is concluded that the high expression of BCRP gene may induce clinical drug resistance, and may be an unfavorable factor for prognosis in adult patients with acute lymphocytic leukemia.