ALBERT

Description: The molecular basis of ineffective hematopoiesis in myelodysplastic syndromes (MDS) has yet to be defined. Elevation of tumor necrosis factor (TNF)-α in bone marrow plasma has been considered to be a cause of apoptosis of hematopoietic progenitor cells, and this could result in ineffective hematopoiesis in MDS. However, the mechanism of TNF-α elevation has not been elucidated. We recently found aberrant regulation of c-fos mRNA stability in neutrophils isolated from MDS patients (Feng et al, PLoS ONE, 2013). Since the expression levels of RNA-binding proteins that regulated c-fos mRNA stability were not altered in the majority of the patients and no mutations were detected in c-fos mRNA, microRNAs were possibly involved in the insufficient stabilization of c-fos mRNA. In this study, we chose 20 microRNAs targeting c-fos according to four different databases and compared their expression levels in neutrophils between MDS patients and healthy volunteers. Among them, miR-34a and miR-155 were significantly increased in MDS (p

Description: Introduction: Nucleated cells differential analysis of body fluid (BF) samples is important diagnostic tool for several diseases including cancer metastasis. Detection of tumor cells in BF requires the manual morphological scanning of slides by the cytopathologists, which is time-consuming, labor-intensive and not always reliable because of a relatively low overall sensitivity rates (ranging 40-90%) with the higher false-negative rates for lymphomas and mesotheliomas. This study aimed to develop the scattergram gating analysis for detection of tumor cells in BF using the automated hematology analyzer Sysmex XN-1000 (Sysmex, Kobe, Japan). Methods: We used a total of 220 BF samples (53 cerebrospinal fluids, 73 pleural or ascitic fluids, and 94 peritoneal dialysis effluent) obtained from patients with cytological diagnoses (papanicolaou stain) including negative and positive of tumor cells, and chronic inflammation with an elevated lymphocyte and histiocyte fractions. As a reference method, morphological manual differential (200 cells counts) was performed by two experienced technologists using cytospin slides stained with the May-Grunwald Giemsa. The BF mode of XN-1000 (XN-BF) determines the differential cell counts of BF samples using side scatter and fluorescence intensity in WDF channel after the nuclear DNA/RNA stain by nucleic acid dye. The polymorphonuclear cells, mononuclear cells and high fluorescence cells (HF-BF), corresponding with a high amount of nucleic acids, are differentiated. Mesothelial cells and macrophages are theoretically categorized as HF-BF cells and included in the total nucleated cell count but not in the WBC count. We selected the tumor cells positive (n=18) and chronic inflammation positive samples (n=108) by morphological manual differential, and reviewed their scattergram patterns determined by XN-BF. Then the novel scattergram gating strategy targeting the tumor cells was evaluated. The gating criteria were based on the WDF scatter plots; #1: detect the cells with larger size and higher fluorescence signal in comparison with general leukocytes, which mainly derived from clustered tumor cells, #2: detect the middle sized mononuclear cells with less granules rather than neutrophils and similar fluorescence signal to monocytes, which targeting hematological malignant cells and solid tumor cells. BF samples that meet at least one criterion were interpreted as positive for tumor cells. Results: The malignant BF samples containing tumor cells showed the different scattergram patterns from the benign ones with chronic inflammation; the malignant BF formed the isolated cellular clusters in our gating system, and the inflammatory BF showed the continuous expansion into the HF-BF area. Our scattergram gating analysis achieved an overall sensitivity of 72.2% and specificity of 98.0% in detecting tumor cells positive samples when screening against all samples outcomes. The positive predictive value was 76.5% and the negative predictive value was 97.5%. For the samples with absence of tumor cells and inflammatory observations (n=94), no false positive was detected. Of notes, each of our gating criterion detected the different type of tumor cells. For example, the scattergram gating analysis #1 achieved an overall sensitivity of 72.7% and specificity of 99.0% in detecting adenocarcinoma with the positive predictive value of 80.0% and the negative predictive value of 98.6%. Conclusions: A simple measurement of BF by automated hematology analyzer in which cells are minimally handled has a potential to reduce costs and allow routine cell screening in clinical applications. For the BF malignancy diagnostics, the scattergram gating analysis is promising to (i) augment diagnostic routines without requiring additional sample preparation procedure, (ii) limit operator bias, and (iii) provide a standardized measurement. Disclosures No relevant conflicts of interest to declare.

