ALBERT

Description: Modern clinical treatments of childhood acute lymphoblastic leukemia (ALL) employ enzyme-based methods for depletion of blood asparagine in combination with standard chemotherapeutic agents. L-asparaginase (L-asp) therapy causes depletion of plasma asparagine followed by the loss of intracellular asparagine. Due to the lack of a rapid up-regulation of asparagine synthetase (ASNS) protein content in ALL cells, they are preferentially killed by L-asp. Elevated expression of ASNS within the leukemia cells causes decreased sensitivity to L-asp. The proof of ASNS deficiency in leukemia cells is considered to predictive for effectiveness of L-asp even in acute myeloid leukemia (AML) other than ALL patients. The establishment of quantitative estimation of ASNS protein content would be useful for the L-asp treatment in leukemia therapy. Objective: Our aim was to set up a flow cytometry system to check ASNS deficiency in leukemia cells and to investigate the sensitivity to L-Asp and the ASNS expression in AML leukemia cells. Methods: AML (KG-1, HL-60, U937) and ALL (MOLT-4, RS4;11) and CML (K562) cell lines were grown in RPMI1640 medium with 10% FCS. Primary leukemic cells from the peripheral blood or bone marrow of 20 AML patients were harvested on EDTA and isolated by Ficoll density gradient within 72h. ASNS expression was evaluated by cytosolic flow cytometry with Z5808 McAb (Hybridoma 31: 325-332.2012) and expressed as a ΔMFI(Difference of Mean Fluorescence Intensity(MFI) between by Z5808 and isotypic control) or MFI ratio(MFI by Z5808/MFI by isotypic control). When a sufficient amount of leukemic cells was available, sensitivity to L-asp (expressed as an IC50 - concentration inhibiting 50% of cell viability) was evaluated in vitro by incubating various concentrations of E. coliL-asp with the cells and by measuring the cell viability with a counting kit (WST1 viability assay) at day 3. Results: Determination of IC50 for the HL-60 (⊿MFI 48 ± 8.01, MFI ratio 1.77 ± 0.03) and U937 (⊿MFI 16.7 ± 0.47, MFI ratio 1.19 ± 0.02) demonstrated that these cells were equally sensitive to L-asp than the ALL cell line MOLT-4 in vitro (0.37 and 0.02IU/mL versus 0.15 IU/mL, respectively). K562 and KG-1 (⊿MFI 135.7 ± 5.66, MFI ratio 2.48 ± 0.09) cells with the highest ASNS expression exhibited resistance to L-asp (〉10 IU/ml). Both of ASNS Expression by ⊿MFI and MFI ratio was inversely correlated with L-asp sensitivity judging from cell line studies. Judging from cell line study, the threshold for ASNS protein expression effective for L-asp treatment was considered to be

Description: Karyotype is an important prognostic factor in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL), but the underlying pharmacogenomics remains unknown. Asparaginase is an integral component in current chemotherapy for childhood BCP-ALL. Asparaginase therapy depletes serum asparagine. Normal hematopoietic cells can produce asparagine by asparagine synthetase (ASNS) activity, while ALL cells are unable to synthesize adequate amounts of asparagine. The ASNS gene has a typical CpG island in its promoter. Thus, methylation of the ASNS CpG island could be one of the epigenetic mechanisms for ASNS gene silencing in BCP-ALL. To gain deep insights into the pharmacogenomics of asparaginase therapy, we investigated the association of ASNS methylation status with asparaginase sensitivity. ASNS CpG island is largely unmethylated in normal hematopoietic cells but is allele-specifically methylated in BCP-ALL cells. The ASNS gene is located at 7q21, an evolutionally conserved imprinted gene cluster. ASNS methylation in childhood BCP-ALL is associated with an aberrant methylation of the imprinted gene cluster at 7q21. Aberrant methylation of mouse Asns and a syntenic imprinted gene cluster is also confirmed in leukemic spleen samples from ETV6-RUNX1 knock-in mice. In three childhood BCP-ALL cohorts, ASNS is highly methylated in BCP-ALL with favorable karyotypes but is mostly unmethylated in BCP-ALL with poor prognostic karyotypes. Higher ASNS methylation is associated with higher l-asparaginase sensitivity in BCP-ALL through lower ASNS gene and protein expression levels. These observations demonstrate that silencing of the ASNS gene due to aberrant imprinting is a pharmacogenetic mechanism for the leukemia-specific activity of asparaginase therapy in BCP-ALL.

Description: Introduction The incidence of irregular erythrocyte allo-antibodies (Abs) increases with the amount of previous erythrocyte transfusions. Blood transfusion has been one of the most important supportive cares in patients with hematological diseases. Therefore, patients with hematological diseases, such as leukemia and myelodysplastic syndrome, have often received considerable amounts of blood transfusion, and have a higher risk for alloimmunization against erythrocyte antigens. On the other hand, patients receiving chemotherapy and immunotherapy exhibit less antibody response than do patients with non-hematological diseases. Several authors reported that the frequency of irregular erythrocyte Abs was unexpectedly low in these patients (Schonewille et al., 2009). In this study, we retrospectively analyzed the frequency and the contents of Abs after blood transfusion in patients with hematological and non-hematological diseases. Materials and Methods We selected patients with hematological or non-hematological diseases, who were transfused in our hospital from 2000 to 2011. We analyzed the patients' profiles including gender, age, the number of blood units previously transfused, and category of transfused products. We also studied the frequencies of irregular erythrocyte Abs. If the same patient was tested more than once, it was counted as one case. If more than two antibodies were detected in the same blood sample, they were tallied separately. If a patient had different antibodies at different times, all of them were summarized. We compared antibody frequencies between the patients with hematological or non-hematological diseases. Statistical analysis was performed by chi-square test and F-test followed by Student's t-test. Results The numbers of patients with hematological or non-hematological diseases were 517 and 4,311 cases, respectively (Table 1). Gender was similar (male / female: 1.35 vs. 1.38, NS). Median age was 64 years (range: 15-93) vs. 75 years (2-82) (p〈 0.001). The median amount of transfused erythrocytes was 18 units (2-358) and 8 units (1-182), respectively. Abs were detected in 24 (4.6%) and 129 cases (2.9%), respectively (p〈 0.05). Frequently determined Abs were as follows: anti-E (63% vs. 34%), anti-Lea (13% vs. 23%), anti-C (4% vs. 5%), anti-Dia (4% vs. 5%), anti-Jka (4% vs. 6%), and anti-E+c Ab(4% vs. 8%, respectively) (Fig. 1). The amount of erythrocyte transfusions until determination of Abs was 19 units (10-100) and 14 units (2-84), respectively. Discussion The frequency of irregular erythrocyte Abs was significantly greater in patients with hematological diseases than in those with non-hematological diseases. The amount of erythrocyte transfusions was greater and age was younger in those with hematological diseases. Anti-E Ab, whose frequency is reportedly less in Japanese, was more frequently detected in those with hematological diseases, while non-Rh Abs were more frequently detected in those with non-hematological diseases. Analyses after the exclusion of perioperative transfusion showed that the amount of erythrocyte transfusion until determination of Abs was greater in those with hematological diseases. These results showed that irregular Abs were more frequently detected in patients with hematological diseases, but the Abs are poorly productive in these patients after the same amount of transfusion. Further studies will solve the detailed mechanisms. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Advances in chemotherapy have improved the outcome of childhood acute lymphoblastic leukemia (ALL). However, leukemia cells in refractory ALL are often resistant to anti-leukemic agents. Although recent studies have focused on the epigenetic changes in refractory leukemia, the relationship between the demethylating agent 2′-deoxy-5-azacytidine (decitabine, DAC) and ALL remains unclear. Here, we examine the combined effects of DAC and anti-leukemic agents such as clofarabine (CLO) and etoposide (ETO) on the ALL cell line CCRF-CEM. Methods and results In vitro drug sensitivity was measured using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. We cultured CCRF-CEM cells for 72 hours with or without DAC, and then removed DAC (when present) prior to culturing CCRF-CEM cells for 48 hours with ETO or CLO, or without chemotherapeutic drugs. After culturing for 48 hours, we removed the chemotherapeutic drugs and measured in vitro drug sensitivity using MTT assay. The MTT assay was performed in triplicate. We then evaluated the inhibitory concentration at 50% (IC50). IC50 for ETO, ETO+DAC, CLO, and CLO+DAC was 3.36, 0.625, 4.96, and 1.92, respectively. The combination Index (CI) was produced with Calcusyn® software, which uses the methodology of Chou and Talalay to perform formal synergy analyses. A CI 〈 1 indicated a synergistic effect. The CI was 0.026 for ETO+DAC and 0.431 for CLO+DAC. We assayed with Annexin-V, PI staining, and caspase-3/7 to detect apoptosis. We observed apoptosis rates of 31.6%, 53.3%, 31.2%, and 52.6% for ETO, ETO+DAC, CLO, and CLO+DAC, respectively. We observed greater caspase-3/7 activity with DAC+CLO and DAC+ETO than with CLO and ETO. Using real-time reverse transcription polymerase chain reaction (RT-qPCR) in CCRF-CEM cells, we examined mRNA expression levels for the pro-apoptotic genes BAK, BID, BAX, BAD, BIM, PUMA, ATM, TP53, and NOXA, as well as those for the anti-apoptotic genes BCL2, BCL2L1, and XIAP. The expression level of each target gene was calculated by normalizing it to the housekeeping gene GAPDH. The RT-qPCR was performed in triplicate. We used Student’s t test to compare the data. We observed DAC increased mRNA expression levels of BAX and NOXA, but decreased those for BAK, BID, PUMA, BCL2L1, ATM, TP53, and XIAP. We then analyzed the methylation status of pro- and anti-apoptotic genes after 48 hours incubation with or without DAC. Methylation status of BAK, NOXA, BCL2L1 and XIAP incubation with DAC was 1.3%, 3.3%, 2.5% and 72.9%, respectively. Methylation status of BAK, NOXA, BCL2L1 and XIAP incubation without DAC was 1.9%, 3.6%, 0.7% and 92.3%, respectively. There was no significant difference. Discussion Our results showed that DAC synergistically enhances CLO and ETO cytotoxicity, and this cytotoxic effect depends on caspase-3/7 activity. We examined mRNA expression levels of pro- and anti-apoptotic genes. We hypothesized that DAC would increase mRNA expression levels of most pro-apoptotic genes, and decrease mRNA levels of most anti-apoptotic genes. We found that DAC decreased some pro-apoptotic genes, such as BAK, BID, PUMA, ATM, and TP53, which disproves our hypothesis. Our present findings are similar to those of Shin et al., who reported that DAC decreased BID mRNA expression levels. However, they provided no explanation for this activity. Our results show that DAC did not demethylate the CpG of BAK, NOXA, BCL2L1, or XIAP. Thus, DAC must demethylate the CpG of other genes. Nevertheless, many genes are involved in apoptosis, and it remains unclear which genes are demethylated by DAC. Disclosures: Sakaguchi: Yakult Honsha Company: Research Funding; Japan Leukemia Research Fund: Research Funding; Japan Society for the Promotion of Science: Research Funding; Sanofi: Research Funding; Teijin Pharma: Research Funding.

Description: Autophagy is an intracellular protein and organelle degradation system, and is upregulated in response to cellular stress such as amino acid starvation. On the other hand, L-asparaginase (L-asp) plays an essential role in acute lymphoblastic leukemia (ALL) therapy by reducing intracellular asparagine and glutamine in ALL cells. However, the relationship of L-asp and autophagy is largely unknown. Here we show that L-asp induced cytoprotective autophagy. Three ALL cell lines of varied genetic background were used for in vitro experiments (REH, ETV6-RUNX1+ B-cell precursor (BCP) ALL; 697, E2A-PBX1+ BCP-ALL; TS-2, MEF2D-DAZAP1+ BCP-ALL). The cells were exposed to chroloquine (CQ) or bafilomycin A1 as autophagy inhibitors for 3 hours. LC3B-II, autophagy flux marker, was significantly increased under L-asp treatment with CQ as compared to only CQ condition, which was confirmed in independent experiments at immunofluorescence staining. Transmission electron microscopy showed that both the number and the area of autophagic vesicles per cell were markedly increased in L-asp with CQ condition. Thus, autophagy was induced by L-asp increasing turnover and clearance of autophagosomes in ALL cells. The toxic effect of 4 groups (control, CQ, L-asp, and L-asp plus CQ) by flow cytometry using Annexin-V staining indicated that combination treatment with L-asp and CQ for 48 hours induced significant cell death in the three ALL cell lines. Furthermore, inhibition of autophagy by CQ comparably sensitize REH cells to L-asp as ATG7 silencing by short interfering RNA. Cell growth assays for 6-9 days showed that L-asp monotreatment suppressed cell growth but did not increase the percentage of dead cells. In contrast, combination treatment with L-asp and CQ decreased the number of living cells and significantly increased the percentage of dead cells in time-dependent manner. Cell cycle analysis showed that cell cycle arrest at G1 phase was induced and the percentage of cells in sub-G1 phase remained a small increase by L-asp monotreatment, indicating leukemia cells endured amino acid deficiency by G1 arrest. In contrast, addition of CQ to L-asp significantly increased the sub-G1 population instead of decreasing G1 population. The apoptosis-related protein expressions using western blot analysis showed that combination treatment with L-asp and CQ induced cleavage of caspase 3 and PERP. In addition, a pan-caspase inhibitor benyloxycarbonyl Val-Ala-Asp (O-methyl)-fluoro-methylketone (z-VAD) significantly reduced the percentage of Annexin-V positive cells in the combination treatment with L-asp and CQ, which suggested that the autophagy inhibition upon L-asp treatment induced apoptotic cell death. We next transduced REH cell line with a luciferase-expressing viral vector. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice were transplanted with these cells via tail vein and 6,000 U/kg L-asp and/or 50 mg/kg CQ were injected intraperitoneally once per day for survival analysis. The combination treatment with L-asp and CQ clearly reduced the leukemia burden as detected by luciferase intensity and improved outcome (L-asp plus CQ vs L-asp at day 28 after administration: 82% vs 0%, respectively. p

Description: Abstract 4962 Juvenile myelomonocytic leukemia (JMML) is an aggressive clonal malignancy and mixed myeloproliferative and myelodysplastic disorder. Although cure in most cases requires hematopoietic stem cell transplantation (HSCT), the major cause of treatment failure is relapse. However, in some cases, symptoms improve without treatment. We report a case of a patient with JMML who sustained remission after graft rejection of an unrelated bone marrow transplantation (UBMT). Case An 18-month-old girl presented with marked splenomegaly and hemorrhagic diathesis. Laboratory blood tests revealed the following: white blood cell (WBC) count 12. 2 × 109/L, monocytes 22. 0%, hemoglobin 7. 6 g/dL, platelets 10. 0 × 109/L, and fetal hemoglobin 12. 8%. A bone marrow aspirate revealed a hypercellular marrow with mild dysplastic changes and 4. 4% blast cells. The BCR–ABL fusion gene was not detected. Following a diagnosis of JMML, she subsequently developed respiratory failure due to leukemic infiltration of the lungs, and was referred to our hospital. On admission, she developed severe thrombocytopenia due to splenic sequestration of platelets, and she needed frequent transfusions. She received chemotherapy with cytarabine and 6-mercaptopurine. Pulmonary leukemic infiltration improved, but transfusion frequency could not be reduced. After she had undergone splenectomy, platelet transfusion was not needed. When her clinical condition had improved, KRAS mutation was investigated by bone marrow aspiration, and the KRAS 13G〉D mutation was detected. Five months after diagnosis, she was transplanted with major mismatch blood type, HLA-A 1-allele mismatch, from an unrelated female donor. The conditioning regimen consisted of busulfan (BU; 16 mg/kg), fludarabine (Flu; 120 mg/m2), and cyclophosphamide (CY; 120 mg/kg). Short-term methotrexate and tacrolimus (FK506) were administered for the prevention of graft-versus-host disease. The level of infused donor marrow cells was 1. 18 × 108/kg. Recovery of peripheral blood count was rapid, and no regimen-related toxicity was observed. Chimerism by short tandem repeat analysis of bone marrow mononuclear cells on day 28 after UBMT was 100% recipient type, indicating graft rejection with autologous hematopoietic cell recovery. FK506 was then discontinued. From day 48 after UBMT until the current day, WBC count has been almost 10. 0 × 109/L. Despite graft rejection, the KRAS 13G〉D mutation was not detected by bone marrow aspiration on day 219, and her peripheral blood counts were normalized. Four years after diagnosis, the KRAS 13G〉D mutation in the peripheral blood, nails, buccal mucosa, and hair was not detected, but the KRAS13G〉D mutation was not. She has been managed without treatment and remained in complete remission for over 5 years since receiving UBMT. Discussion In JMML patients with specific RAS mutations, spontaneous improvement in hematologic abnormalities has been reported. HSCT was needed in this case because the patient developed respiratory failure due to pulmonary infiltration of JMML cells. In JMML patients with gene mutation, JMML-specific gene mutations could not be detected after engraftment of HSCT. In contrast, most JMML cases relapse and need a second HSCT after rejection of the first. However, this patient's condition normalized after rejection of UBMT. Nowadays, minimal residual disease in JMML is analyzed by detection of JMML cell-specific gene mutations. The KRAS mutation can be detected in spontaneously regressed JMML following hematological improvement. We suggest that a myeloablative conditioning regimen including BU, Flu, and CY could eradicate JMML clones, and in some JMML cases, this could prevent the need for a second HSCT after rejection of the first. Disclosures: No relevant conflicts of interest to declare.