ALBERT

Description: NNKY5-5, an IgG monoclonal antibody directed against the von Willebrand factor-binding domain of glycoprotein (GP) Ibα, induced weak but irreversible aggregation (or association) of platelets in citrate-anticoagulated platelet-rich plasma. This phenomenon was defined as small aggregate formation (SAF ). Platelets in hirudin-anticoagulated plasma or washed platelets showed little response to NNKY5-5 alone, but the antibody potentiated aggregation induced by low concentrations of adenosine diphosphate or platelet-activating factor. NNKY5-5 did not induce granule release or intracellular Ca2+ mobilization. However, NNKY5-5 caused tyrosine phosphorylation of a 64-kD protein and activation of a tyrosine kinase, p72syk. An anti-FcγII receptor antibody had no effect on SAF, suggesting that NNKY5-5 activated platelets by interacting with glycoprotein Ib. Fab′ fragments of NNKY5-5 did not induce SAF, but potentiated aggregation induced by other agonists. The Fab′ fragment of NNKY5-5 induced the activation of p72syk, suggesting that such activation was independent of the FcγII receptor. Cross-linking of the receptor-bound Fab′ fragment of NNKY5-5 with a secondary antibody induced SAF. GRGDS peptide, chelation of extracellular Ca2+, and an anti-GPIIb/IIIa antibody inhibited NNKY5-5-induced SAF, but had no effect on 64-kD protein tyrosine phosphorylation or p72syk activations. Various inhibitors, including aspirin and protein kinase C, had no effect on SAF, protein tyrosine phosphorylation, or p72syk activation. In contrast, tyrphostin 47, a potent tyrosine kinase inhibitor, inhibited NNKY5-5–induced SAF as well as tyrosine phosphorylation and p72syk activation. Our findings suggest that binding of NNKY5-5 to GPIb potentiates platelet aggregation by facilitating the interaction between fibrinogen and GPIIb/IIIa through a mechanism associated with p72syk activation and tyrosine phosphorylation of a 64-kD protein.

Description: Background Tyrosine kinase inhibitors (TKIs) currently represent the main therapy for chronic myeloblastic leukemia (CML). Although they are therapeutically effective, some TKI-related events such as pleural effusion and elevation of large granular lymphocytes (LGL) have been reported. In addition, these events itself may affect the therapeutic response to TKIs. In the present study, we measured the levels of some cytokines, chemokines, soluble factors and coagulation markers in patients with CML, and investigated the relationship between these markers and TKI-related events. Methods The subjects were 48 patients with CML. Blood samples were collected and levels of cytokines (interleukin (IL)-6, tumor necrosis factor-α, high-mobility group box 1), chemokines (monocyte chemotactic protein (MCP)-1, RANTES), soluble molecules (soluble vascular cell adhesion molecule-1, soluble E-selectin, angiopoietin-2, vascular endothelial growth factor, and soluble thrombomodulin) and coagulation markers (plasminogen activator inhibitor (PAI)-1, platelet-derived microparticles (PDMP)) were measured by ELISA. Results Levels of all markers except IL-6 and MCP-1 were significantly increased in patients with de novo CML. Level of LGL/lymphocyte was significantly higher in dasatinib-treated CML patients compared to other TKI-treated patients. LGL/lymphocyte and many biomarkers exhibit the time-dependent increase after dasatinib treatment. In addition, LGL/lymphocyte was already increasing in patients with pleural effusion before dasatinib treatment. Conclusion These results suggest that LGL and endothelial-cell-related factors play an important role in the pathophysiology of CML and its response to TKIs. Disclosures: No relevant conflicts of interest to declare.

Description: In 1998, Gasbarrini et al reported that in ITP cases with Helicobacter pylori (H.pylori) infection, elevation of platelet counts was observed by eradication of this bacterium. Since then, several reports from Italy and Japan confirmed the elevation of platelet counts after eradication. However, the characteristic background in the H.pylori positive ITP and eradication effects on platelet counts is unclear. On the other hand, reports from Spain, North Europe and USA could not show the evidence that eradication is effective on elevating platelet counts in H.pylori positive ITP. Therefore, we designed a nationwide retrospective study in Japan to evaluate the incidence of H.pylori positive ITP cases and the effects of eradication on platelet counts and to clear above problems. Four hundred and thirty-five ITP cases were enrolled over a period of one and half years (2002. 7~2003.12) from 12 hospitals. H. pylori infection was found in 300 cases(65%), who were significantly (P

Description: Background: Thrombotic microangiopathy (TMA) and sinusoidal obstruction syndrome (SOS) as well as acute GVHD are major complications after allogeneic hematopoietic stem cell transplantation (alloHSCT) to overcome to achieve better overall survival. These complications are closely relevant to inflammation and coagulation involving endothelium, which are named as transplantation-associated coagulopathy (TAC). Recombinant thrombomodulin (rTM) is a new drug for treating DIC approved by Japanese Ministry of Health, Labour and Welfare in 2008. Membranous TM has many aspects to inhibit inflammation via activated Protein C (APC), lectin-like domain, and activated thrombin-activatable fibrinolysis inhibitor as well as to inhibit coagulation by binding factor IXa and Xa through APC. Biomarkers such as inflammatory cytokines and endothelial molecules are expected to be clue in elucidating TAC. We established SIGHT study group to explore above complications following alloHSCT. In the present study, we investigated the effects of rTM on levels of the biomarkers and whether we could prevent TAC using rTM among registered patients in the SIGHT study group. Patients and methods: SIGHT study group covered nationwide institutes more than 70 in Japan. Patients whoever were eligible for alloHSCT to treat hematopoietic disorders by physicians decision were permitted and written informed consent was requested to register in the study. Blood samples were collected from the patients before and after transplantation through day 28. Levels of cytokines (IL-6, TNF-α, HMGB1, MCP-1, RANTES) and soluble molecules (VCAM-1, E-selectin, PAI-1, PDMP) were measured by ELISA. rTM was administered as a prophylactic therapy for TAC in day 4-14 after alloHSCT. Control group was received heparin or no anti-coagulation therapy as prophylaxis during same schedule as rTM group. Patients were not randomized to these three arms but allowed to select one by their physicians decision. The primary endpoint was a comparison of fluctuation of these biomarkers in three arms. Secondary endopoint was a comparison of the rate of developing TAC. Results: The subjects were 293 patients who underwent alloHSCT in SIGHT study group between June 2010 and February 2014. 271 patients out of them were analyzed to measure level of these biomarkers. There was no significant difference in all characteristics but GVHD prophylaxis between two groups. rTM group received more cyclosporine than control group (p=0.05). MCP-1 and IL-6 exhibited more significant elevations on day 7 after alloHSCT, while HMGB-1 did on the day of alloHSCT. In contrast, the levels of TNF-α, E-selectin, VCAM-1, PAI-1 and PDMP exhibited increment since around day 7 after alloHSCT. Significant improvements in TNF-α, E-selectin, VCAM-1, HMGB-1, PAI-1 and PDMP was shown after rTM-treatment, but not shown in control group. Regarding complications following alloHSCT, the rate of developing acute GVHD and SOS was significantly lower in rTM group than without it (36.5% vs 62.2%, p=0.000; 12.5% vs 24.5%, p=0.032, respectively), while TMA did not differ (8.4% vs 10.2%, p=0.961). Stepwise multivariate logistic regression analyses revealed that anti-coagulation therapy without rTM and the level of PAI-1 on day 28 after HSCT were independent risk factor for acute GVHD (p=0.000, odds ratio=3.006; p=0.000, odds ratio=1.068, respectively). Also anti-coagulation therapy without rTM and the level of HMGB-1 on day 28 after HSCT were significantly predictive for risk of SOS (p=0.015, odds ratio=2.650; p=0.001, odds ratio=1.433, respectively). Tacrolimus was significantly good risk of SOS (p=0.022, odds ratio=0.404). The level of VCAM-1 on day 28 after HSCT was significantly associated with risk of TMA (p=0.040, odds ratio=1.001). Conclusions: We identified predictive biomarkers for TAC following alloHSCT, which were PAI-1 for acute GVHD, HMGB-1 for SOS, and VCAM-1 for TMA, respectively in this study. The present findings suggest that rTM has the possibility to play a therapeutic role for acute GVHD and SOS induced by suppression on these biomarkers. Endothelial and pro-coagulant biomarkers were released delayed compared with inflammatory biomarkers. TMA is reported to develop around day 44 in median after alloHSCT. Further studies are needed whether rTM should administer later or longer to improve TMA than the present duration after alloHSCT. Disclosures No relevant conflicts of interest to declare.

