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This 3+3 dose-escalation phase I multicenter study investigated the optimal dose of azacitidine (AZA) for post-hematopoietic stem cell transplantation (HSCT) maintenance, which remains unknown in Japan. Recipients of a first HSCT for high-risk myelodysplastic syndromes (MDS, n = 12) or acute myeloid leukemia (AML) with antecedent MDS (n = 3) received post-HSCT AZA maintenance in 2015-2019. The optimal AZA dose was defined as the dose at which 50-70% of patients can complete four cycles without dose-limiting toxicity (DLT). The initial dose level 1 was set as 30 mg/m

Busulfan (Bu) has been used in combination with fludarabine (Flu; BuFlu) or cyclophosphamide (Cy; BuCy) as conditioning for allogeneic hematopoietic stem cell transplantation (HSCT). This multi-institutional prospective study compared pharmacokinetic (PK) parameters of Bu between BuFlu and BuCy. Plasma Bu concentrations were measured by high-performance liquid chromatography at the first dose of the first and fourth days of intravenous Bu administrations (total of 16 doses of 0.8 mg/kg). Thirty-seven patients were evaluable (BuFlu, N = 18; BuCy, N = 19). The median age was significantly higher in BuFlu. In BuFlu, the median area under the blood concentration–time curve of Bu on the fourth day was 1183 μmol min/L (range 808–1509), which was significantly higher than that on the first day [1095 μmol min/L (range 822–1453), P

Systemic chronic active Epstein-Barr virus infection (sCAEBV) was defined as a T- or NK-cell neoplasm in the 2017 World Health Organization (WHO) classification. To clarify the clinical features of sCAEBV under this classification and review the effects of chemotherapy, we performed a nationwide survey in Japan from 2016 through 2018 of patients with sCAEBV newly diagnosed from January 2003 through March 2016. One hundred cases were evaluated. The patients were aged 1 to 78 years (median, 21) and included 53 males and 47 females. Spontaneous regression was not observed in patients with active disease. In the childhood-onset group (age, 45 years) were female. The prognosis of the childhood-onset group was better than those of the adolescent/adult- and elderly-onset groups. The main chemotherapies used were a combination of cyclosporine A, steroids, and etoposide (cooling therapy) in 52 cases and cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP) in 45 cases. The rate of complete response (CR), defined as complete resolution of disease activity, was 17% for cooling therapy and 13% for CHOP. Virological CR was not observed. The 3-year overall survival rates in patients treated with chemotherapy only (n = 20), chemotherapy followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT; n = 47), and allo-HSCT only (n = 12) were 0%, 65%, and 82%, respectively. Distinct characteristics were observed between childhood- and elderly-onset sCAEBV, and they appeared to be different disorders. Chemotherapy is currently insufficient to resolve disease activity and eradicate infected cells. The development of an effective treatment is urgently needed.

A 45-year-old woman was diagnosed with myelodysplastic syndrome (MDS) with trisomy 8 and Behcet-like disease (BLD) with multiple colorectal ulcers. Nonspecific inflammatory cells were infiltrated in the intestinal mucosa, whereas fluorescence in situ hybridization (FISH) analysis revealed only sporadic trisomy 8-positive cells. She presented massive lower gastrointestinal bleeding early after bone marrow transplantation but achieved long-term remission of both MDS and BLD. This is the first report of massive gastrointestinal bleeding after transplantation for MDS with BLD. Based on FISH analysis, dysregulation of systemic inflammation may be involved in BLD rather than direct invasion by trisomy 8-positive MDS clones.

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

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 POEMS syndrome is a rare systemic disorder characterized by various symptoms, including polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy (M-protein), and skin changes, which is based on the presence of monoclonal plasma cells (PC). We previously reported the analysis of the clonal immunoglobulin λ light-chain variable region (IGLV) gene rearrangements in POEMS syndrome using next-generation sequencing (NGS), and the clone identification rate was 36.7% (Kawajiri-Manako C, Am J Hematol. 2018;93(9):1161). EuroFlow-based next-generation flow (EuroFlow-NGF) has also been standardized for detecting minimal residual disease in multiple myeloma (MM). However, multiparameter flow cytometry (MFC) utility in detecting clonal PC of POEMS syndrome remains unknown. Therefore, to clarify the feasibility of detecting the clonality with MFC, we analyzed PC of bone marrow samples in patients with POEMS syndrome by MFC and validated gating strategies of PC in POEMS syndrome. Methods Patients with newly diagnosed or relapsed POEMS syndrome (n=25) at Chiba University Hospital from April 2019 to July 2021 were included in this study. Primary bone marrow aspirations were performed after obtaining informed consent following the Declaration of Helsinki. The collected bone marrow samples were analyzed by the SRL-Flow protocols (single-tube 8-color: CD138/CD27/CD38/CD56/CD45/CD19/CyIgκ/CyIgλ), which is highly correlated with EuroFlow-NGF (Takamatsu H, Int J Hematol. 2019;109(4): 377). Furthermore, 21 cases for which data files were available from SRL Inc. were reanalyzed by gating PC with lower expression of CD45 regarding the previous study (Dao LN, Blood. 2011;117(24):6438). This study was approved by the ethics committee of Chiba University Graduate School of Medicine. Results Among 25 patients, the median age was 59 (25-77) years, and 13 (52.0%) were men. The types of M-proteins identified by immunofixation electrophoresis (IFE) were IgA-λ in 12 (48.0%), IgG-λ in 9 (36.0%), IgA-κ in 1 (4.0%), IgG-κ in 1 (4.0%), and negative in 2 patients (8.0%), respectively. The median serum VEGF level was 3,240 (747-9,060) pg/ml. SRL-Flow identified the clonal PC in 9 of 25 (36.0%, λ in 7 and κ in 2): 7 of 23 IFE-positive cases (30.4%) and 2 of 2 IFE-negative cases (100%). The median clone size was 0.03 (0.01-3.78)%, smaller than MM. Interestingly, the clone size was not correlated with the level of serum VEGF (r = -0.17). Aberrant expression of clonal PC in POEMS syndrome was similar to MM: CD19-56+ in 5 (55.6%), CD19-56- in 2 (22.2%), CD19dim56- in 1 (11.1%), and CD19-56dim in 1 (11.1%) patient, respectively. No clone was detected in PC with CD19+56-. The clone identification rate by single-tube 8-color SRL-Flow protocols was lower than expected. Therefore, we reanalyzed the data by gating PC with lower expression of CD45 and CD38. In the reanalysis, we gated broadly for CD38 dim-to-bright and CD138 dim-to-bright cells and narrowly for CD38 dim and CD45 negative-to-dim cells. As a result, we identified the clonal PC in 15 of 21 cases (71.4%, λ in 13 and κ in 2): 13 of 19 IFE-positive cases (68.4%) and 2 of 2 IFE-negative cases (100%)). Of the 15 cases in which monoclonal PC were identified by reanalysis, 8 were negative by SRL-Flow. Of these, 6 were identified by selective gating of CD45- and CD38 dim, and 2 were identified by selective gating of CD45- and CD38+. The median clone size was 0.008 (0.001-3.27)%, smaller than that of MM, and the clone size was not correlated with the level of serum VEGF (r = -0.15). The aberrant expression of the clonal PC in POEMS remained similar to MM for CD19 and CD56 expression: CD19-56+ in 3 (20.0%), CD19-56- in 7 (46.7%), CD19-56dim in 4 (26.7%), and CD19dim 56- in 1 (6.7%) patient, respectively. No clone was detected in PC with CD19+56-. Conclusions EuroFlow-based SRL-Flow protocols detected the clonal PC in about one-third of POEMS patients. Aberrant immunophenotype of clonal PC in POEMS syndrome was similar to MM for CD19 and CD56 expression; however, CD45 and CD38 expression of POEMS PC tended to be downregulated. Selective gating of PC with CD38dim and CD45 negative-to-dim detected clonal PC in 71.4% of cases. This novel gating strategy is more accessible and might improve the identification rate of clonal PC in POEMS syndrome. Disclosures Tsukamoto: Daiichi Sankyo: Honoraria. Yokote: Kowa Company, Ltd.: Honoraria, Other: Scholarship; MSD K.K.: Honoraria, Other: Scholarship, Courses endowed by company; Astellas Pharma Inc.: Honoraria, Other: Scholarship; Mitsubishi Tanabe Pharma Corporation: Honoraria, Other: Scholarship; Amgen K.K.: Honoraria; Taisho Pharmaceutical Co., Ltd.: Honoraria, Other: Scholarship, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Honoraria, Other: Scholarship; Janssen Pharmaceutical K.K.: Honoraria; Kao Corporation: Other: Schlarship; Novo Nordisk Pharma Ltd.: Honoraria, Other: Scholarship; Teijin Pharma Limited: Other: Scholarship; