Abstract: Background: After long-term analysis of the JALSG-APL204 study we recently reported that maintenance therapy with tamibarotene was more effective than all-trans retinoic acid (ATRA) by reducing relapse in APL patients. Here, the clinical significance of other important prognostic factors was evaluated with multivariate analyses. Patients and Methods: Newly diagnosed acute promyelocytic leukemia (APL) patients were registered with the study. Induction was composed of ATRA and chemotherapy. Patients who achieved molecular remission after consolidation were randomly assigned to maintenance with tamibarotene or ATRA. Results: Of the 344 eligible patients, 319 (93%) achieved complete remission (CR). After completing consolidation, 269 patients underwent maintenance random assignment—135 to ATRA, and 134 to tamibarotene. By multivariate analysis, overexpression of CD56 in blast was an independent unfavorable prognostic factor for relapse-free survival (RFS) (p = 0.006) together with more than 10.0 × 109/L WBC counts (p = 0.001) and the ATRA arm in maintenance (p = 0.028). Of all phenotypes, CD56 was related most clearly to an unfavorable prognosis. The CR rate, mortality rate during induction and overall survival of CD56+ APL were not significantly different compared with CD56− APL. CD56 is continuously an independent unfavorable prognostic factor for RFS in APL patients treated with ATRA and chemotherapy followed by ATRA or tamibarotene maintenance therapy.

Abstract: Triglyceride-rich, low-density lipoproteins (TG-rich LDL) have been reported as an oxidized lipoprotein species in patients with severe liver disease. Using TG-rich LDL as an immunogen, we obtained a monoclonal antibody (G11-6) that reacted with TG-rich LDL from patients with liver disease and with metal-oxidized LDL only in the early process of the oxidation reaction. This study determined the G11-6-reactive lipoproteins in hypertriglyceridemic serum. Methods Serum samples from healthy volunteers ( n = 12) and hypertriglyceridemic patients ( n = 9) were fractionated by gel filtration and subjected to a sandwich enzyme-linked immunosorbent assay (ELISA) using G11-6 and polyclonal anti-apolipoprotein B antibodies. Results Small LDL and larger lipoproteins reacted with G11-6. G11-6-reactive small LDL was identified in both the healthy subjects and hypertriglyceridemic patients, whereas G11-6-reactive larger lipoproteins were found only in the hypertriglyceridemic patients. Conclusions G11-6 is a useful tool for detecting increased large oxidized lipoproteins in hypertriglyceridemic patients.

Abstract: The size of lipoprotein particles is relevant to the risk of coronary artery disease (CAD). Methods We investigated the feasibility of atomic force microscopy (AFM) for evaluating the size of large low-density lipoprotein (LDL) and small dense LDL (sd-LDL) separated by ultracentrifugation. The measurements by AFM in tapping mode were compared to those by electron microscopy (EM). Results There was a significant difference in particle sizes determined by AFM between large LDL (20.6 ± 1.9 nm, mean ± SD) and sd-LDL (16.2 ± 1.4 nm) obtained from six healthy volunteers ( P  〈  0.05). The particle sizes determined by EM for the same samples were 23.2 ± 1.4 nm for large LDL and 20.4 ± 1.4 nm for sd-LDL. The difference between large LDL and sd-LDL detected by EM was also statistically significant ( P  〈  0.05). In addition, the particle sizes of each lipoprotein fraction were significantly different between AFM and EM: P  〈  0.05 for large LDL and P  〈  0.05 for sd-LDL. Conclusions AFM can differentiate between sd-LDL and large LDL particles by their size, and might be useful for evaluating risk for CAD.

Abstract: Background: Anti-CD38 monoclonal antibodies (MoAbs), including daratumumab and isatuximab, are introduced in the treatment of multiple myeloma (MM). However, CD38 is also expressed on red blood cells (RBCs), so that free anti-CD38 MoAbs in patient sera sometimes binds to test RBCs in pretransfusion blood-compatibility tests. Consequently, panreactive agglutination arises in the standard indirect antiglobulin test (IAT) used for antibody screening and cross-matching. Several methods, including pretreatment of RBCs with dithiothreitol (DTT), have been introduced to overcome the interference. However, the optimum concentration and treatment time for these reagents have not been well elucidated (Chapuy et al, Hosokawa et al). One reason is that evaluation for RBC agglutination during the IAT relies on visual judgement. In this study, we quantified CD38 on RBCs before and after DTT treatment using a newly-devised flow cytometric antibody binding assay (FABA; Takeshita et al) by employing the D-value in the Kolmogorov-Smirnov (K-S) test, and compared with the results from IAT and a conventional flow cytometric method using fluorescent labelled anti-CD38 antibody and isotype-matched control antibody. We then assessed the optimum DTT concentration and treatment time for CD38 inactivation on RBCs to enhance MoAb therapy for MM. Methods: RBCs from healthy donors were treated with 0.2-0.0001 mol/L of DTT for 0-30 min at 37°C. The effect of DTT treatment was evaluated using flow cytometetry (FCM, Navious, Beckman Coulter, Tokyo) and IAT. For conventional FCM analysis, untreated or DTT-treated RBCs were incubated with fluorescein isothiocyanate (FITC)-labeled anti-CD38 antibody or anti-IgG1 antibody for 30 min, and then analyzed. For FABA, untreated or DTT treated RBCs were incubated with FITC-labelled anti-CD38 antibody, in the presence or absence of a 100-fold or more excess of unlabeled anti-CD38 antibody and then analyzed by FCM. Dissociation of CD38 positive and control histograms were determined from the mean fluorescence intensity (MFI) and the D-value in the K-S test, which is the maximum vertical displacement between two cumulative frequency distributions for two histograms. Statistical analyses were performed by paired t-test (SPSS, Tokyo), with significance set at p & lt;0.05. The amount of CD38 following DTT treatment was also evaluated by Western blotting. Results: In conventional FCM using FITC-labelled isotype-matched antibody as control, the difference of measured MFIs between CD38 positive and control were considerably volatile in repeated analysis (Fig. A). By contrast, in FABA using an excess concentration of unlabeled anti-CD38 antibody as control, the values were more consistent than those from conventional FCM (Fig. B). The D-value in the K-S test is reliable by numerical modeling in the analysis of the difference between CD38 positive and control histograms. Analysis showed that 0.005 mol/L DTT for 30 min is sufficient to inactivate CD38 on RBCs. These results correlated with those of IAT (Fig. C). Western blotting identified CD38 at DTT concentrations of 0.01-0.001 mol/L, but denaturation of CD38 was observed at 0.2 mol/L of DTT (Fig. D). Conclusions: Our newly-devised flow cytometric antibody binding assay, FABA, facilitates the detection of small numbers of cell surface antigens. The method is also useful as an objective way of evaluating the efficacy of DTT treatment for CD38 on RBCs. Our results show 0.005 mol/L DTT for 30 min is sufficient to inactivate CD38 on RBCs in plasma sampled after treatment with the anti-CD38 MoAbs. Figure Disclosures Takeshita: Chugai Pharmaceutical Co.Ltd.: Research Funding; Pfizer Japan Inc: Research Funding; Astellas Pharma Inc.: Research Funding; Takeda Pharmaceutical Co.Ltd.: Research Funding; Bristol-Myers Squib Co.: Research Funding.

Abstract: Background: CD56 expression is reported to be associated with adverse prognosis in patients with acute promyelocytic leukemia (APL) treated with all-trans retinoic acid (ATRA) and chemotherapy (Murray et al, 1999, Ferrara et al, 2000, Montesinos et al, 2011, Ono T et al, 2014). However, the prognostic significance of CD56 has not been elucidated, particularly when more potent agents are used. We recently reported long term analysis of the Japan Adult Leukemia Study Group (JALSG) APL204 study and concluded that maintenance therapy with tamibarotene was more effective than ATRA by reducing relapse in APL patients (Takeshita et al, 2018). In this study, the clinical significance of CD56 was evaluated with other surface markers on APL cells. Patients and Methods: Newly diagnosed APL patients with documented cytogenetic and/or molecular evidence of t(15;17)/PML-RARA were registered to the APL204 study from April 2004 to December 2010. The eligibility criteria included age between 15 and 70 years, ECOG performance status between 0 and 3, and sufficient function of organs. Induction therapy was composed of ATRA and chemotherapy whose dose and duration were based on initial white blood cell (WBC) count. Patients who achieved molecular remission after three courses of consolidation therapy were randomly assigned to maintenance therapy with tamibarotene 6 mg/day for 14 days or ATRA 45 mg/day for 14 days, which was repeated every 3 months for 2 years. The primary endpoint was hematological or molecular relapse-free survival (RFS). Surface markers, including CD56, were defined as positive if more than 10% of the CD45-gated cells expressed a specific antigen. Clinical characteristics were compared by the chi-square test or the Fisher's exact test for categorical data and the Wilcoxon rank-sum test for continuous data. RFS, overall survival (OS) and event-free survival (EFS) were estimated by the Kaplan-Meier method, and compared using the log-rank test. Cumulative incidence of relapse (CIR) was compared by Gray's test. Multivariate analyses were also performed by the Cox-proportional-hazards-model. Clinical outcomes were renewed between January 2016 and June 2017 and the median follow-up period was 7.3 years. This study is registered at the University Hospital Medical Information Network Clinical Trials Registry as C000000154. Results: Of the 344 eligible patients, 319 (93%) achieved CR. After completing consolidation chemotherapy, 269 patients underwent maintenance random assignment; 135 to ATRA, and 134 to tamibarotene. Among 344 eligible patients, 325 were assessable for CD-phenotypes, and 45 (14%) were CD56-positive (CD56+). Among 269 patients who underwent the maintenance assignment, 34 (13%) were CD56+. CD56 expression was significantly associated with obvious bleeding (p 〈 0.001). The CR rate and mortality during induction therapy were not significantly different compared with CD56- APL. RFS and CIR was significantly inferior in CD56+ APL (77% vs. 91%, HR 3.04, 95% CI 1.34-6.90, p=0.005 and 24% vs. 8%, p=0.004, respectively), whereas OS was not significantly different between the two groups 80% vs. 89%, p=0.069). In patients whose initial WBC counts were more than 3.0 x 109/L, RFS for the CD56+ group (n=14) was significantly inferior (64% vs. 87%, p=0.028), while in patients whose initial WBC count was under 3.0 x 109/L (n=20), RFS was not different (85% vs. 93%, p=0.164). Other surface markers such as CD13 and CD33 did not show any prognostic significance except for CD34 (p=0.040). By multivariate analysis, CD56 expression was an independent unfavourable prognostic factor for RFS (HR=3.19, 95% CI 1.40-7.25, p=0.006) together with more than 3.0 x 109/L WBC counts (p=0.001) and the ATRA arm in maintenance therapy (p=0.028). Conclusions: CD56 expression is an independent unfavorable prognostic factor for RFS in APL patients treated with ATRA and chemotherapy followed by ATRA or tamibarotene maintenance therapy, especially in patients whose initial WBC count was more than 3.0 x 109/L. The present study supports the prognostic significance of CD56 in the treatment of APL using more potent agents. Figure. Figure. Disclosures Takeshita: Chugai Pharmaceutical Co. Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Astellas Pharma Inc.: Research Funding; Takeda Pharmaceutical Co. Ltd.: Research Funding; Bristol-Myers Squibb Co.: Research Funding; Kyowa Hakko Kirin Co. Ltd.: Research Funding. Asou:Asahi Kasei Pharma Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; SRL Inc.: Consultancy; Yakult Honsha Co., Ltd.: Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Astellas Pharma Inc.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Sawa:Celgene Corporation: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis International AG: Honoraria; CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria; Mundipharma K.K.: Honoraria. Dobashi:Celgene Co.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co. Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Sysmex Co.: Research Funding. Kobayashi:Pfizer: Research Funding; Ohtuka: Research Funding; Astellas: Research Funding. Kiyoi:Kyowa Hakko Kirin Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria; FUJIFILM Corporation: Research Funding; Celgene Corporation: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Sanofi K.K.: Research Funding; Astellas Pharma Inc.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding.

Abstract: Triglyceride-rich low-density lipoproteins (TG-rich LDLs) in the plasma of patients with severe liver disease are reported to change macrophages into foam cells in vitro. Methods Male BALB/c mice were immunized with TG-rich LDLs isolated from the plasma of a patient with severe liver disease. The resulting monoclonal antibody (G11-6) was used in a sandwich enzyme-linked immunosorbent assay (ELISA) in combination with polyclonal anti-apolipoprotein B antibodies. The time course of copper-mediated LDL oxidation was monitored using this ELISA. The results were compared with those of the two commercial ELISAs for oxidized LDLs using DLH or ML25, thiobarbituric acid reactive substances and the optical absorbance for the conjugated dienes generated in lipid peroxides. Furthermore, the lipoprotein fractions separated by gel filtration were tested with this ELISA in healthy volunteers ( n = 11) and patients ( n = 3) with liver disease. Results G11-6 reacted with oxidized LDLs during only the early phase of copper oxidation, being distinct from the other monoclonal antibodies and methods. G11-6 was confirmed to react with TG-rich LDLs in patients, while it reacted with small LDL particles in normal controls. Conclusions The monoclonal antibody G11-6 is useful for detecting oxidized small LDLs in normal controls and oxidized TG-rich LDLs in patients with severe liver disease.