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Objectives: Procalcitonin (PCT) is an early diagnosis marker of sepsis/bacteremia. However, some reports refer to its lower responsiveness to gram-positive bacteremia. We retrospectively evaluated the PCT values at the onset of bacteremia in relation to severity index. Methods: Patients with bacteremia caused by two gram-negative bacteria (46 E. coli and 50 Klebsiella pneumoniae) and three gram-positive bacteria (45 S. aureus, 56 S. epidermidis, and 10 S. mitis) were studied. The plasma PCT and C-reactive protein (CRP) levels were compared between species and different Sequential Organ Failure Assessment (SOFA) score groups. Results: The median PCT level was higher in gram-negative than in gram-positive bacteremia in overall (13.09 vs. 0.50 ng/mL, p

Due to an increased incidence of copper deficiency, we investigated adult patients who had low serum levels of copper with cytopenia at our hospital from March 2014 to March 2021.We retrospectively reviewed the clinical data of patients who had been diagnosed with cytopenia due to copper deficiency at the Aichi Medical University Hospital from March 2014 to March 2021.In the 15 patients with cytopenia secondary to low serum copper level, 11 had cytopenia of two to three lineages; three (27%) had pancytopenia, and eight (73%) had bicytopenia. Of the 15 patients, nine (60%) underwent bone marrow examinations; three (30%) showed typical morphologic features associated with copper deficiency, such as multiple clear cytoplasmic vacuoles in erythroblasts and myeloid cells, and three (30%) showed dysplastic features as observed in myelodysplastic syndrome. Among the 14 (93%) patients who were treated with copper supplements, had cessation of zinc supplements, or both, 11 (73%) and eight (53%) showed normal copper levels and hematological improvement, respectively.Copper deficiency is more common than expected and should be considered in patients with unexplained cytopenia.

Linezolid has been the common antimicrobial treatment for Gram-positive infection even in neonates and infants. Major adverse events associated with linezolid treatment is cytopenia. However, there were few reports about the relationship between cytopenia and gestational age. Primary objective of this study was to compare the relationship between cytopenia in infants and neonates treated with linezolid therapy and gestational age.In total, 44 patients were divided into two groups depend on their gestational age [180 days; low gestational age group (20 patients);180 days group; high gestational age group (24 patients)]. All patients treated with linezolid from April 2014 to March 2018 at NICU or GCU of Aichi Medical University Hospital. Investigation items were as follows; sex, age, weight, duration of treatment, Apgar score, laboratory data, rate of patients with blood transfusion, concomitant medications, hematologic abnormalities during linezolid treatment.The incidence of overall cytopenia in low gestational age group was significantly higher than high gestational age group (65.0 % vs. 25.0 %; p0.05). Of note, the incidence of thrombocytopenia in low gestational age group showed significantly higher than high gestational age group (45.0% vs. 8.3%, p 0.05). Then, the proportion of patients occurred thrombocytopenia who received linezolid 10 mg/kg every 8 h were higher than 10 mg/kg every 12 h in both groups.In cases linezolid is administered three times a day should be more carefully of thrombocytopenia in patients with gestational days less than 180 days.

We herein report a case of a combined crystalline light chain tubulopathy, podocytopathy, histiocytosis, and cast nephropathy in a patient with monoclonal gammopathy of renal significance (MGRS). A 66-year-old female with impaired renal function was referred to our department. Despite intravenous fluid resuscitation, the kidney function worsened progressively; thus, a kidney biopsy was performed. The kidney biopsy revealed light chain proximal tubulopathy (LCPT) with crystals, light chain crystal podocytopathy (LCCP), crystal-storing histiocytosis (CSH), and light chain cast nephropathy (LCCN). Of note, LCCP and CSH were diagnosed via electron microscopy. Serum and urine immunoelectrophoresis (IEP) revealed the presence of monoclonal Bence–Jones protein and free κ light chains. Bone marrow aspiration showed

Unbalanced translocation der(1;7)(q10;p10) is a characteristic chromosomal abnormality in myelodysplastic syndrome (MDS). The current study revealed that among 13 MDS patients with der(1;7)(q10;p10), seven cases with no apparent dysplasia also had low numbers of myeloblasts in the bone marrow and a 3-year survival rate of 86%; in contrast, the remaining six cases had a 3-year survival rate of 0% (

We report the case of a patient with fulminant pneumococcal infection along with the presence of Howell-Jolly bodies (HJBs) and splenic hypoplasia at the onset. A 71-year-old man developed fever during outpatient chemotherapy for IgG-κ multiple myeloma and was diagnosed with septic shock due to invasive pneumococcal infection. HJBs were observed on peripheral blood smears at this visit. Computed tomography revealed marked hypoplasia of spleen, suggesting the presence of hyposplenic function. Antibacterial therapy was initiated and the pneumococcal infection was cured; however, there was no notable change in his splenic hypoplasia. Splenic hypoplasia can be associated with fatal infections; hence, care should be taken when it is found in the elderly and in patients with cancer and those receiving immunosuppressive treatment. Even today, when automated hematology analyzers have become common, not all patients with hematological diseases have peripheral blood smears checked with a normal optical microscope. This study suggests that HJBs may be useful for simple and rapid screening of splenic hypofunction. The importance of detecting HJBs in peripheral blood smears with a normal optical microscope should be re-recognized.

