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The standard cryoprotectant for human cellular products is dimethyl sulfoxide (DMSO), which is associated with hematopoietic cell infusion-related adverse events (HCI-AEs) in hematopoietic stem cell transplantation including peripheral blood stem cell (PBSC) transplantation (PBSCT). DMSO is often used with hydroxyethyl starch (HES), which reduces DMSO concentration while maintaining the postthaw cell recovery. The cryoprotectant medium CP-1 (Kyokuto Pharmaceutical Industrial) is widely used in Japan. After mixture of a product with CP-1, DMSO and HES concentrations are 5% and 6%, respectively. However, the safety profile of CP-1 in association with HCI-AEs has not been investigated.To compare CP-1 with other cryoprotectants, we conducted a subgroup analysis of PBSCT recipients in a prospective surveillance study for HCI-AEs. Moreover, we validated the toxicity of CP-1 in 90 rats following various dose administration.The PBSC products cryopreserved with CP-1 (CP-1 group) and those with other cryoprotectants, mainly 10% DMSO (non-CP-1 group), were infused into 418 and 58 recipients, respectively. The rate of ≥grade 2 HCI-AEs was higher in the CP-1 group, but that of overall or ≥grade 3 HCI-AEs was not significantly different, compared to the non-CP-1 group. Similarly, after propensity score matching, ≥grade 2 HCI-AEs were more frequent in the CP-1 group, but the ≥grade 3 HCI-AE rate did not differ significantly between the groups. No significant toxicity was detected regardless of the CP-1 dose in the 90 rats.Infusion of a CP-1-containing PBSC product is feasible with the respect of HCI-AEs.

Purpose: EZH2 and EZH1, the catalytic components of polycomb repressive complex 2 (PRC2), trigger trimethylation of H3K27 (H3K27me3) to repress the transcription of target genes and are implicated in the pathogenesis of various cancers including multiple myeloma and prostate cancer. Here, we investigated the preclinical effects of UNC1999, a dual inhibitor of EZH2 and EZH1, in combination with proteasome inhibitors on multiple myeloma and prostate cancer.Experimental Design: In vitro and in vivo efficacy of UNC1999 and the combination with proteasome inhibitors was evaluated in multiple myeloma cell lines, primary patient cells, and in a xenograft model. RNA-seq and ChIP-seq were performed to uncover the targets of UNC1999 in multiple myeloma. The efficacy of the combination therapy was validated in prostate cancer cell lines.Results: Proteasome inhibitors repressed EZH2 transcription via abrogation of the RB-E2F pathway, thereby sensitizing EZH2-dependent multiple myeloma cells to EZH1 inhibition by UNC1999. Correspondingly, combination of proteasome inhibitors with UNC1999, but not with an EZH2-specific inhibitor, induced synergistic antimyeloma activity in vitro. Bortezomib combined with UNC1999 remarkably inhibited the growth of myeloma cells in vivo. Comprehensive analyses revealed several direct targets of UNC1999 including the tumor suppressor gene NR4A1. Derepression of NR4A1 by UNC1999 resulted in suppression of MYC, which was enhanced by the combination with bortezomib, suggesting the cooperative blockade of PRC2 function. Notably, this combination also exhibited strong synergy in prostate cancer cells.Conclusions: Our results identify dual inhibition of EZH2 and EZH1 together with proteasome inhibition as a promising epigenetics-based therapy for PRC2-dependent cancers. Clin Cancer Res; 23(16); 4817–30. ©2017 AACR.

Supplementary Materials and Methods Supplementary References Supplementary Tables Supplementary Figures and legends Supplementary Table S1 Combination index (CI) values. Supplementary Table S2 Significantly upregulated PRC2 gene sets in UNC1999-treated MM.1S cells (FDR q-value < 0.05) Supplementary Table S3 Significantly upregulated PRC2 gene sets in combination-treated MM.1S cells (FDR q-value < 0.05) Supplementary Table S4 Top 74 PRC2 genes that are significantly altered in UNC1999-treated MM.1S cells Supplementary Table S5 Significantly upregulated gene sets in RPMI8226 cells transduced with EZH2- overexpressing versus empty vectors. Supplementary Table S6 List of primers used for Quantitative RT-PCR Supplementary Table S7 List of primers used for ChIP-PCR Supplementary Figure S1. Pharmacological and lentiviral suppression of EZH2 and EZH1 in MM cell lines. Supplementary Figure S2. Proteasome inhibitors downregulate EZH2 in MM cell lines. Supplementary Figure S3. EZH2 suppression by bortezomib is mediated through E2F1. Supplementary Figure S4. MM patients with higher levels of EZH2 expression have poorer response to bortezomib. Supplementary Figure S5. EZH2 overexpression in MM cells confers resistance to bortezomib through upregulation of gene sets related to MYC, cell cycle and metabolism. Supplementary Figure S6. UNC1999 enhances the cytotoxicity induced by bortezomib in MM cell lines including resistant ones but not in PBMNCs from healthy donors. Supplementary Figure S7. The combination of UNC1999 and bortezomib induces significant apoptosis in vitro and is tolerated in vivo. Supplementary Figure S8. The expression of NR4As in MM. Supplementary Figure S9. Dual inhibition of EZH2 and EZH1, but not specific EZH2 inhibition, is critical for the synergy with bortezomib in MM cell lines. Supplementary Figure S10. UNC1999 exhibits synergy with bortezomib in DU145 prostate cancer cells.

Comparative studies between total body irradiation (TBI)-based and busulfan-based myeloablative conditioning (MAC) regimens for cord blood transplantation (CBT) have been limited. We retrospectively analyzed the results of single-unit CBT in 333 adult patients who received either TBI-based (

An intensified myeloablative conditioning regimen, involving the addition of granulocyte colony-stimulating factor (G-CSF)-combined high-dose cytarabine (12 g/m2) to standard total body irradiation and cyclophosphamide, has been performed for adult patients with myeloid malignancies in single-unit cord blood transplantation (CBT) since 1998 in our institute. We update the results of CBT, as the first allogeneic hematopoietic cell transplantation after this conditioning regimen, in 169 patients with a median long-term follow-up of 10.4 years. The median age was 43 years (range, 16 to 59 years). Ninety-four patients (56%) were in non-remission at the time of CBT, and 124 patients (73%) were acute myeloid leukemia. The median cryopreserved cord blood total nucleated cell dose and CD34+ cell dose was 2.40 × 107/kg and 0.93 × 105/kg, respectively. The cumulative incidence of neutrophil recovery at 42 days was 94.4% (95% confidence interval [CI]: 88.6–97.3%). Among the whole cohort, 105 patients were still alive at the end of the study period. The cumulative incidences of relapse and non-relapse mortality at 10 years were 26.0% (95% CI: 19.5–33.0%) and 16.9% (95% CI: 11.4–23.4%), respectively. There was an overall survival probability of 62.5% (95% CI: 54.3–69.7%) at 10 years. Higher disease risk index alone significantly affected higher overall mortality (hazard ratio 2.21, P = 0.003) in multivariate analysis. These outcomes demonstrate that G-CSF-combined myeloablative conditioning could have favorable long-term remission rates for adult patients with myeloid malignancies undergoing single-unit CBT.

Background Hematopoietic cell infusion-related adverse events (HCI-AEs) in hematopoietic stem cell transplantations (HSCTs) have been largely attributed to toxicity of dimethyl sulfoxide (DMSO) for cryopreservation, but HSC products also contain various cells and plasma components. Our recent prospective study of 1125 HSCT recipients revealed the highest overall HCI-AE rate in bone marrow transplantation (BMT) using fresh/noncryopreserved products, although products of peripheral blood stem cell transplantation and cord blood transplantation (CBT) are generally cryopreserved with DMSO containing smaller plasma volumes. We aimed to clarify if product volume and component effects are more substantial in small recipients including children. Study design and methods We performed subgroup analysis on 219 recipients of 45 kg or less body weight (whole small recipients), including 90 children (pediatric recipients), from the original cohort (general recipients). Results Whereas overall HCI-AE rates did not differ among hematopoietic stem cell sources in the general recipients, bradycardia most often occurred after CBT in whole small recipients. Conversely, whole small and general recipients shared the same trend of having the highest rate of hypertension in BMT. The overall HCI-AE rate was higher in allogeneic HSCT compared with autologous HSCT. Notably, pediatric recipients showed a 10-fold higher incidence of nausea and vomiting in allogeneic HSCT compared with autologous HSCT, suggesting a possible role of allogeneic antigens. Multivariate analysis identified a relatively large infusion volume per body weight as a significant factor correlating with HCI-AE in whole small recipients. Conclusions We should be aware of product volume and specific HCI-AEs such as nausea and vomiting in small patients including children.

Anagrelide is used worldwide to treat essential thrombocythemia (ET) by reducing platelet counts. Cardiomyopathy and heart failure (HF) are rare but serious complications associated with anagrelide use, although no cases were reported during Japanese Phase I to III studies. A 46-year-old, otherwise healthy, Japanese ET patient developed HF with reduced ejection fraction after 18 months of treatment with 1.0-3.5 mg of anagrelide daily. HF was stabilized with anagrelide withdrawal and guideline-directed HF therapy. The cardiac function returned to normal after six months. This case suggests that anagrelide can cause cardiomyopathy and HF in ET patients, regardless of nationality, comorbid cardiovascular conditions, or therapy duration.

Myeloid/lymphoid neoplasms with PDGFRB rearrangement are a distinct type of myeloid neoplasms that occur in association with rearrangement of PDGFRB at 5q32. The hematological features most often show prominent eosinophilia. We herein report a patient with myeloid/lymphoid neoplasms with PDGFRB rearrangement with t (5;10) (q33;q22) who showed atypical chronic myeloid leukemia-like clinical features without eosinophilia and achieved an optimal response to imatinib. A sequence analysis showed a CCDC6-PDGFRB fusion gene with a new break point in the PDGFRB gene. This is the sixth case of myeloid/lymphoid neoplasm with PDGFRB rearrangement harboring a CCDC6-PDGFRB fusion gene, and it has a new breakpoint in the PDGFRB fusion gene.

The efficacy of pharmacokinetically (PK) guided, once-daily administration of busulfan (BU) was evaluated in elderly patients with acute myeloid leukemia/myelodysplastic syndrome (AML/MDS). Twenty-one patients (median age 61) received 30 mg/m

The novel small molecule PTC596 inhibits microtubule polymerization and its clinical development has been initiated for some solid cancers. We herein investigated the preclinical efficacy of PTC596 alone and in combination with proteasome inhibitors in the treatment of multiple myeloma (MM). PTC596 inhibited the proliferation of MM cell lines as well as primary MM samples in vitro, and this was confirmed with MM cell lines in vivo. PTC596 synergized with bortezomib or carfilzomib to inhibit the growth of MM cells in vitro. The combination treatment of PTC596 with bortezomib exerted synergistic effects in a xenograft model of human MM cell lines in immunodeficient mice and exhibited acceptable tolerability. Mechanistically, treatment with PTC596 induced cell cycle arrest at G2/M phase followed by apoptotic cell death, associated with the inhibition of microtubule polymerization. RNA sequence analysis also revealed that PTC596 and the combination with bortezomib affected the cell cycle and apoptosis in MM cells. Importantly, endoplasmic reticulum stress induced by bortezomib was enhanced by PTC596, providing an underlying mechanism of action of the combination therapy. Our results indicate that PTC596 alone and in combination with proteasome inhibition are potential novel therapeutic options to improve outcomes in patients with MM.

Anti-platelet agents or anticoagulants are administered for patients with multiple myeloma (MM) receiving immunomodulatory drugs (IMiDs) to prevent thrombotic events (TEs). However, there is a discrepancy between current guidelines and clinical practice in thromboprophylaxis and the varied incidence of TEs depending on patient cohort. Therefore, a consensus on the optimal thromboprophylactic strategy is needed. To determine an appropriate strategy for the prevention of TEs in MM patients receiving IMiDs, we performed a retrospective single-institution analysis. In total, 95 MM patients (62% male, median age 65 years, range 30–85 years) from November 2008 to January 2018 were recruited, and 140 cases were analyzed in the medical-record-based study. Thromboprophylactic drugs were given to 69% of patients, anti-platelet agents to 66%, and anticoagulants to 3.0%. Seven TEs (5.0%) and six bleeding events (4.3%) were observed, but no patients died from thrombohemorrhage. The median follow-up period was 184 days (range 21–2224), and the cumulative TE incidence was 1.7% at 3 months, 7.0% at 1 year, and 12.5% at 3 years. Multivariate analysis determined that age > 70 years (p = 0.012) and BMI

Adverse events (AEs) associated with blood transfusions, including component-specific red cell, platelet, and plasma products, have been extensively surveyed. In contrast, surveillance of AEs associated with hematopoietic stem cell (HSC) products in HSC transplantation (HSCT) has been less rigorous, even though HSC products include a diversity of immature and mature hematopoietic cells, substantial plasma, and dimethyl sulfoxide (DMSO) in the case of cryopreserved HSC products. HSC infusion-related AEs have been attributed to DMSO toxicity, but AEs associated with the infusion of noncryopreserved HSC products are not uncommon. To quantify the frequencies, types, and risk factors of HSC infusion-related AEs, we implemented national surveillance for AEs observed within 24 hours after infusion. Herein we report on 1125 HSCTs, including 570 peripheral blood stem cell transplantations (PBSCTs) (290 autologous [auto-] and 280 allogeneic [allo-]), 332 allo-bone marrow transplantations (allo-BMTs) and 223 allo-cord blood transplantations (allo-CBTs). Unexpectedly, incidences of grade ≥ 2 AEs were most frequent in allo-BMTs (37.7%) with no DMSO in any product compared with auto-/allo-PBSCTs (20.9%, P .001) and allo-CBTs (19.3%, P .001) typically cryopreserved with DMSO. Hypertension was most often noted in BMTs, whereas nausea/vomiting, fever, and allergic reactions were most frequent in allo-PBSCTs. In a multivariate analysis, a history of transfusion reactions was a risk factor for overall AEs in all HSCTs (odds ratio [OR] = 1.459, P = .045). For grade ≥ 2 AEs in allo-HSCTs, a history of transfusion reactions (OR = 1.551, P = .044) for overall AEs, and high infusion volume (OR = 7.544, P = .005) and allo-PBSCTs (versus BMTs, OR = 9.948, P = .002) for allergic reactions were identified as risk factors. These findings suggest that some factors unrelated to DMSO, such as allo-antigens, contribute to HSC infusion-related AEs. As severe AEs, a total of 117 grade ≥ 3 AEs were reported in 1125 HSCTs, including life-threatening complications in 3 (0.3%) HSCTs: 1 allo-CBT (anaphylaxis) and 2 allo-PBSCTs (hypoxia, kidney injury) with cryopreserved product. Our data show that HSC infusion risks vary by product, can be severe, and should be monitored with the same rigor as modern transfusion hemovigilance programs.

Mediastinal gray zone lymphoma (MGZL) is a provisional entity with intermediate features between classical Hodgkin lymphoma (cHL) and diffuse large B-cell lymphoma. Outcomes for patients with MGZL are reportedly poorer than those for patients with cHL or primary mediastinal large B-cell lymphoma. Additionally, no standard management guidelines for patients with MGZL are available, primarily due to its recent identification, rarity, and challenges in diagnosis. Although recent several studies have suggested dose-adjusted EPOCH-R (etoposide, doxorubicin, vincristine, cyclophosphamide, prednisolone, and rituximab) may improve outcomes in patients with MGZL, numerous patients still suffer from relapsed/refractory MGZL, and the optimal management for such patients remains uncertain. Here, we report the first case of successful treatment of refractory MGZL by tandem high-dose chemotherapy supported by autologous stem cell transplantations (auto-SCTs) and consolidative radiotherapy (RT). To date, the patient remains in CR 33 months after the completion of RT, with no significant complications. This case suggests that tandem auto-SCTs may be a promising therapeutic option for relapsed/refractory MGZL.

Introduction: Recent studies based on next-generation sequencing revealed the genetic landscape of diffuse large B cell lymphoma (DLBCL). A new genetic classification system was proposed, including four groups; the BCL2 and SGK1 groups that were both associated with germinal center B-cell like (GCB) DLBCL, an MYD88 group that was associated with non-GCB DLBCL, and a NOTCH2 group that was not associated with a specific cell-of-origin (COO). Unfortunately, this classification method is somewhat complex and thus not fully accepted in clinical practice. Furthermore, part of all cases remained unclassified; as such, it is clear that the diversity of the genetic landscape of DLBCL is not yet fully understood. In this study, we performed a retrospective analysis of genetic abnormalities and their correlations with clinical characteristics in a Japanese cohort of patients diagnosed with DLBCL. Materials and Methods: We collected clinical data from 224 of a total of 234 patients who were diagnosed with DLBCL between 2013 and 2018 at Chiba University Hospital. We also collected formalin-fixed paraffin-embedded (FFPE) specimens obtained from 204 patients via standard diagnostic procedures. The COO for each sample was assessed using the Hans algorithm with immunohistochemical staining; rearrangements of BCL2, BCL6, and MYC were evaluated by fluorescence in situ hybridization. Targeted DNA sequencing was performed using a 144-gene custom panel. Results: Of the 224 DLBCL patients evaluated in our study, the median age at diagnosis was 69 years (range, 18 - 92 years). The median follow-up time was 34 months; three-year progression-free survival (PFS) was 63.2%, and three-year overall survival was 78.0%. COO was determined in 218 cases; 93 of these cases (41.5%) were classified as GCB type and 125 cases (55.8%) as non-GCB type. We successfully performed targeted DNA sequencing in 167 of the 204 FFPE samples. Compared to previous reports, our cohort included relatively higher frequencies of mutations in PIM1 (44.3%), KMT2D (40.7%), MYD88L265P (37.7%), and CD79B (24.5%); by contrast, we detected comparatively lower mutation frequencies in B2M (6.6%), ETV6 (5.4%), CDKN2A (4.7%), and TNFAIP3 (4.1%). Multivariate adjustment of genes extracted by univariate analysis and factors identified by International Prognostic Index score revealed that genetic mutations of MYD88L265P (Hazard ratio [HR] = 2.045), IL16 (HR = 4.848), and BCOR (HR = 6.295) were significant prognostic factors. Mutations in IL16 and BCOR are novel factors that will be used to predict poor prognosis. In the 167 sequenced cases, 110 were classified into the aforementioned four subtypes based on mutations detected in 23 genes together with rearrangements of BCL2 and BCL6; specifically, 47 cases were classified as members of the MYD88 group, 28 in the BCL2 group, 19 in the NOTCH2 group, and 16 in the SGK1 group. We identified a trend toward differential PFSs among the four groups (p = 0.18); three-year PFS was 51.2% among patients with MYD88 group, 59.6% in the BCL2 group, 63.2% in the NOTCH2 group, and 75.0% for the SGK1 group. When compared outcomes of patients in the BCL6 group, patients in the MYD88 group had significantly inferior PFS (p = 0.043) among the non-GCB DLBCL patients; those in the BCL2 group revealed a trend toward inferior PFS compared with those in the SGK1 group (p = 0.34) among the GCB DLBCL patients. We next focused on 28 cases of CD5-positive DLBCL; this is a distinct DLBCL subtype with poor prognosis that is identified more frequently among Asians. We observed significantly higher frequencies of frameshift or nonsense mutations in CD58 and missense mutations in MYD88L265P. Furthermore, evaluation of gene expression data for CD5-positive DLBCL reported in a published database (Miyazaki K et al. Int J Hematol. 2015) revealed diminished expression of CD58 (p = 0.01) and augmented expression of MYD88L265P (p < 0.001). These results suggest that there may be a specific correlation between genetic mutations and gene expression. Intriguingly, these specific genes are key regulators of the immune response, suggesting that inflammation contribute to the unfavorable outcome of CD5-positive DLBCL. Conclusion: Our study provides support for the genetic classification of DLBCL using targeted DNA sequencing to evaluate outcomes in our Japanese cohort. We also identified mutations in novel candidate genes that may be used to predict clinical outcomes. Disclosures Nakaseko: Novartis Pharma KK: Speakers Bureau; Pfizer Japan Inc.: Speakers Bureau. Kaneda:SRL Inc.: Research Funding; SCRUM Inc.: Research Funding; Tsubakimoto Chain Co.: Research Funding; Takeda Foundation: Research Funding; Princess Takamatsu Cancer Research Fund.: Research Funding; Wedge Co Ltd: Research Funding.

Engraftment syndrome (ES) is an immune-mediated complication of allogeneic stem cell transplantation (SCT) characterized by fever, rash and vascular leak [1]. Pre-engraftment immune reactions (PIR)...

Myeloid/lymphoid neoplasms with PDGFRB rearrangement are a distinct type of myeloid neoplasms that occur in association with rearrangement of PDGFRB at 5q32. The hematological features most often show prominent eosinophilia. We herein report a patient with myeloid/lymphoid neoplasms with PDGFRB rearrangement with t (5;10) (q33;q22) who showed atypical chronic myeloid leukemia-like clinical features without eosinophilia and achieved an optimal response to imatinib. A sequence analysis showed a CCDC6-PDGFRB fusion gene with a new break point in the PDGFRB gene. This is the sixth case of myeloid/lymphoid neoplasm with PDGFRB rearrangement harboring a CCDC6-PDGFRB fusion gene, and it has a new breakpoint in the PDGFRB fusion gene.