Description: Background: Recent transcriptome-wide analyses have revealed an overwhelming amount of transcribed, but not translated, non-coding RNAs capable of influencing diverse cellular processes such as proliferation, apoptosis, and motility. Long non-coding RNA (lncRNAs), which are commonly defined as transcripts 〉200 nt in length, have emerged as a class of key regulatory RNA. LncRNAs are deregulated in diverse human cancers and associated with disease progression; however, little is known about its role in multiple myeloma (MM). To elucidate the role of lncRNAs in MM, we studied the expression patterns of several well-known lncRNAs in the plasma cells of MM, MGUS and plasmacytoma patients and the function in MM cell lines in vitro. Moreover, to reveal the distinct lncRNA signature comprehensively, we performed next-generation sequencing-based RNA sequencing. Methods: CD138+ plasma cells from bone marrow (BM) mononuclear cells were obtained from 110 MM patients, 48 MGUS patients, 19 control subjects and 1 patient with extramedullary plasmacytoma of the liver and analyzed after obtaining informed consent from all the patients. The expression levels of lncRNAs MALAT1, ANRIL, HOTAIR, HOTTIP, and XIST were determined by a RQ-PCR analysis. RNase H-activating LNA™ GapmeR antisense oligonucleotides were used to knockdown lncRNA in vitro in MM cell lines. The cell lines were then treated with bortezomib, MG132, doxorubicin and hypoxic conditions to evaluate the effects of cytotoxic stress on the lncRNA expression. This study was approved by the IRB of Gunma University Hospital in accordance with the Declaration of Helsinki. Results: A significant higher level of MALAT1 expression was observed in BM plasma cells of MM patients (4.49) compared to MGUS patients (1.51) and control subjects (0.55) (p

Description: Multiple myeloma (MM) is a neoplasm of plasma cells that often remains fatal despite the use of high-dose chemotherapy with hematopoietic stem cell transplantation. In the clinical setting, the introduction of novel agents, such as proteasome inhibitors and immunomodulatory drugs, has improved the clinical outcomes of both patients with newly diagnosed MM and patients with advanced MM. However, most patients eventually relapse and develop drug resistance. T-LAK cell-originated protein kinase (TOPK), also known as PDZ-binding kinase (PBK), is a mitogen-activated protein kinase kinase (MAPKK)-like serine/threonine kinase that plays a critical role in many cellular functions, such as cell proliferation, apoptotic cell death, and inflammation, in normal tissues. Because the expression of TOPK is up-regulated during mitosis and is activated by the Cdk1/cyclin B1 complex, TOPK is thought to have a role in cytokinesis. While the expression of TOPK is very low in most normal human tissues except for testis and placenta, it is overexpressed in various malignant neoplasms, indicating its crucial role in tumorigenesis. Phosphorylation of TOPK leads to the activation of the MAPK signaling pathway including p38 and Ras extracellular signal-regulated kinase (ERK). Moreover, TOPK interacts with p53 tumor suppressor protein and inhibits its function. Ribosomal protein S6 kinase (RSK2) is a downstream target of the ERK/MAPK signaling cascade and it has a pivotal role in cell survival and proliferation. Recent studies suggest that RSK2 inhibition induces apoptotic cell death and sensitizes MM cells to lenalidomide. Suppression of p53 function is also involved in MM progression. Taken together, these data suggest that TOPK might be an attractive target for new therapeutic agents against this incurable hematological malignancy. HI-TOPK-032, which is a potent and specific inhibitor of TOPK, occupies the ATP-binding site of TOPK and thereby suppresses TOPK kinase activity. In the present study, we investigated the role of TOPK/PBK in MM as a potential therapeutic target by using HI-TOPK-032. MTSand trypan blue dye exclusion assays showed that HI-TOPK-032 inhibited the proliferation of various MM cell lines, including U266, RPMI8226, MM1.S, OPM-2, and KMS-11, in a dose- (0 to 10 mM) and time- (0 to 72 h) dependent manner. To examine the mechanisms behind the growth inhibition effect induced by HI-TOPK-032, assays for apoptotic cell death were performed; these assays demonstrated that HI-TOPK-032 induced both early and late apoptosis in MM cells. To investigate the molecular mechanisms of HI-TOPK-032-induced cell death in MM cells, the expression of various cell death-associated proteins and down-stream