Description: Background Levels of cytokines, chemokines and soluble molecules fluctuate after allogeneic hematopoietic stem cell transplantation (HSCT). These biomarkers are possible to be a diagnostic and prognostic value for transplantation-associated coagulopathy (TAC). In the present study, we investigated the effects of recombinant thrombomodulin (rTM) on levels of the cytokines, chemokines, and soluble factors registered in the ‘SIGHT’ research. SIGHT comprises the capital letters of five complications after HSCT, namely sinusoidal obstruction syndrome (SOS same as VOD), infection, GVHD, hemophagocytic syndrome and thrombotic microangiopathy. Methods The subjects were 159 patients who underwent allogeneic HSCT (bone marrow = 83, peripheral blood stem cells = 31, cord blood = 45). Blood samples were collected before and after transplantation. Levels of cytokines (interleukin-6, tumor necrosis factor-α, high-mobility group box (HMGB) 1), chemokines (monocyte chemotactic protein (MCP)-1, RANTES), and soluble molecules (soluble vascular cell adhesion molecule (VCAM)-1, soluble E-selectin, plasminogen activator inhibitor-1, platelet-derived microparticles (PDMP)) were measured by enzyme-linked immunosorbent assay. The rTM was administered as a therapy for transplantation-associated coagulopathy (TAC). This protocol was completed in day 4-14 after HSCT and consisted of day doses of 380 unit/kg with every days. Control group was also used heparin or no anti-coagulation therapy. Results MCP-1, IL-6, and TNF-a exhibited more significant elevations on days 7–14 after HSCT. In contrast, the levels of HMGB1, sE-selectin, sVCAM-1, PAI-1 and PDMP exhibited significant changes on days 14–28. There were significant improvements in TNF-a, sE-selectin, sVCAM-1, HMGB1, PAI-1 and PDMP after rTM-treatment (n=73), but not after rTM-untreatment patients (n=86). Conclusion We believe one of causes for TAC is pro-inflammatory cytokine including HMGB1. For this reason, it is thought that the direct anti-inflammatory effect of rTM’s lectin domein plays an important role in therapeutic mechanism for TAC. The present findings suggest the possibility that rTM can play a therapeutic role for TAC after allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

Description: Idiopathic thrombocytopenic purpura (ITP) is one of the major causes of thrombocytopenia. Currently, the diagnosis of ITP is principally based on the exclusion of other possible concurrent causes of thrombocytopenia. In the guidelines proposed by the American Society of Hematology, the panel recommended that no specific laboratory tests are considered necessary for the diagnosis. However, availability of reliable laboratory assays should be helpful in supporting the diagnosis of ITP. Recently, 2 of us (MK and YI) reported that erythrocyte count, leukocyte count, anti-GPIIb/IIIa antibody-producing B cells, platelet-associated anti-GPIIb/IIIa antibodies, reticulated platelets and thrombopoietin measured at first visit were useful to predict a future diagnosis of chronic ITP (manuscript submitted). To confirm this, we conducted a multicenter prospective study involving 113 patients with thrombocytopenia and a normal peripheral blood film at first visit. Patients with clinically apparent associated conditions that can cause thrombocytopenia were excluded. Each patient underwent physical examination and routine laboratory tests, and was prospectively followed for 〉6 months. Anti-GPIIb/IIIa antibody-producing B cells, platelet-associated anti-GPIIb/IIIa antibodies, reticulated platelets and plasma thrombopoietin were also examined at first visit. Clinical diagnosis was made in a blinded fashion based on bone marrow findings and the clinical course. Eighty-nine patients were diagnosed as having chronic ITP, and 24 had a non-ITP disorder, including 11 with aplastic anemia (AA), 10 with myelodysplastic syndrome (MDS), and one each with Fanconi anemia, May-Hegglin anomaly and myelofibrosis. Six laboratory findings were identified as initial parameters that discriminated future diagnosis of chronic ITP from non-ITP. These included the absence of anemia, absence of leukopenia, increased anti-GPIIb/IIIa antibody-producing B cell frequency, increased platelet-associated anti-GPIIb/IIIa antibodies, elevated reticulated platelet percentage and normal or slight increase of thrombopoietin (all for P 〈 0.001). Stepwise multiple regression analysis revealed that anemia, anti-GPIIb/IIIa antibody-producing B cell frequency, reticulated platelet percentage and thrombopoietin were factors that independently contributed to the later diagnosis of chronic ITP. Three or more of 6 ITP-associated laboratory findings were present at presentation in 78 (93%) patients later diagnosed as chronic ITP, compared with 6 (25%) patients whose disorder was non-ITP (P 〈 10−5). All 6 “false-positive” patients were diagnosed as AA or MDS, but 4 of them probably had overlapping immune thrombocytopenia. In summary, this multicenter prospective study confirms usefulness of 6 laboratory tests for the diagnosis of chronic ITP. The identification of ITP-associated laboratory findings encourages the future development of reliable diagnostic criteria for chronic ITP.