Expression of the tumor suppressor gene NR4A3 is silenced in the blasts of acute myeloid leukemia (AML), irrespective of the karyotype. Although the transcriptional reactivation of NR4A3 is considered to have a broad-spectrum anti-leukemic effect, the therapeutic modalities targeting this gene have been hindered by our minimal understanding of the transcriptional mechanisms regulating its expression, particularly in human AML. Here we show the role of intragenic DNA hypermethylation in reducing the expression of NR4A3 in AML. Bisulfite sequencing analysis revealed that CpG sites at the intragenic region encompassing exon 3 of NR4A3, but not the promoter region, are hypermethylated in AML cell lines and primary AML cells. A DNA methyltransferase inhibitor restored the expression of NR4A3 following a reduction in DNA methylation levels at intragenic CpG sites. The in silico data revealed an enrichment of H3K4me1 and H2A.Z at exon 3 of NR4A3 in human non-malignant cells but that was excluded specifically in leukemia cells with CpG hypermethylation. This suggests that exon 3 represents a functional regulatory element involved in the transcriptional regulation of NR4A3. Our findings improve the current understanding of the mechanism underlying NR4A3 silencing and facilitate the development of NR4A3-targeted therapy.

We describe two cases of polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, and skin changes (POEMS) syndrome patients with deteriorated extravascular volume overload without increased levels of vascular endothelial growth factor after the administration of cyclophosphamide + granulocyte colony-stimulating factor for stem cell mobilization. We then measured the serum levels of 27 cytokines from these cases using a multiplex suspension array system. The analysis revealed the changes of cytokine profiles before cyclophosphamide + granulocyte colony-stimulating factor and after the development of capillary leak symptoms in both cases. This may improve our current level of understanding of the pathogenesis of POEMS syndrome not driven by vascular endothelial growth factor.

A 60-year-old man with chronic hepatitis C was referred to our hospital with significantly elevated total protein and serum IgM (9,500 mg/dl) levels identified via a routine checkup. Blood examination revealed increased serum IgM-monoclonal protein and serum-soluble IL-2 receptor (sIL2R) levels. Computed tomography and fluorodeoxyglucose positron emission tomography revealed pulmonary masses, abnormal soft tissue masses surrounding the bilateral kidneys, and thickened mucous membrane of the bladder with high fluorodeoxyglucose uptake. Pathological examination of the pulmonary mass revealed infiltration of medium-sized lymphocytes and plasma cells. Immunohistochemical analysis revealed tumor cells positive for CD138 and IgM, with a low positive rate of Ki-67 expression. Notably, the tumor cell-surrounding lymphocytes were positive for CD20. Although the patient was initially regarded as having Waldenström's macroglobulinemia owing to the significantly increased serum IgM levels, based on positive IgH-MALT1 translocation and negative MYD88 L265P mutation findings, he was further diagnosed with extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). Complete remission was achieved following six cycles of rituximab + CHOP therapy. This study data suggest that analysis of the MYD88 L265P mutation in tumor cells is suitable for accurately diagnosing hematopoietic malignancies with increased IgM monoclonal protein.

POEMS syndrome is a rare paraneoplastic disease associated with monoclonal plasma cells; however, the pathogenic importance of plasma cells remains unclear. We performed comprehensive genetic analyses of plasma cells in 20 patients with POEMS syndrome. Whole exome sequencing was performed in 11 cases and found a total of 308 somatic mutations in 285 genes. Targeted sequencing was performed in all 20 cases and identified 20 mutations in 7 recurrently mutated genes, namely KLHL6, LTB, EHD1, EML4, HEPHL1, HIPK1, and PCDH10. None of the driver gene mutations frequently found in multiple myeloma (MM) such as NRAS, KRAS, BRAF, and TP53 was detected. Copy number analysis showed chromosomal abnormalities shared with monoclonal gammopathy of undetermined significance (MGUS), suggesting a partial overlap in the early development of MGUS and POEMS syndrome. RNA sequencing revealed a transcription profile specific to POEMS syndrome when compared with normal plasma cells, MGUS and MM. Unexpectedly, disease-specific VEGFA expression was not increased in POEMS syndrome. Our study illustrates that the genetic and transcriptional profiles of plasma cells in POEMS syndrome are distinct from MM and MGUS, indicating unique function of clonal plasma cells in its pathogenesis.

TO THE EDITOR: Polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome is a rare plasma cell disorder additionally characterized by extravascular volume overload, sclerotic bone lesions, and plasmacytomas. Although high-dose melphalan followed by

Polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes (POEMS) syndrome is a rare plasma cell dyscrasia characterized by polyneuropathy, organomegaly, endocrinopathy, extravascular fluid overload, M protein, and a myriad of skin changes. The pathogenesis is poorly understood, but monoclonal plasma cells are λ-restricted and these immunoglobulin λ light chain variable (IGLV) region genes are derived from only two germlines, either IGLV1-44 or 1-40. Here we analyzed the clonal IGLV gene rearrangements of genomic DNA samples of bone marrow mononuclear cells using next-generation sequencing (NGS) to understand the clonal composition of IGLV genes in patients with POEMS syndrome (n = 30). The dominant IGLV gene rearrangement of POEMS syndrome-specific germline sequences were significantly increased in 11 POEMS patients (36.7%; IGLV1-44: n = 9, IGLV1-40: n = 2). In some cases, IGLV gene rearrangement clone was not detected as significant increase but was detected using cDNA samples by heteroduplex (HD) analysis and Sanger sequencing, suggesting that the quite small number of monoclonal plasma cells may produce large quantity of mRNA of monoclonal proteins. However, significant increase of dominant clone sizes was not directly linked to the initial disease status. On the other hand, in cases with significantly increased dominant clones, they decreased and increased accompanying with disease remission and relapse. These data demonstrate that monoclonal plasma cells are related to the pathogenesis of POEMS syndrome.

A 60-year-old man was referred to us with high levels of aspartate aminotransferase and lactate dehydrogenase (LDH). He did not complain of any symptoms; however, he had been diagnosed with lumbar disc herniation, even though his back pain improved only to half of its previous level with pregabalin. Thus, we asked about the red flag of back pain and confirmed that he had involuntary body weight loss, which led us to diagnose truly non-secretory multiple myeloma, a variant of multiple myeloma that is associated with M-protein negativity and a normal serum free light chain level.

Severe acute lung injury is a rare but life-threatening complication associated with bortezomib. We report a patient with multiple myeloma who developed a severe diffuse alveolar hemorrhage (DAH) immediately after the first bortezomib administration. The patient was suspected to have pulmonary involvement of myeloma, which caused DAH after rapidly eradicating myeloma cells in the lungs with bortezomib. Rechallenge with bortezomib was performed without recurrent DAH. In patients with multiple myeloma who manifest abnormal pulmonary shadow, we should be aware of early-onset severe DAH after bortezomib administration, which might be due to pulmonary involvement of myeloma cells.