molecules of TOPK was examined. Western blotting analysis showed that HI-TOPK-032 arrested cell growth and induced apoptotic cell death in MM cells in a dose-dependent manner by reducing t he phosphorylation of ERK and RSK2, thereby reducing the expression of the target molecules of RSK2, i.e., MCL1 and c-Myc. Moreover, HI-TOPK-032 induced p53 expression in a dose-dependent manner. We next examined the effects of HI-TOPK-032 on bortezomib (BTZ)-resistant MM cells, which represent an urgent issue in clinics and for which a therapeutic solution is important. Interestingly, HI-TOPK-032 inhibited the proliferation of both BTZ-sensitive wild-type KMS cells and BTZ-resistant KMS cells, suggesting that BTZ resistance can be overcome by targeting TOPK. Because our results showed that HI-TOPK-032 reduced the phosphorylation of RSK2, and previous studies have suggested that RSK2 inhibition sensitized MM cells to lenalidomide, we next studied the effects of HI-TOPK-032 in combination with lenalidomide on MM cell growth. HI-TOPK-032 and lenalidomide synergisticallyinduced growth arrest in not only lenalidomide-sensitive MM cells, but also in lenalidomide-resistant cells. To determine whether HI-TOPK-032 can re-sensitize BTZ-resistant cells to the anti-MM activity of BTZ, the effects of the combination of HI-TOPK-032 and BTZ were tested using an MTS assay. Interestingly, HI-TOPK-032 was able to re-sensitize BTZ-resistant MM cells to BTZ. These results indicate that the inhibition of TOPK may serve as an attractive therapeutic option for both patients with BTZ- or lenalidomide-resistant MM. In conclusion, these data suggest that TOPK/PBK can be a promising molecular target for the treatment of MM. Disclosures Kizaki: Nippon Shinyaku Co., Ltd.: Research Funding; Ono Phranacutical Co., Ltd.: Consultancy; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Chugai Phrarmaceutical Co., Ltd.: Research Funding.

Description: Background: Donor selection is one of key factors for better outcomes after hematopoietic stem cell transplantation (HSCT). It is shown that increased donor age is associated with high mortality. In the era of high-resolution typing of HLA and various methodological options in HSCT (e.g. conditioning regimens or cell source), however, the significance of donor age in the selection of donor or cell source is unclear. Here we examined the impact of donor age on clinical outcome after unrelated bone marrow transplantation (UBMT) and compared to the outcome after unrelated cord blood transplantation (UCBT). Patients and methods: For this retrospective cohort study, clinical data of donors and recipients were obtained from the registry data of the Japan Society of Hematopoietic Cell Transplantation (JSHCT). This study included 6035 adult patients 16 years of age or older with AML, MDS, ALL, or CML who received the first HSCT between 2000 and 2010. Among them, 3304 recipients received UBMT from 8/8 HLA-matched or 7/8 HLA-matched for HLA-A, -B, -C, and -DRB1 allele-level donor and 2731 recipients received single-unit UCBT from maximum 2-antigen (HLA-A, -B, -DR antigen-level) mismatched donor. Risk factors for overall mortality and other endpoints were analyzed using Cox proportional hazards models and Fine and Gray's proportional hazards models, respectively. Results: The median ages of UBMT and UCBT recipients were 42 years (range, 16-77) and 50 years (range, 16-82), respectively. The median age of UBMT donor was 34 years (range, 20-55). Among 3304 UBMT recipients, older donor age (≥40 years) was a significant risk factor for overall mortality (adjusted HR, 1.14; 95% CI, 1.03-1.27, p=0.015) and grades II-IV acute graft-versus-host disease (aGVHD) (adjusted HR, 1.27; 95% CI, 1.13-1.43, p

Description: Background In Japan, the phase II study of bortezomib-melphalan-prednisolone (VMP) for NDMM using the same protocol of VISTA trial revealed that there were more treatment discontinuation in this study than in the VISTA study. We planned five cycles of reduced intensity VMP therapy as an induction. And it is reported that lenalidomide plus low dose dexamethasone (Rd) therapy is very effective regimen after VMP therapy. In order to further improve its outcome, patients are treated with six cycles of Rd and maintenance of lenalidomide after VMP induction. Patients and methods We included 82 pts with NDMM. Pts were received 5 cycles of VMP followed by 6 cycles of Rd. After Rd, pts received maintenance of lenalidomide. VMP included the IV or SQ administration of weekly bortezomib at 1.3 mg/m2 in combination with oral melphalan 6 mg/m2 and prednisone 60 mg/m2 once daily on days 1-4 of a 35-day cycle. Rd treatment consisted of lenalidomide 25 mg daily on days 1-21 plus dexamethasone 40 mg weekly of a 28-day cycle. Lenalidomide maintenance therapy consisted of lenalidomide 10 mg daily on days 1-21 of a 28-day cycle. Results In total, 82 pts were recruited in the trial by 27 Japanese centers between 10/2012 and 8/2014. Median age were 73.5 years (range 61-84), 37.8% were 75 years of age or older, 45.1% were male, 48.8% had International Staging System (ISS) stage II and 20.7% had ISS stage III. Fifty four patients had IgG-type myeloma (65.8%), 19 had IgA-type (23.2%), and 9 had Bence Jones-type (11%). Of the cases analyzed by FISH (N=80), 16.3% had t(4;14), 10% had del 17p and 41.3% had +1q21. Eight patients (10%) had t(4;14) and +1q21, five patients (6.3%) had del 17p and +1q21 and only one patient (1.3%) had t(4;14), del 17p and +1q21. The best response during VMP therapy (the maximum treatment number was 5 cycles) could be evaluated. After five course of VMP therapy, the rates of partial response (PR) or better were 68% including sCR (5%), CR (6%),VGPR (20%) and PR (37%). The best response rate after VMP+Rd (maximum treatment number of Rd was 6 cycles) was also evaluated. The rates of PR or better were 90% including sCR (6%), CR (16%), VGPR (39%) and PR (29%). The most commonly observed grade 3 or higher adverse events during VMP therapy were anemia (30%), neutropenia (16%), thrombocytopenia (5%) and GI toxicity (6%). Summary The induction therapy of reduced intensity VMP was safe and effective. The best response rate after VMP+Rd was very effective. However, we need to evaluate consolidation of Rd and maintenance of lenalidomide after longer follow up. Disclosures Ishida: Takeda: Honoraria, Research Funding; Celgene: Honoraria; Janssen: Honoraria. Sunami:Ono: Research Funding; Takeda: Research Funding.

Description: Background: MicroRNAs (miRs) are small non-coding RNAs of 19-25 bases in length that have the ability to modulate gene expression. Some miRs are involved in carcinogenesis and act as tumor suppressor genes (TSG). It has been shown that epigenetics and miRs play an important role in multiple myeloma (MM) progression; however, precise mechanism underlying miR dysregulation has not yet been fully elucidated. Transcriptional silencing of TSG in cancer cells is often associated with DNA methylation and carried out by DNMTs. miR-29 family directly targets DNMTs and promoters of miR-34 family are also methylated in cancers. In this study, we attempted to clarify the interaction between miR and epigenetics focusing on the miR-29 and miR-34 families and their associated genes to understand mechanism of miR dysregulation in MM. Methods: Bone marrow plasma cells from 123 MM patients, 57 MGUS patients, 20 control subjects and 9 MM cell lines were analyzed. This study was approved by the IRB of Gunma University and all patients provided their informed consent prior to enrollment. MiRs and their target gene mRNA values were determined by RQ-PCR. DNA methylation status was determined by methylation-specific PCR. Decitabine, nutlin-3, c-myc inhibitor 10058-F4, and miRNA-mimicTM were used. Result: We found a significantly reduced expression of miR-29a, -29b, -29c, -34a, -34b, and -34c in MM patients compared with MGUS patients and the control subjects (all: p

Description: Fetal hemoglobin (HbF) induction with hydroxyurea (HU) remains the only specific pharmacological treatment for sickle cell disease (SCD) patients. Compliance is key to achieve high HbF levels, but assessing patient adherence to long-term treatment is difficult. Since patient interviews are often unreliable, most hematologists rely on mean corpuscular volume (MCV) and HbF levels to monitor compliance, but red blood cell indices change slowly. Hypersegmented neutrophils (HN) have been for long recognized in patients taking HU, and automated digital cell morphology platforms allow routine peripheral blood smears to be conveniently photographed and stored for analysis. We have designed a protocol to determine the percentage of circulating HN and have studied how this parameter compares with others commonly used in clinical practice during HU therapy, such as HbF, MCV, and reticulocyte count. We collected blood samples from 38 patients with SCD in steady state, receiving an average dose of 20.5mg/kg/day of HU (range 10.2-33.3) at our Hematology outpatient clinic. Complete blood counts and HN percentage