Description: Background: The international prognostic index(IPI) was widely used to predict aggressive lymphoma patients' outcome and to choose the best therapeutic treatment. However, this scale is unsatisfactory in identifying patients who would receive best benefit from rituximab containing regimens. Recently, NLR has been recognized as a poor prognostic indicator in various solid tumors. Here we quantify the prognostic impact of NLR in de novo DLBCL patients. However, various studies of the usefulness of the NLR have used different cut-off values, and the methods of selecting these NLR cut-offs were unclear. Therefore, we verify the adaptive cut-off value. Methods: We retrospectively analyzed 543 patients with de novo DLBCL who diagnosed at Kansai Medical University Hospital and Kansai Medical University Medical Center from January 2003 to December 2017. The prognostic value of NLR at diagnosis was assessed. We put the cut-off of NLR; 3, 4, 5, 6, and evaluate which the most predictive cut-off value is. Results: The median age was 69(20-95) years old, and male was 59%. The Ann Arbor stage III and IV was 60%. The proportion of patients with IPI, Low, Low-intermediate, High-intermediate, High was 36%, 19%, 21%, 24%, respectively. The optimal cutoff for NLR was 6. NLR(6) was associated with overall survival(OS)(HR 1.76, 95%CI: 1.23-2.51, p= 0.002) and progression free survival(PFS) (HR 2.66, 95%CI: 1.65-4.28, p

Description: Introduction Allogeneic hematopoietic stem cell transplantation (aHSCT) is a curative treatment option for hematopoietic malignancies. In addition to relapse mortality, non-relapse mortality (NRM) such as severe graft-versus-host disease(GVHD) is a major cause of aHSCT. Pre-transplant clinical risk factors for acute GVHD include the degree of human leukocyte antigen (HLA) match between donor and recipient, recipient age, donor type, and conditioning regimen intensity. Although GVHD prophylaxis is usually decided by these risk factors, excessive immunosuppression induces opportunistic infection. Laboratory test cannot predict the risk of NRM or severe GVHD after aHSCT prior to the onset of GVHD symptoms; however, 2-biomarker (ST2 and REG3α) algorithm, namely MAGIC algorithm, may predict severe GVHD as has been shown recently (Hartwell et al. JCI Insight 2017). We retrospectively analyzed these 2 biomarkers in our patients to try whether the 2-biomarker algorithm is available to identify high risk of lethal GVHD and non-relapse mortality in Japanese patients. Patients and Methods We retrospectively analyzed consecutive 47 patients who received aHSCT for hematologic malignancies at Kansai Medical University Hospital from January 1, 2010 until April 30, 2016.Overall survival (OS), non-relapse mortality, cause of death, and GVHD clinical staging were investigated. Non-relapse deaths were considered related to GVHD if the patient died from either GVHD itself or from an infection that developed while receiving systemic steroids (at least 10 mg prednisone daily or equivalent) for the treatment of GVHD. Bloodsamples were obtained 7 days after transplantation. ST2 and REG3α concentrations were analyzed by ELISA, and were used for the 2-biomarker algorithm (Hartwell et al. JCI Insight 2017). Results Of the 47 patients (male 24, female 23), median age was 52 years (range, 22-69 years). Eleven patients received PBSCT, 27 patients received bone marrow transplantation, and 9 patients received cord blood transplantation. Of 27 patients who received bone marrow transplantation, 3 patients were transplanted allografts from HLA mismatched donor. Twenty-seven patients underwent reduced intensity conditioning regimen. The median survival time and 1-year OS rate were 375 days (range, 7-3236 days) and 53.2 %, respectively.The overall cumulative incidences of 6-month NRM was 23.4%. The incidence of grade II-IV and III-IV acute GVHD were 17.0%, and 13.2%, respectively. Twelve patients were identified as high risk (HR), and 35 patients were identified as low risk (LR) by the MAGIC algorithm. OS was 33.3% in HR and 71.4% in LR (p