Thrombopoietin-receptor agonists have been recently introduced for a second-line treatment of immune thrombocytopenia (ITP). Splenectomy has tended to be avoided because of its complications, but the response rate of splenectomy is 60–80% and it has still been considered for steroid-refractory ITP. We performed partial splenic embolization (PSE) as an alternative to splenectomy. Between 1988 and 2013, 91 patients with steroid-resistant ITP underwent PSE at our hospital, and we retrospectively analyzed the efficacy and long-term outcomes of PSE. The complete response rate (CR, platelets > 100 × 109/L) was 51% (n = 46), and the overall response rate (CR plus response (R), > 30 × 109/L) was 84% (n = 76). One year after PSE, 70% of patients remained CR and R. The group with peak platelet count after PSE ≥ 300 × 109/L (n = 29) exhibited a significantly higher platelet count than the group with platelet count

Infections of the central nervous system (CNS) with varicella zoster virus (VZV) is a rare occurrence after allogeneic hematopoietic stem cell transplantation. We herein report a case of VZV meningitis, radiculitis and myelitis that developed 8 months after cord blood transplantation, shortly after the cessation of cyclosporine and low-dose acyclovir. Although treatment with acyclovir did not achieve a satisfactory response, the patient was successfully treated with foscarnet. Our report indicates that VZV infection should be considered in allo-hematopoietic stem cell transplantation (HSCT) patients with CNS symptoms and that foscarnet may be effective for the treatment of acyclovir-resistant VZV infections of the CNS. The development of optimal prophylactic strategies and vaccination schedules may eradicate post-transplant VZV disease.

Granulocyte colony-stimulating factor (G-CSF) induces the mobilization of leukocytes from the bone marrow (BM) to the circulation by a yet incompletely understood mechanism. Here, we describe that the membrane-bound receptor LR11 is highly expressed in human myeloid cells and that the shed soluble form of LR11 (sLR11) is a modifier of myeloid cell migration. In the process of leukocyte mobilization by G-CSF treatment, circulating sLR11 levels are transiently elevated in humans and mice. Moreover, following G-CSF treatment, the sLR11 levels in patients show significant positive correlation with the numbers of mobilized leukocytes. The changes of LR11 levels in BM cells and of sLR11 released into the BM fluid of mice correlate tightly with the changes in circulating sLR11 levels. G-CSF dose-dependently enhanced sLR11 release from HL-60 cells, which in turn accelerated cell migration. Finally, cooperatively with tumor necrosis factor-α (TNF-α) and G-CSF, sLR11 increased the attachment of floating cells (HL-60 and U937) to endothelial cells. We propose that sLR11 is a novel candidate modifier of G-CSF-mediated mobilization of hematologic cells. Identification of sLR11 as a regulatory component of G-CSF-mediated hematologic cell mobilization may facilitate further improvement of hematologic stem cell collection for clinical applications.

Introduction: A novel tubulin binding agent PTC596, which is currently in clinical trials for solid tumors, was originally identified by its ability to kill cancer stem cells and to reduce BMI1 activity. PTC596 treatment results in hyperphosphorylation of the BMI1 protein and loss of BMI1 function as demonstrated by a reduction in H2A ubiquitination levels in a range of solid tumor lines. Subsequent studies have shown that the down-regulation of BMI1 protein is due to a G2/M arrest. In this study, we aimed to investigate the in-vitro and in-vivo anti-tumor activities of PTC596 and the combination with bortezomib in multiple myeloma (MM). Methods: For in-vitro evaluation, MTS and BrdU ELISA assays were performed using human MM cell lines. Approved by the Institutional Review Committee at Chiba University, primary myeloma cells and bone marrow stromal cells (BMSCs) were collected from the bone marrow of MM patients with informed consent. For in-vivo evaluation, the MM.1S subcutaneous xenograft model in NOG mice was used. To understand the mechanisms of action and target genes of the treatments, flow cytometry (FCM), western blotting, RNA-seq, and ChIP-seq were performed. Results: PTC596 induced significant cytotoxicity in all MM cell lines tested, including bortezomib-resistant OPM-2/BTZ and KMS-11/BTZ cells (CC50: 24-98 nM). PTC596 also suppressed cell proliferation when these cell lines were co-cultured with BMSCs. As expected, PTC596 reduced the levels of BMI1 protein and uH2A in a dose-dependent manner. Of note, PTC596 induced cell cycle arrest as detected by a BrdU FCM assay in MM cells and apoptosis as detected by annexin-V FCM in MM cell lines and primary myeloma cells. Moreover, oral administration of PTC596 twice a week for three weeks significantly inhibited the growth of MM.1S tumors implanted in immunodeficient mice and improved the survival of mice as compared with mice treated with vehicle only (p=0.0021). Of interest, bortezomib appeared to transcriptionally repress the expression of BMI1 and reduce the levels of uH2A. We then tested the efficacy of the combination of PTC596 with bortezomib in MM cells and found additive or synergistic effects when MM cell lines were co-cultured with BMSCs. Reductions in the levels of BMI1 protein and uH2A by PTC596 or bortezomib alone were significantly enhanced in the combination treatment. Furthermore, apoptosis induced by bortezomib was significantly enhanced by the combination with PTC596 as evidenced by increased annexin-positive cells detected in flow cytometric analysis and increased cleavage of caspases with reduction in MCL1 protein in western blotting. RNA-seq of MM.1S cells treated with PTC596 alone or in combination with bortezomib demonstrated repression of gene sets related to the cell cycle in either setting and enrichment of gene sets related to apoptosis in the combination. Ongoing analysis of our ChIP-seq data will reveal the direct targets of BMI1 in MM cells. Remarkably, oral administration of PTC596 combined with subcutaneous injection of bortezomib twice a week for five weeks significantly reduced MM.1S tumor growth in comparison to the control or either single treatment (p Conclusions: Our results demonstrate that PTC596 alone and in combination with proteasome inhibition are potential novel therapeutic options in MM. The results of this study support the clinical evaluation of this promising therapeutic approach to improve the outcome of MM patients. Disclosures Sheedy: PTC Therapeutics: Employment. Weetall:PTC Therapeutics: Employment.

[Introduction] AL amyloidosis is caused by the deposition of abnormally folded clonal immunoglobulin (IG) light chains (LCs, λ:κ = 3:1) made by malignant plasma cells in the bone marrow (BM), which leads to multi-organ dysfunction, often involving the heart, kidney, liver, skin, and nerves. However, little is known about what regulates organ tropism of amyloid deposition in this disease. In addition, no study has analyzed the repertoire of IG germlines of plasma cells in the BM in AL amyloidosis using next generation sequencing (NGS). In this study, we aimed to identify the clonal composition of IG λ light-chain variable region (IGLV) genes in BM cells in patients with AL amyloidosis using NGS. [Material and method] BM cells were obtained at diagnosis from 38 patients with AL amyloidosis and those with other plasma cell disorders: multiple myeloma (MM, n = 7), and monoclonal gammopathy of undetermined significance (MGUS, n = 11) with λ-type monoclonal paraprotein. Seven normal control (NC) patients had either immune thrombocytopenia or malignant lymphoma without BM invasion. Genomic DNA was extracted from the BM mononuclear cells preserved in LABO Banker1 or BM clots in O.C.T compound using QIAamp DNA Blood Mini kit. The IGLV1 and IGLV2 genes were amplified by polymerase chain reaction using a 5′ primer for the IGLV1/2 framework 3 (FR3) region and 3′ consensus primers for the IGLJ1/2/3 joining regions. Multiple samples were pooled, and paired-end 2 × 250 base pair sequencing reactions were performed using an Illumina MiSeq sequencer and then analyzed by an open-source program called Vidjil. All subjects provided written informed consent to participate in the study, in accordance with the Declaration of Helsinki. This study was approved by the ethics committee of the Chiba University Graduate School of Medicine and Japanese Red Cross Medical Center. [Results] Clinical and laboratory features of 38 patients with AL amyloidosis were as follows: primary AL amyloidosis (n = 31); 15 and 20 patients had cardiac and renal dysfunctions, respectively, and secondary AL amyloidosis with MM (n = 7); 4 and 1 patient had cardiac and renal dysfunctions, respectively. In patients with AL amyloidosis, the median plasma cell count in BM aspirate smears was 3.3% (0.1%-50.4%), and the median difference in involved and uninvolved light chains (dFLC) was 104.5 mg/L (28.5mg/L -2673.3mg/L). Representative results of the Vidjil analysis in NC, MGUS, AL amyloidosis, and MM are shown in Figure 1. The most abundant IGLV gene accounted for not >1% of the reads, and there was no dominant germline in NC samples. Therefore, we defined the dominant clone as >1% of IG germlines in plasma cells. According to this definition, clonal IG germlines were found in 27 of 31 patients with primary AL amyloidosis (87%), 5 of 7 with secondary AL amyloidosis (71%), 7 of 7 with MM (100%), and 8 of 11 with MGUS (73%). However, the size of clones in AL amyloidosis (median 3.1%, 0.38%-14.3%) was significantly smaller than that in MM (median 17.8%, 2.2%-17.9%) (P [Conclusion] We successfully identified the clonal composition of IGLV genes in the BM of most patients with AL amyloidosis using NGS, according to the differences in the V and J region recombination and CDR3 sequences using the Vidjil program. In AL amyloidosis, the clonal size of plasma cells in the BM is small and small malignant clones of plasma cells may secret FLC and cause LC depositions in AL amyloidosis. Figure 1 Disclosures Suzuki: Ono: Research Funding; BMS: Honoraria, Research Funding; Takeda: Honoraria; Janssen: Honoraria; Celgene: Honoraria.