determinations were performed on a Sysmex XE-5000/Cellavision DM96 equipment. HbF levels were determined by HPLC (Bio-Rad). Statistical analysis was performed using GraphPad Prism 6.0 software. Standard protocol for image acquisition initially recorded 100 cells per smear and HN count was performed independently by two experienced laboratory personnel using the same image database, by counting cells with 5 or more nuclear segments. To increase precision due to the relatively low numbers of neutrophils in some samples, analysis was subsequently improved by acquiring 300 images per smear aiming to picture 100 neutrophils. Mean hemoglobin level in our population was 8.6±1.3g/dL (mean±SD), MCV 103.8±14.2fL, reticulocyte count 222,950±129,090/uL, and HbF was 13.1±7.8% (range 2.1-30.9%). HN percentage as determined with 100 images per smear displayed only borderline correlation with HbF levels (P =0.094), but acquisition of 300 images per smear yielded over 90 neutrophil images in 35/38 samples. Average HN percentage was 14,5% (range 2,0-45,0%) and correlated positively with HbF (r=0.4172, P =0.009) and MCV (r=0.4301, P =0.007). As expected, HbF also correlated with MCV (r=0.5777, P =0.0001) and reticulocyte count (r=-0.489, P =0.003). Despite our limited number of patients, ROC curve analysis showed that HN percentage had an area under curve of 0.7241 to detect patients with HbF〉20% (P =0.047). Patients with more than 6 segments per neutrophil were also more likely to have higher HbF. Average daily HU dose did not correlate with HbF or any of the parameters analyzed. Lack of correlation between dose and HbF supports that medication adherence may indeed be suboptimal in the study population, but that may also be caused by individual differences in HU metabolism. While MCV still displayed the strongest correlation with HbF in our study, HN percentage performed similarly, with higher counts associated with higher HbF. Neutrophils have a much shorter half-life than erythrocytes, with an estimated half-life of hours rather than the 20 days calculated for sickle red blood cells. Therefore, digital cell morphology analysis enables clinical laboratories to determine HN counts that can change more quickly than HbF, MCV, or reticulocyte counts during HU therapy. Monitoring the number of HN may allow more timely assessment of compliance in patients starting HU or in those in need for HU in combination with sporadic blood transfusions, since neutrophil hypersegmentation should not be affected by changes in red blood cell mass. Further studies should investigate HN percentage as a potential surrogate marker of response to HU and of patient compliance. Financial support: FAPESP Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by the production of platelet antibodies, resulting in the destruction of platelets and inhibition of their production. Many cytokine profile study have revealed a clear T helper type1 (Th1) cytokine polarization in chronic ITP patients, using quantitative RT-PCR and flow cytometry method. However, it remains unclear whether genetic factors of Th1/Th2 cytokine and cytokine receptor affect chronic ITP. We investigated the impact of IFN-γ+874T/A, IFN-γ receptor (R) -611G/A, IL-4 -590C/T, and IL-4 receptor (R) a Q576R polymorphisms on the susceptibility and clinical feature of chronic ITP and Th1/Th2 ratio in peripheral blood of ITP patients, Patients and methods: Genotyping was determined by PCR based technique and direct sequencing. The diagnosis and response criteria of the ITP were defined according to International Working Group criteria. We evaluated Th1/Th2 ratio in peripheral blood of 15 normal donors and 25 ITP patients by intracellular flow cytometry. Intracellular IL-4 (Th2 cytokine) and IFN-γ (Th1 cytokine) production was assessed in CD4+ T lymphocytes activated by phorbol 12-myristate 13-acetate and ionomycin by flow cytometry. This study was approved by the IRB of our hospital. Results: The platelet count ranged from 1´109/L to 98´109/L with a mean count of 32´109/L at the initial diagnosis. Eighty-three patients (56.1%) had bleeding tendency and 24 patients (16.2%) had severe thrombocytopenia (〈 10 ´109/L). Steroid treatment was given to 86 patients (58.1%), and eradication of Helicobacter pylori was performed in 38 patients (25.7%), while splenectomy was performed in only 18 patients (12.2%). As compared to control group, chronic ITP patients had significantly higher frequency of the IL-4R576 non-QQ (low function type) than QQ (high function type) (29.7% vs 15.2%, P