Chromosome band 8q24 is the most frequently amplified locus in various types of cancers. MYC has been identified as the primary oncogene at the 8q24 locus, whereas a long noncoding gene, PVT1, which lies adjacent to MYC, has recently emerged as another potential oncogenic regulator at this position. In this study, we established and characterized a novel cell line, AMU-ML2, from a patient with diffuse large B-cell lymphoma (DLBCL), displaying homogeneously staining regions at the 8q24 locus. Fluorescence in situ hybridization clearly detected an elevation in MYC copy numbers corresponding to the homogenously staining region. In addition, a comparative genomic hybridization analysis using high-resolution arrays revealed that the 8q24 amplicon size was 1.4 Mb, containing the entire MYC and PVT1 regions. We also demonstrated a loss of heterozygosity for TP53 at 17p13 in conjunction with a TP53 frameshift mutation. Notably, AMU-ML2 cells exhibited resistance to vincristine, and cell proliferation was markedly inhibited by MYC-shRNA-mediated knockdown. Furthermore, genes involved in cyclin D, mTOR, and Ras signaling were downregulated following MYC knockdown, suggesting that MYC expression was closely associated with tumor cell growth. In conclusion, AMU-ML2 cells are uniquely characterized by homogenously staining regions at the 8q24 locus, thus providing useful insights into the pathogenesis of DLBCL with 8q24 abnormalities.

This retrospective study is to evaluate the efficacy and safety of daptomycin (DAP) intermittent doses and the effectiveness of DAP loading dose in renal failure patients received DAP intermittent doses. One hundred and ninety-seven patients received DAP for at least 3 days from 2014 to 2017. Clinical and microbiological outcomes and the safety were assessed. A total of 183 patients (93, 60 and 30 patients received DAP daily dose, every 48 h dose and thrice per week dose) were included. DAP intermittent doses, such as every 48 h dose (28.3%) and thrice per week dose (30.0%), showed significantly higher mortality rates than that of DAP daily dose (6.5%) (p = 0.0320). Especially for bacteremia patients, significantly higher mortality was admitted, compared with patients received DAP daily doses (p = 0.0160). Moreover, patients received DAP intermittent doses were admitted slower improvements of their inflammation after DAP therapy started, compared with patients received daily dose. Additionally, DAP loading dose for renal failure patients decreased their mortality and improved patients' inflammation early. Especially for patients received DAP thrice per week dose, they showed significantly lower mortality than patients received non-loading dose (p = 0.0306). Additionally, these clinical enhancements of DAP therapy with loading dose were admitted without any enhancements of its adverse effect risks, except alkaline phosphatase elevation, compared with non-loading dose. In conclusion, DAP intermittent doses showed poor clinical outcomes, compared with daily dose. Then, DAP loading dose would be better clinical option for patients received DAP intermittent doses.

Background/purpose: Acinetobacter is an aerobic, gram-negative coccobacillus, which causes nosocomial infections including bacteremia. Recent development of molecular techniques has made classification of the Acinetobacter genomospecies possible, but there are still only a few studies comparing clinical features of the subspecies. We investigated bacteremia caused by Acinetobacter, isolated subspecies, and compared clinical features for each group. Methods: A retrospective analysis of Acinetobacter bacteremia cases was made in a 900-bed hospital in Japan. In addition to conventional procedures, subspecies identification based on rpoB sequence was made, and comparison of clinical characteristics between each subspecies were analyzed. Results: We collected 35 cases (Acinetobacter baumannii 14, A. nosocomialis 12, Acinetobacter ursingii 6, and A. seifertii 3). All of the A. seifertii bacteremia cases were blood stream infection occurring in cerebrovascular disease patients, showing particularly higher incidence of shock (100%) and high Pitt bacteremia score (PBS) (6.33 ± 2.52) in comparison to A. baumannii (43% and 2.86 ± 2.25, respectively). Sequential Organ Failure Assessment (SOFA) score and the PBS were slightly higher in A. nosocomialis in comparison to A. baumannii, and the 7 day mortality rate was higher in A. nosocomialis (25%) than in A. baumannii (7%), though this difference was not found to be significant. Conclusions: A.seifertii, the recently defined novel species, showed distinctive clinical features of bacteremia. And, in contrast to previous studies, the severity of A. nosocomialis infection was not lower than that of A. baumannii, which might suggest the influence of local epidemiology. Further characterization of these subspecies should be continued. Keywords: Acinetobacter seifertii, Acinetobacter nosocomialis, Acinetobacter baumanii, Bacteremia, rpoB, Genomospecies

The shift in energy metabolism from oxidative phosphorylation to glycolysis can serve as a target for the inhibition of cancer growth. Here, we examined the metabolic changes induced by 2-deoxyglucose (2-DG), a glycolysis inhibitor, in leukemia cells by metabolome analysis. NB4 cells mainly utilized glucose as an energy source by glycolysis and oxidative phosphorylation in mitochondria, since metabolites in the glycolytic pathway and in the tricarboxylic acid (TCA) cycle were significantly decreased by 2-DG. In THP-1 cells, metabolites in the TCA cycle were not decreased to the same extent by 2-DG as in NB4 cells, which indicates that THP-1 utilizes energy sources other than glucose. TCA cycle metabolites in THP-1 cells may be derived from acetyl-CoA by fatty acid β-oxidation, which was supported by abundant detection of carnitine and acetylcarnitine in THP-1 cells. 2-DG treatment increased the levels of pentose phosphate pathway (PPP) metabolites and augmented the generation of NADPH by glucose-6-phosphate dehydrogenase. An increase in NADPH and upregulation of glutathione synthetase expression resulted in the increase in the reduced form of glutathione by 2-DG in NB4 cells. We demonstrated that a combination of 2-DG and inhibition of PPP by dehydroepiandrosterone (DHEA) effectively suppressed the growth of NB4 cells. The replenishment of the TCA cycle by fatty acid oxidation by carnitine palmitoyltransferase in THP-1 cells, treated by 2-DG, might be regulated by AMPK, as the combination of 2-DG and inhibition of AMPK by compound C potently suppressed the growth of THP-1 cells. Although 2-DG has been effective in preclinical and clinical studies, this treatment has not been fully explored due to concerns related to potential toxicities such as brain toxicity at high doses. We demonstrated that a combination of 2-DG and DHEA or compound C at a relatively low concentration effectively inhibits the growth of NB4 and THP-1 cells, respectively. These observations may aid in the identification of appropriate combinations of metabolic inhibitors at low concentrations which do not cause toxicities.

HIV陽性のため当院を紹介された31歳男性.入院後HAART (AZT, 3TC, NFV)療法を開始. 4病日に間質性肺炎を合併し,ステロイドパルス療法施行. 14病日,サイトメガロウイルス(CMV)抗原陽性のためガンシクロビル(GCV)の点滴を開始. 19病日,大腸穿孔し緊急手術にて人工肛門造設.病理検査でCMV腸炎の診断を得た. GCV点滴するもCMV抗原の陰性化得られず,ホスカビルにて抗原陰性化得られたが,休薬後に抗原陽性化.次はGCV点滴にて効果が得られたが,点滴終了後に再度陽性化したため, GCV点滴後維持量として3.000mg/日を経口で開始した. CMV抗原が陰性化して退院した. CMV抗原陰性化の維持にGCV経口療法は有用であった.

We examined the effects of diet nutrients on xenotransplanted leukemia cells, THP-1 or NB4. THP-1 tumors showed more growth when fed with high fat diet, while NB4 tumors grew more with high carbohydrate diet. Then, administration of 2-deoxyglucose (a glycolysis inhibitor) showed a significant antitumor effect on both tumors: NB4 tumor showed large necrotic areas, while THP-1 tumor did not, but had augmented expression of enzymes for fatty acid oxidation. 2-Deoxyglucose inhibited the growth of NB4 by cell death because main energy producing pathway (glycolysis) was abolished, while 2-deoxyglucose slowed the growth of THP-1 by shifting energy metabolism to fatty acid β-oxidation.

Introduction: Primary refractory DLBCL is an extremely difficult condition to treat and represent an unmet medical need. The clarification of the molecular pathogenesis of this disease can contribute the development of new therapeutic possibilities. PVT1 is located adjacent to MYC at 8q24 and a non-protein coding gene. PVT1 produces a variety of long non-coding RNAs (lncRNAs) and has now emerged as a potential regulator in the pathogenesis of a lot of cancers. However, the precise biological and clinical significance of PVT1 remains largely unknown. In this study we established a novel human DLBCL cell line with hsr (homogeneous staining region) of 8q24 inducing high expression of PVT1 lncRNAs, named AMU-ML2. The cell line was established from a patient with primary refractory DLBCL before initiation of chemotherapy. We analyzed the genetic characteristics and investigated the drug sensitivity of the cell line in comparison with other B-cell lymphoma cell lines which had 8q24 abnormalities. Case: A 64-year-old man was diagnosed as DLBCL involved with bone marrow and pleural effusion. He was refractory to initial R-CHOP and subsequent R-hyper-CVAD/MA therapy and died of Trichosporon asahiisepsis 6 months after diagnosis. His lymphoma cells at diagnosis were positive for CD19, CD20, BCL6 and HLA-DR, and negative for CD3, CD5, CD10, cyclinD1, BCL2, MUM1, TP53 and EBER by flow cytometry and/or immunohistochemical staining, indicative of germinal center B-cell-like (GCB) DLBCL. The representative karyotype of cells was 46,XY, including add(6)(p11), add(8)(q24), hsr 8q24, add(9)(p13) and add(17)(p11.2). Establishment of the cell line: After 2 weeks of culture, the cells in pleural effusion collected before chemotherapy started to grow in suspension. The cell line was designated as AMU-ML2 after confirmation that the cells started growing again after the conventional freeze-thaw procedure. Materials and methods: B-cell lymphoma cell lines; AMU-ML2, SU-DHL-10, Raji, P3HR-1 and VAL were used in the present study. These cell lines had t(8;14)(q24;q32) or 8q24 amplification. The genomic aberration of AMU-ML2 was analyzed by cytogenetics including G-banding and FISH (fluorescence in situ hybridization) combined with array-CGH (SurePrint G3, Agilent). The TaqMan real time RT-PCR was used for measurement of expression levels of MYC and PVT1. MTT assay was used for the cell proliferation to analyze the drug sensitivities of cyclophosphamide, doxorubicin, vincristine (VCR) and prednisolone. Results: AMU-ML2 cells showed same immunophenotypic feature and karyotype as the primary sample from the patient. FISH using a MYC/IGH probe set showed no fusion signal for IGH and MYC; however, the MYC copy number was extremely increased, corresponding to hsr on chromosome 8q24. Array-CGH revealed that a 1,462 kb region, containing both the entire MYC and PVT1 genes at 8q24.21, was amplified, with greater than 20 copies present in cells (Figure 1). In addition to amplification of the MYC/PVT1 region at 8q24, 14 additional copy number alterations were detected. These included segment losses on 6p22.1-6p21.31 and 17p13 led to the LOH of human leukocyte antigen (HLA) loci and TP53, respectively (Figure 1). Using the real time RT-PCR, the expression level of the PVT1 lncRNAs were highest in AMU-ML2 among other B-cell lymphoma cell lines that we used, while the expression level of MYC in AMU-ML2 was relatively low. The proliferation rate of AMU-ML2 was significantly higher after 72-h exposure to VCR (100, 500 and 1,000 nM) compared with other cell lines (Figure 2). Discussion: AMU-ML2 was established in 2-week culture from a refractory patient before starting the chemotherapy, therefore this cell line seems to reflect the real nature of primary refractory DLBCL, not related to chemotherapy and/or cell culture. MYC amplification and the LOH of TP53 and HLA may contribute to the development of lymphoma in our case. High expression of PVT1 lncRNAs was likely to be more related with drug resistance to VCR than MYC expression, although AMU-ML2 had co-amplification of MYC and PVT1. Thus, AMU-ML2 can provide insight into the genetic and biological features and the therapeutic approaches in primary refractory DLBCL. Disclosures Ueda: Mundipharma KK: Consultancy; Kyowa Hakko Kirin: Research Funding.

In this study, we established and analyzed a novel human myeloid leukemia cell line, AMU-AML1, from a patient with acute myeloid leukemia with multilineage dysplasia before the initiation of chemotherapy. AMU-AML1 cells were positive for CD13, CD33, CD117, and HLA-DR by flow cytometry analysis and showed a single chromosomal abnormality, 46, XY, t(12;22)(p13;q11.2), by G-banding and spectral karyotyping. Fluorescent in situ hybridization analysis indicated that the chromosomal breakpoint in band 12p13 was in the sequence from the 5' untranslated region to intron 1 of TEL and that the chromosomal breakpoint in band 22q11 was in the 3' untranslated region of MN1. The chimeric transcript and protein of MN1-TEL could not be detected by reverse-transcriptase polymerase chain reaction or Western blot analysis. However, the MN1 gene was amplified to three copies detected by array comparative genomic hybridization analysis, and the expression levels of the MN1 transcript and protein were high in AMU-AML1 cells when compared with other cell lines with t(12;22)(p13;q11-12). Our data showed that AMU-AML1 cells contain t(12;22)(p13;q11.2) without chimeric fusion of MN1 and TEL. The AMU-AML1 cells gained MN1 copies and had high expression levels of MN1. Thus, the AMU-AML1 cell line is useful for studying the biological consequences of t(12;22)(p13;q11.2) lacking chimeric MN1-TEL.

For generation of energy, cancer cells utilize glycolysis more vigorously than oxidative phosphorylation in mitochondria (Warburg effect). We examined the energy metabolism of four leukemia cell lines by using glycolysis inhibitor, 2-deoxy-d-glucose (2-DG) and inhibitor of oxidative phosphorylation, oligomycin. NB4 was relatively sensitive to 2-DG (IC(50): 5.75 mM), consumed more glucose and produced more lactate (waste product of glycolysis) than the three other cell lines. Consequently, NB4 was considered as a "glycolytic" leukemia cell line. Dependency on glycolysis in NB4 was confirmed by the fact that glucose (+) FCS (-) medium showed more growth and survival than glucose (-) FCS (+) medium. Alternatively, THP-1, most resistant to 2-DG (IC(50): 16.14 mM), was most sensitive to oligomycin. Thus, THP-1 was recognized to be dependent on oxidative phosphorylation. In THP-1, glucose (-) FCS (+) medium showed more growth and survival than glucose (+) FCS (-) medium. The dependency of THP-1 on FCS was explained, at least partly, by fatty acid oxidation because inhibitor of fatty acid β-oxidation, etomoxir, augmented the growth suppression of THP-1 by 2-DG. We also examined the mechanisms by which THP-1 was resistant to, and NB4 was sensitive to 2-DG treatment. In THP-1, AMP kinase (AMPK), which is activated when ATP becomes limiting, was rapidly phosphorylated by 2-DG, and expression of Bcl-2 was augmented, which might result in resistance to 2-DG. On the other hand, AMPK phosphorylation and augmentation of Bcl-2 expression by 2-DG were not observed in NB4, which is 2-DG sensitive. These results will facilitate the future leukemia therapy targeting metabolic pathways.

Summary Several anti-angiogenic drugs have recently been clinically tested for haematological malignancies. To improve the efficacy of molecular target therapy against angiogenic molecules in acute myeloid leukaemia (AML), we examined the dependency of AML cells on the vascular endothelial growth factor (VEGF)/VEGF receptor type2 (VEGFR2) system by using VEGFR2 kinase inhibitor. Nineteen patient AML samples were cultured with or without VEGFR2 kinase inhibitor. All four t(8;21) viable AML cells showed significant reductions when treated with VEGFR2 kinase inhibitor, although VEGFR2 kinase inhibitor did not affect the cell proliferation of five t(15;17) AML samples. Other AML cases showed variable responses. VEGFR2 kinase inhibitor greatly suppressed the growth of Kasumi-1, a t(8;21) cell line in a dose-dependent manner through induction of apoptosis, but did not show any significant influence on NB4, a t(15;17) cell line. In addition, VEGFR2 kinase inhibitor potentiated the growth inhibitory effect of cytarabine in Kasumi-1. Finally, it was shown that the Akt phosphorylation was augmented by VEGF165 in Kasumi-1, which was abrogated by VEGFR2 kinase inhibitor. NB4 showed undetectable Akt phosphorylation even with VEGF165. These data demonstrated that t(8;21) AML cells are dependent on VEGF through VEGFR2, resulting in the phosphorylation of Akt.

Various angiogenic factors, such as vascular endothelial growth factor (VEGF) and an associated molecule, placenta growth factor (PlGF), are thought to be important for normal and malignant hematopoiesis. This study examined mRNA expression of VEGF, PlGF and receptors for these molecules in AML cells and identified the disease-specific patterns of expression. AML M3 having t(15;17) abnormality showed highest expression of VEGF and VEGF receptor type 1 (VEGFR1), suggesting the autocrine pathway of VEGF-VEGFR1. Then, t(8;21) AML demonstrated augmented expression of VEGF and VEGF receptor type 2 (VEGFR2), suggesting VEGF-VEGFR2 autocrine pathway. Then, addition of VEGFR2 kinase inhibitor in Kasumi-1, a t(8;21) AML cell line, resulted in marked inhibition of cell growth, although growth inhibitory effect of R2 kinase inhibitor to HL-60 was marginal. In addition, cell cycle analysis study showed S-phase cell population reduction by R2 kinase inhibitor in Kasumi-1, but not in HL-60. This observation is thought to be the rationale for novel molecular target therapy directed to angiogenic molecules.

P-glycoprotein (P-gp)/multi-drug resistance 1 (MDR1) gene is recognized to be, at least in part, responsible for the refractoriness to chemotherapy of leukemia. The transcriptional mechanism of MDR1 gene is largely unknown. However, recent reports have clarified that early growth response 1 gene (Egr1) positively regulates MDR1 transcription, while Wilms' tumor suppressor gene (WT1) does negative regulation of MDR1 gene expression in 12-O-tetradecanoylphorbol-13-acetate treated K562 cells. In addition, Egr1 and WT1 are structurally related transcription factors and bind to quite similar DNA sequences. Our study of mRNA expression profile of Egr1, WT1 and MDR1 in fresh AML samples demonstrated that there are disease-specific patterns. Egr1 mRNA was frequently and strongly expressed in monocytic leukemia cells, especially in AML M4 cells. WT1 mRNA was undetectable in t(8;21) AML cells. mRNA expression of MDR1 was frequent in AML M1 and t(8;21) AML cells, in which the expression level was highest in AML M1 and was low in monocytic leukemia (M4 and M5). Then, expression level of MDR1 was inversely correlated with Egr1. By liquid culture of leukemia cell lines and fresh AML cells with the addition of all-trans retinoic acid (ATRA), modulation of P-gp/MDR1 and Egr1 was observed and the pattern of modulation was divided into four groups: (1) blastic AML type, in which distinct expression of P-gp/MDR1 and CD34 was not influenced by ATRA; (2) t(8;21)AML type, in which P-gp/MDR1 expression was augmented by ATRA, while CD34 was kept high; (3) AML M3 type, in which P-gp/MDR1 expression was reduced with granulocytic differentiation by ATRA; (4) monocytic AML type, in which P-gp/MDR1 expression was augmented by ATRA, while CD34 expression decreased, and strong Egr1 expression was downregulated just prior to the augmentation of P-gp/MDR1 expression. WT1 expression was not influenced by the addition of ATRA in each group. Previous reports have suggested that P-gp/MDR1 plays an important role in resistance to chemotherapy, and is recognized as one of the stem cell marker. However, P-gp/MDR1 expression augmented by ATRA, which was observed in monocytic AML, was recognized as a functional molecule of mature monocyte/macrophage, because CD34 expression decreased and CD13 expression increased by ATRA. Finally, expression of P-gp/MDR1 in monocytic leukemia, which was functionally confirmed by Rh123 efflux study, was thought to be closely related to the characteristic modulation of Egr1 expression by ATRA.

Recently, it has been clarified that interaction between hematopoietic cells and endothelial cells is important in normal hematopoiesis and leukemogenesis. In this study, we examined the relationship between AML cells and endothelial cells by analyzing the expression profile of angiogenic factors, angiopoietin-1 (Ang-1), Ang-2, Tie-2 (a receptor for angiopoietins) and vascular endothelial growth factor (VEGF). Our results demonstrated that CD7(+)AML expressed Ang-2 mRNA frequently and integrin-family adhesion molecules (CD11c and CD18) intensively, suggesting the close correlation with endothelial cells. On the other hand, in t(8;21) AML cells, expression of Ang-2 was infrequent and expression of integrin-family adhesion molecules (CD11b, CD11c and CD18) was weak, suggesting the sparse association with endothelial cells. As for CD7(+)AML cells, despite the frequent and intense expression of endothelial cell-associated molecules (such as Ang-2, CD11c and CD18), intensity of Tie-2 expression was quite low (P < 0.05). Ang-2 expressed in CD7(+)AML cells is not considered to act in an autocrine fashion, but to work on endothelial cells to "feed" leukemic cells. Although Ang-2 is recognized as a natural antagonist for Tie-2, our data presented here suggested the alternative role of Ang-2 in the relationship between endothelial cells and leukemia cells, at least in a subset of leukemia such as CD7(+)AML. These results were supported by the study using AML cell lines, KG-1 (CD7 negative) and its subline KG-1a (CD7 positive); KG-1 had mRNA expression profile of Ang-1(+)Ang-2(-)Tie-2(+), while KG-1a showed Ang-1(+)Ang-2(+)Tie-2(-). These difference in the expression profile of angiogenic factors between CD7(+)AML and t(8;21)AML may explain the characteristic morphological features of these leukemias (CD7(+)AML as blastic type and t(8;21)AML as differentiative type).

Abstract 5210 Mature B-cell lymphomas with both BCL2 and MYC translocations to IG loci are rarely identified and most of them are classified as B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (IL) in the new World Health Organization classification. In this study, we established a novel human lymphoma cell line, AMU-ML1, from pericardial effusion (PE) of a patient with IL before the initiation of chemotherapy and analyzed its characters. A 60-year-old male was admitted to our hospital because of PE and diagnosed as having IL from the atypical lymphocytes in PE. He was treated with rituximab plus hyper-CVAD and other regimens but died of lymphoma approximately 10 months after diagnosis without reaching complete remission. The cells from patient's PE at diagnosis were cultured in RPMI 1640 supplemented with 20% heat-inactivated fetal bovine serum (FBS). After 5 months of culture, cell proliferation became continuous with 10% FBS and the cell line was designated as AMU-ML1 (Aichi Medical University, malignant lymphoma, no. 1) after confirmation that the cells began growing again after the conventional freeze-thaw procedure. AMU-ML1 cells were positive for CD10, CD19, CD20, CD79a, HLA-DR and cytoplasmic lambda chain and negative for CD3, CD4, CD5, CD8, CD13, CD23, CD33 and CD56 by flow cytometry analysis and showed a complex karyotypes including t(2;18)(p11.2;q21) and t(3;8;14)(q27;q24;q32) by G-banding analysis. This profile is consistent with the profile of the patient's cells. Spectral karyotyping and fluorescent in situ hybridization analysis of AMU-ML1 cells revealed that t(2;18)(p11.2;q21) was IGL/BCL2 and t(3;8;14)(q27;q24;q32) was BCL6/MYC, MYC/IGH and IGH/BCL6. Subcutaneous transplantation of AMU-ML1 cells into NOD/scid mice treated with anti-asialo GM1 antibody resulted in formation of primary tumors. Thus, the AMU-ML1 cell line is useful for studying the biological consequences of IL with triple hit of BCL2, BCL6 and MYC, and possibly invasion to PE of lymphoma. Disclosures: No relevant conflicts of interest to declare.

Abstract 4991 Bortezomib improved the survival in patients with multiple myeloma (MM), however, cannot cure this disease even when it is combined with autologous stem cell transplant(s) and other new drugs. Myeloma cells cannot be excluded completely from a patient due to drug refractoriness and/or inefficiency of drug delivery in extramedullary sites such as cerebrospinal fluid (CSF). Therefore, it is important to clarify the mechanisms of bortezomib resistance and invasion to central nerve system (CNS) of myeloma for the progress of myeloma therapy. In this study, we established a novel human myeloma cell line from a myeloma patient involved with CNS and analyzed its characters including sensitivity to bortezomib. The patient had been treated with bortezomib for 1.5 years when her CNS was involved with myeloma. The cells from patient's CSF were cultured in RPMI 1640 supplemented with 20% heat-inactivated fetal bovine serum (FBS) and rhIL-6. After 3 months of culture, cell proliferation became continuous with 10% FBS and without rhIL-6. The cell line, named AMU-MM1 was established and negative for EBV. A doubling time of AMU-MM1 cells was about 48 hours. AMU-MM1 cells were positive for CD38, CD54, CD138 and cytoplasmic kappa chain and negative for CD19, CD20, CD33 and CD56 by flow cytometry analysis while those in patient's bone marrow were positive for CD56. AMU-MM1 showed hypo- and pseudodiploid karyotypes with t(4;14), t(8;13), t(1;19), del13q, amp1q21 and others but without del17p by cytogenetic analyses including FISH. The G322A mutation in the proteosome beta 5 subunit (PSMB5) gene, which is reported as a mutation found in bortezomib-resistant cell lines induced via repeated drug selection, was not detected in AMU-MM1 by direct sequencing. Apoptosis analysis using Annexin V/PI assay indicated that AMU-MM1 was sensitive to bortezomib. Our data suggest that AMU-MM1 was derived from the cells that invaded to CSF in which bortezomib concentration was very low and not resistant to bortezomib, and the downregulation of CD56 might play a role in the pathogenesis of CNS involvement as reported before. In addition, we are now focusing on new chimera or dysregulated genes in t(8;13), t(1;19) and other sites as well. In conclusion AMU-MM1 is a useful cell line for analysis of mechanisms of CNS involvement and also possibly the acquired resistance to bortezomib. Disclosures: No relevant conflicts of interest to declare.

Abstract 4375 Leukemia cell lines are ubiquitous powerful research tools that are available to many investigators. In balanced chromosomal aberration in leukemia, a chimeric fusion gene formed by genes existing on breakpoints is frequently related to leukemogenesis. Cytogenetic abnormalities of chromosome band 12p13 are detected non-randomly in various hematological malignancies and usually involved TEL, which encodes a protein of the ETS transcription factor family. Chromosome band 22q11-12 is one of partners of translocation 12p13 and t(12;22)(p13;q11-12) results in fusion of TEL and MN1 or in just the partial inactivation of TEL. It is important to analyze precisely the breakpoint in a non-random translocation such as t(12;22)(p13;q11-12) and in addition it contributes to the better understanding of the molecular pathogenesis of leukemogenesis. In this study, we established a novel human myeloid leukemia cell line, AMU-AML1, having t(12;22) from a patient with acute myeloid leukemia with multilineage dysplasia and analyzed its characters. Mononuclear cells were isolated by Ficoll-Hypaque sedimentation from patient's bone marrow before initiation of chemotherapy and cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS). After 3 months, cell proliferation became continuous. The cell line, named AMU-AML1, was established. In AMU-AML1, the following pathogens were negative for EBV, CMV, HBV, HCV, HIV-1, HTLV-1 and mycoplasma. A doubling time of AMU-AML1 cells was about 96 hours. Proliferation of the cells was stimulated by rhG-CSF (10 ng/ml), rhGM-CSF (10 ng/ml), M-CSF (50 ng/ml), rhIL-3 (10 ng/ml) and rhSCF (100 ng/ml) but not by IL-5 (10 ng/ml), rhIL-6 (10 ng/ml), and rhEPO (5 U/ml). AMU-AML1 was positive for CD13, CD33, CD117 and HLA-DR, negative for CD3, CD4, CD8 and CD56 by flow cytometry analysis. G-banding combined with SKY analysis of AMU-AML1 cells showed single structural abnormality; 46, XY, t(12;22)(p13;q11.2). Double-color FISH using PAC/BAC clones listed in NCBI website and array CGH analyses indicated that the breakpoint in 12p13 was within TEL or telomeric to TEL and it of 22q11 was centromeric to MN1. A chimeric MN1-TEL transcript and fusion protein of MN1-TEL could not be detected by RT-PCR and western blot analysis. The wild type of MN1 protein was strongly expressed in AMU-AML1 compared with other leukemic cell lines with t(12;22), MUTZ-3 and UCSD/AML1. Our data suggest that AMU-AML1 had a t(12;22)(p13;q11.2) without fusion of MN1-TEL and the expression level of MN1 protein was relatively high, which might have some effects on leukemogenesis. In conclusion, AMU-AML1 is a useful cell line to analyze the biological consequences of the leukemic cells with t(12;22)(p13;q11.2) but no fusion of MN1-TEL. Disclosures: No relevant conflicts of interest to declare.

Cancer cells are more dependent on glycolysis than oxidative phosphorylation in the mitochondria for generation of ATP as energy source. By using 2-deoxy-D-glucose (2-DG: glycolysis inhibitor) and oligomycin (inhibitor of oxidative phosphorylation), we examined the energy metabolism of various leukemia cell lines. The growth of the cell lines was measured by MTS assay, which detects viable cells in proliferation. 2-DG suppressed the growth of all leukemia cell lines examined in dose-dependent manners. The IC50 of each cell line was as follows: Kasumi-1 0.5±0.1mM, KG-1a 1.8±0.6mM, HL-60 3.3±0.1mM, NB4 3.8±0.4mM, and THP-1 23.1±3.8mM. The concentration of lactic acid (the final product of glycolytic pathway) in the culture supernatant was greatly reduced by the treatment with 0.2mM 2-DG for 24 hours in Kasumi-1 (54.5% of the control), compared with THP-1 (92.2%). It is suggested that the growth of Kasumi-1 was strongly suppressed by 2-DG through inhibition of glycolysis, which is supposed to be a main metabolic pathway in this cell line. On the other hand, treatment with oligomycin (1μg/ml) for 48 hours potently suppressed the growth of THP-1 (44.7%), then Kasumi-1 (72.1%). The growth of NB4, KG-1a and HL-60 was minimally suppressed (more than 90%) by oligomycin. Cell cycle was analyzed after 24 hours treatment with 2-DG or oligomycin. Sub-G1 fraction (apoptosis) was greatly increased by 2-DG (5mM) in Kasumi-1 (56.5%) and NB4 (30.6%), compared with THP-1 (7.6%). The apoptosis inducing effect was confirmed by annexinV staining. Oligomycin treatment (1μg/ml) increased apoptosis (subG1) in THP-1 (35.8%), then Kasumi-1 (16.6%) and NB4 (12.2%). Oligomycin treatment also increased G1 population (G1 arrest) in THP-1 (35.9% to 69.4%). AMP-activated protein kinase (AMPK) is activated by an elevated AMP/ATP ratio, which means the energy-deprived status of the cell. Western blot analysis using phospho-AMPK α (Thr172) antibody revealed that treatment with 2-DG or oligomycin induced prompt (30 min) phosphorylation of AMPK in leukemia cell lines. The extent of AMPK phosphorylation was almost proportional to the suppression of the growth. Collectively, it is suggested that leukemia cells are dependent almost exclusively on either glycolysis or oxidative phosphorylation in the mitochondria for energy production. Then, inhibition of glycolysis by 2-DG or oxidative phosphorylation by oligomycin results in growth suppression by inducing apoptosis and/or cell cycle arrest through activation of AMPK. Our data clarified the characteristics of the energy metabolism of each leukemia cell, and showed the key to produce novel therapeutic approach targeting metabolic pathway.

Most human leukemia cell lines are dependent on serum supplementation (usually fetal calf serum (FCS)), although the extent of serum dependency differs among each cell line. Kasumi-1, a t(8;21) AML cell line is one of the most serum-dependent cell lines. Since growth and survival of many leukemia cell lines are associated with phosphorylation of Akt, we examined the Akt phosphorylation by FCS treatment. In Kasumi-1, Akt was phosphorylated by culture with FCS in a dose-dependent manner, although no such Akt phosphorylation was observed in NB-4, a t(15;17) cell line. By FCS stimulation, Akt (Thr308, Ser473) was phosphorylated from 0.5 hr and the phosphorylation sustained until 48 hours in Kasumi-1. Then, we tested the effect of VEGF/VEGFR signaling in phosphorylation of Akt by FCS. The addition of VEGFR1/Fc and VEGFR2/Fc (which bind external VEGF and abrogate its function) inhibited the Akt phosphorylation from 2 hours until 10 hours, although the growth of Kasumi-1 was not inhibited. The addition of VEGFR2 kinase inhibitor (which inhibits internal VEGF signal) inhibited the Akt phosphorylation from 0.5 hr until 2 hours, and the growth of Kasumi-1 was greatly inhibited. Taken together, it is suggested that serum dependency of Kasumi-1 is at least in part attributed to VEGF/VEGFR pathway. Then, both external and internal VEGF/VEGFR pathways work in Kasumi-1, which in turn phosphorylate Akt. However, blockade of only internal VEGF signal (by VEGFR2 kinase inhibitor) inhibit the early Akt phosphorylation (0.5 hr), which resulted in growth inhibition, indicating the importance of early Akt phosphorylation.