Although Hodgkin lymphoma (HL) is readily cured with conventional chemoradiotherapy, patients still develop refractory or relapsed HL even after allogeneic stem cell transplantation (allo-SCT) [1]....

Myeloid sarcoma (MS) is an extramedullary myeloid tumor that sometimes presents with antedating systemic leukemia, leading physicians to the misdiagnosis of lymphoma. CD25 is expressed in 13% of patients with acute myeloid leukemia (AML), and its expression is associated with FLT3-ITD mutations, an elevated serum soluble interleukin 2 receptor (sIL-2R) level and a lower survival rate. However, there are no reports concerning the relationship between MS and the CD25 expression. We herein report a case of AML accompanied by thoracic epidural MS with a high CD25 expression, the FLT3-ITD mutation and an extremely elevated serum sIL-2R level in a 59-year-old man who presented with paraplegia.

Purpose: EZH2 and EZH1, the catalytic components of polycomb repressive complex 2 (PRC2), trigger trimethylation of H3K27 (H3K27me3) to repress the transcription of target genes and are implicated in the pathogenesis of various cancers including multiple myeloma and prostate cancer. Here, we investigated the preclinical effects of UNC1999, a dual inhibitor of EZH2 and EZH1, in combination with proteasome inhibitors on multiple myeloma and prostate cancer. Experimental Design: In vitro and in vivo efficacy of UNC1999 and the combination with proteasome inhibitors was evaluated in multiple myeloma cell lines, primary patient cells, and in a xenograft model. RNA-seq and ChIP-seq were performed to uncover the targets of UNC1999 in multiple myeloma. The efficacy of the combination therapy was validated in prostate cancer cell lines. Results: Proteasome inhibitors repressed EZH2 transcription via abrogation of the RB-E2F pathway, thereby sensitizing EZH2-dependent multiple myeloma cells to EZH1 inhibition by UNC1999. Correspondingly, combination of proteasome inhibitors with UNC1999, but not with an EZH2-specific inhibitor, induced synergistic antimyeloma activity in vitro. Bortezomib combined with UNC1999 remarkably inhibited the growth of myeloma cells in vivo. Comprehensive analyses revealed several direct targets of UNC1999 including the tumor suppressor gene NR4A1. Derepression of NR4A1 by UNC1999 resulted in suppression of MYC, which was enhanced by the combination with bortezomib, suggesting the cooperative blockade of PRC2 function. Notably, this combination also exhibited strong synergy in prostate cancer cells. Conclusions: Our results identify dual inhibition of EZH2 and EZH1 together with proteasome inhibition as a promising epigenetics-based therapy for PRC2-dependent cancers. Clin Cancer Res; 23(16); 4817–30. ©2017 AACR.

Although autologous stem cell transplantation can achieve excellent responses in patients with POEMS syndrome, the optimal regimen for peripheral blood stem cell (PBSC) collection is still controversial. We retrospectively investigated the safety and efficacy of 41 PBSC collecting procedures in 37 patients with POEMS syndrome. PBSC mobilization was performed using cyclophosphamide + granulocyte colony–stimulating factor (G-CSF) (CG, n = 14) or G-CSF alone (G, n = 27). Twelve (85.7%) patients in the CG group and all (100%) patients in the G group received induction chemotherapy before PBSC collection. The proportions of good mobilizers (≥2.0 × 106 CD34+ cells/kg) were comparable between the 2 groups (CG versus G: 78.6% versus 70.4%, P = .71). Two (14.3%) patients in the CG group developed severe capillary leak symptoms during the PBSC mobilization period, whereas no patient in the G group experienced severe adverse events. Appropriate induction therapies followed by the G-CSF monotherapy compose an optimal strategy for PBSC collection.

A 34-year-old man who had been referred to our hospital was diagnosed with acute promyelocytic leukemia (APL). All-trans retinoic acid (ATRA), oral administration, was initiated. On day 25, he developed fever and respiratory distress with bilateral pulmonary infiltrates, suggesting differentiation syndrome (DS) caused by ATRA. These symptoms showed amelioration after discontinuing ATRA and initiating methylprednisolone. ATRA was re-started on day 29 at half the original dose because of residual APL blasts. The patient subsequently developed fever, severe stomatitis, and oropharyngeal ulcers, which persisted even after discontinuing ATRA. On day 48, he suddenly developed severe abdominal pain with free air, observable on an abdominal X-ray, and underwent emergency ileocecal resection. Pathological examination of the resected ileocecal intestines revealed multiple ulcers and perforations. No leukemic cell infiltration was observed. In this case, only ATRA was administered for APL treatment. These findings suggest that ileocecal ulcerations and perforations, as well as oropharyngeal ulcers, might have been caused by DS or ATRA. Furthermore, DNA typing of the HLA-B locus revealed that the patient had HLA-B51 associated with Behçet's disease. Therefore, hypercytokinemia with DS might have induced Behçet's disease-like symptoms, including stomatitis and ileocecal perforation, complications that are particularly observed in patients with HLA-B51.

Posterior reversible encephalopathy syndrome (PRES) has been reported in childhood leukemia patients increasingly frequently. However, the development of PRES in adult leukemia patients during chemotherapy is very rare. We present a case of PRES in an adult patient with acute lymphoblastic leukemia (ALL) after remission induction chemotherapy. A 28-year-old woman with ALL was administered remission induction chemotherapy consisting of cyclophosphamide, daunorubicin, vincristine, prednisone, and L-asparaginase. After initiation of chemotherapy, the patient developed paralytic ileus and hypertension, and on day 30, she suddenly developed generalized convulsions, loss of visual acuity, and muscle weakness in the legs. Magnetic resonance imaging findings and her signs and symptoms were typical of PRES. The symptoms gradually improved following treatment with an anticonvulsant and an antihypertensive agent, and the patient underwent allogeneic bone marrow transplantation. She has completely recovered from PRES and has been asymptomatic without leukemia relapse. During remission induction chemotherapy for ALL, PRES may be caused by multiple drugs, such as L-asparaginase, vincristine, and corticosteroids, with different mechanisms of action. PRES should be recognized as an important complication, which will occur more frequently with the increased intensity of chemotherapy for adult ALL patients.

Introduction: Dysfunction of epigenetic pathways has been frequently implicated in hematological malignancies. In multiple myeloma (MM), EZH2, a methyltransferase that induces histone H3 lysine 27 trimethylation (H3K27me3), acts as an oncogene as evidenced by its overexpression, which was found to be positively correlated with disease progression (Kalushkova et al. PloS One. 2010). We have shown that inhibition of both EZH2 and its homolog EZH1 is effective in eradicating MM cells in vitro and in vivo (Rizq et al. Clin Cancer Res. 2017). In addition, inactivating somatic mutations in UTX/KDM6A, an X-linked histone demethylase that removes di- and tri-methyl groups from H3K27, are found in 3 - 10% of MM patients (van Haaften et al. Nat Genet. 2009 and Pawlyn et al. Clin Cancer Res. 2016), indicating a tumor suppressive role for UTX, which has yet to be delineated. Up till now, no mouse model has been generated to test Utx insufficiency in post germinal center (GC) B cells and plasma cells. On the other hand, an activating mutation V600E in the BRAF kinase gene is closely associated with aggressive disease features such as extramedullary disease and shorter overall survival in MM patients (Andrulis et al. Cancer Discov. 2013) and could accelerate induction of myeloma in mice. Methods: To investigate whether loss of Utx cooperates with Braf V600E in myelomagenesis in mice, we generated and analyzed mice with conditional knock-out allele of Utx and/or knock-in allele of Braf V600E combined with Cγ1-Cre allele, in which Cre is activated by immunization in post GC B cells. Results: Loss of Utx and Braf V600E synergistically induced post GC B-cell lymphoma and plasma cell neoplasms in mice and significantly shortened the survival of mice compared with control mice and either allele alone. Utx-/-Braf V600E females succumbed to death earlier than Utx-/YBraf V600Emales and Utx-/+Braf V600Efemales. Of note, plasma cell neoplasms developed at a high frequency in Utx-/YBraf V600Emales and Utx-/+Braf V600Efemales and, less frequently, in Utx-/-Braf V600E females. Mice with plasma cell neoplasms showed expansion of CD138+ plasma cells in bone marrow as well as spleen and/or lymph nodes, exhibiting extramedullary disease. Loss of Utx alleles and expression of Braf V600E were confirmed by genomic PCR of plasma cells. Importantly, the clonality of plasma cells was demonstrated by genomic PCR detecting rearrangements of immunoglobulin heavy and light chain genes. In addition, M protein was detected by serum protein electrophoresis (SPEP) at a high frequency. Notably, we were able to establish murine myeloma cell lines from moribund compound mice. These cells readily engrafted in the bone marrow of NOG mice after transplantation and caused myeloma-associated phenotypes including paraplegia in recipient mice. Interestingly, Utx-/-Braf V600E cells were sensitive to dual inhibition of EZH2 and EZH1 but not to specific inhibition of EZH2 in culture. They also showed decreased susceptibility to proteasome inhibitors when compared with human MM cell lines. To gain insight into the changes in the transcriptional landscape following Utx loss, we performed RNA sequencing (RNA seq) and then gene set enrichment analysis (GSEA). We found positive enrichment of gene sets related to Myc, implying that Myc is one of the main drivers of myelomagenesis in our mouse model. In addition, gene sets related to MM were significantly enriched following Utx loss. We are now working on ChIP sequencing (ChIP seq) of UTX-related histone modifications to evaluate the epigenetic impact of Utx loss on myelomagenesis. Conclusion: Utx insufficiency cooperates with Braf V600E in the induction of myeloma in mice. Our mouse model is a promising tool for understanding the role of epigenetic dysregulation in the pathogenesis of MM and evaluating novel anti-myeloma agents. Disclosures Okuno: Celgene: Research Funding. Tamaru:Nichirei Bioscience INC.: Research Funding; Takeda Pharmaceutical Company Limited: Speakers Bureau.

Background : Anagrelide is a widely used therapeutic agent for patients with essential thrombocythemia. While other cytoreductive agents, such as hydroxyurea, influence multi-lineage blood cells, anagrelide exerts less effect on the white and red blood cell lineages. Although the clinical efficacy of anagrelide has been reported, the exact mechanism of action is unclear. Recently, immortalized megakaryocyte progenitor cell lines (imMKCLs) were established from human induced pluripotent stem (iPS) cells by the introduction of doxycycline-inducible lentiviral vectors harboring c-MYC, BMI1, and BCL-XL for the clinical application of artificially generated platelets. In this study, we aimed to elucidate the molecular mechanism of anagrelide on the inhibition of platelet production using imMKCLs as an ideal model for human megakaryogenesis and platelet formation. Materials and Methods : imMKCLs, established at Center for iPS Cell Research and Application, Kyoto University, Japan, were cultured in Iscove's modified Dulbecco's medium with thrombopoietin (TPO), stem cell factor (SCF), and doxycycline. The differentiation of imMKCLs and platelet generation were induced by doxycycline removal. The generation of mature platelets was observed approximately 7 days after the differentiation was initiated. Both undifferentiated and differentiated imMKCLs were treated with several different concentrations of anagrelide. The cell proliferation and number of generated platelets following anagrelide treatment were analyzed by BrdU cell proliferation assay and flow cytometry, respectively. To explore the molecular mechanism of anagrelide treatment in imMKCLs, we performed mRNA sequencing in imMKCLs treated with or without anagrelide followed by gene ontology (GO) analysis and gene set enrichment analysis (GSEA). The expression of genes related to megakaryogenesis and platelet formation was also analyzed utilizing quantitative real-time PCR. Results : Anagrelide exposure caused morphologically suppressive changes in the differentiation of imMKCLs. Anagrelide treatment also suppressed the mRNA expression of the megakaryocytic surface markers CD41 and CD61 in both undifferentiated (P < 0.01 and P < 0.001, respectively) and differentiated (P < 0.01 and P < 0.001, respectively) settings. The BrdU incorporation rate in differentiated imMKCLs decreased significantly following anagrelide treatment (P < 0.001, anagrelide 0 vs. 1 or 10 µM). The resultant generation of mature platelets (double positive for CD41 and CD42b) was significantly decreased by exposure to anagrelide, as analyzed by flow cytometry (P < 0.001). Regarding the molecular mechanism of anagrelide treatment on imMKCLs, GO analysis following RNA sequencing demonstrated that gene sets related to platelet activation and degranulation were significantly downregulated in both undifferentiated and differentiated conditions. Moreover, GSEA revealed that gene sets related to the cell cycle, such as mitosis and DNA replication, were decreased as well as platelet-specific genes. The mRNA expression levels of genes related to megakaryogenesis and platelet-formation, such as FLI1, TAL1, GATA1, and PF4, were significantly downregulated, especially in differentiated imMKCLs, by anagrelide treatment (P < 0.001, P = 0.013, P < 0.01, and P < 0.01, respectively). Conclusions : We successfully reproduced the platelet-lowering effect of anagrelide by using imMKCLs from human iPS cells that could generate functional platelets in culture. Our RNA sequencing results revealed that anagrelide specifically suppressed megakaryogenesis and platelet formation-related genes. Additional studies including an apoptosis assay and cell cycle analysis of imMKCLs following anagrelide exposure are ongoing to elucidate further molecular mechanisms of anagrelide treatment. Disclosures Takayama: Megakaryon co. Ltd.: Research Funding. Eto:Megakaryon co. Ltd.: Research Funding.

Adverse events (AEs) associated with blood component transfusion have been widely surveyed. In contrast, surveillance of AEs associated with hematopoietic stem cell (HSC) infusion in HSC transplant (HSCT),including bone marrow transplant (BMT), peripheral blood stem cell transplant (PBSCT), and cord blood transplant (CBT),has been less rigorous, even though HSC products contain cells of diverse maturity and viability,plasma with various antigens, cytokines and antibodies, and dimethyl sulfoxide (DMSO) in the case of cryopreserved products. In fact, HSC infusion is associated with several AEs, e.g., allergic reactions, flushing, hypo- or hypertension, and respiratory distress, which have been attributed to toxicity of dead cells and DMSO (Otrock et al, Transfusion, 2017). However, our recent prospective surveillance revealed that HSC infusion-related AEs often occurred in each HSC type and the overall rates of AEs were greater in allo-BMT with no DMSO, compared with auto-PBSCT, allo-PBSCT, and allo-CBT typically cryopreserved with DMSO. Hypertension was the most common AE in each HSC source, with the highest rate in BMT, while allergic reactions were the most frequent in allo-PBSCT. A multivariate analysis identified a history of transfusion reactions as a risk factor of HSC infusion-related AEs (Ikeda et al, Transfus Med Rev, 2018). These findings suggest that some DMSO-independent factor(s), such as plasma components, may contribute to HSC infusion-related AEs. Thus, we asked if HSC volume and component effects were more substantial in small recipients, and if age-related factors alter susceptibility to HSC infusion-related AEs in pediatric patients. So far, data on HSC infusion-related AEs in pediatric and low body-weight recipients are lacking. Here, to address these issues, we investigated AEs due to HSC infusions in 219 recipients of The rates of overall HSC infusion-related AEs were quite similar among HSC sources among low body-weight/pediatric recipients (Table 1) andexclusivepediatric recipients (P= .158 in auto-/allo-HSCT and .867 in allo-HSCT), in contrast to general recipientswhose rate of AEs was highest in BMT. In addition, bradycardia was more often reported in CBT compared with PBSCT and BMT (Table 1), especially in pediatric recipients (30.8% in CBT and 0% in others, P< .001). On the other hand, there were some similar trends between low body-weight/pediatric recipients and general recipients: PBSCT and CBT recipients, but not BMT recipients, complained of malodor, whereas the rate of hypertension was highest in BMT (Table 1). Next, we compared HSC infusion-related AEs between auto- and allo-HSCT using cryopreserved products (Table 2). This comparison showed higher overall rates of AEs in allo-HSCT compared with auto-HSCT, especially in pediatric recipients, suggesting that plasma componentsrather than DMSO contribute to HSC infusion-related AEs in low body-weight/pediatric recipients as well as in general recipients. Of note, pediatric recipients showed a 10-foldhigher incidence of nausea/vomiting in allo-HSCT versus auto-HSCT, instead of allergic reactions, for which the incidence was significantly higher in allo-HSCT than auto-HSCT in general recipients. Subsequently, we sought factors that correlate with HSC infusion-related AEs in allo-HSCT using multivariate analysis with logistic regression, which identified lymphoid neoplasms over myeloid neoplasms as a factor for overall AEs (OR 3.013, P= .026), while history of transfusion reactions did not reach statistical significance (OR 2.368, P= .066). Notably, in a multivariate analysis for any grade ≥2 AEs, there were some factors that did not correlate with general recipients but did with low body-weight/pediatric recipients, including complications before HSCT (OR 5.764, P= .019), without plasma or red blood cell removal for ABO mismatch (OR 3.815, P= .032), and >10 ml/kg infusion volume (OR 5.306, P= .027). In conclusion, our data quantifysome specific symptoms associated with HSC infusionin low body-weight and pediatric recipients. We should be mindful ofinfusion volume and preexisting complications when small recipients receive HSC infusion. Disclosures Fujiwara: Astellas: Consultancy; Kyowa-Hakko: Consultancy; Kirin: Consultancy; Chugai: Consultancy; Pfizer: Consultancy; Shire: Consultancy. Muroi:JCR: Speakers Bureau; Becton: Speakers Bureau; Dickinson and Company: Speakers Bureau; Japanese Red Cross Society: Speakers Bureau. Mori:Astella Pharma: Honoraria; Kyowa Hakko Kirin: Honoraria; Japan Blood Products Organization: Honoraria; MSD: Research Funding; Ono: Honoraria; Novartis Pharma: Honoraria; Eisai: Honoraria; Janssen: Honoraria; Asahi Kasei: Research Funding; Novartis Pharma: Research Funding; Celgene: Honoraria; Taisho Toyama Pharmaceutical Co: Honoraria; Shire Japan: Honoraria; Pfizer: Honoraria; CHUGAI: Honoraria; MSD: Honoraria; SHIONOGI: Honoraria. Nagai:Ono Pharmaceutical Co.Ltd.: Consultancy; Kaneka Corporation: Research Funding; Kawasumi Laboratories Inc.: Research Funding.

We serially determined serum erythropoietin (Epo) and the reticulocyte count in patients with various types of leukemia during chemotherapy. Serum Epo increased soon after the initiation of chemotherapy and decreased after the termination of therapy irrespective of the types of leukemia or treatment regimen. However, it did not stay at low level but fluctuated. The reticulocyte count, on the other hand, showed a transient rise while serum Epo level descended. The value of serum Epo when increased was higher than the value expected from hemoglobin concentration; this finding was similar to that in aplastic anemia. These results suggest that myelosuppression is a major factor for the increase in serum Epo level during leukemia chemotherapy.

Increasing recipient age is a well-known risk factor for graft-versus-host disease (GVHD) and treatment-related mortality (TRM) and has a negative impact on allogeneic hematopoietic stem cell transplantation. Since the incidence of severe GVHD after cord blood transplantation (CBT) is lower than that after transplants using bone marrow or mobilized peripheral blood grafts from adult cells, we should expect better outcomes from CBT in older patients. To evaluate the feasibility and efficacy of myeloablative unrelated CBT in patients aged between 50 and 55 years, we performed a retrospective comparison of 100 patients with acute leukemia who received cord blood grafts at our institution. Nineteen older patients (median age, 52; range, 50-55) and 81 younger patients (median, 36; range, 16-49) received a myeloablative conditioning regimen including 12 Gy of total body irradiation and chemotherapy. GVHD prophylaxis included cyclosporine with (n = 96) or without (n = 4) methotrexate. There were no significant differences in the incidences of grades II to IV acute GVHD, extensive-type chronic GVHD, TRM, and the probability of overall and disease-free survival between these groups. These results suggest that, in patients with acute leukemia, myeloablative CBT might be as safe and effective in patients aged between 50 and 55 years as in younger patients.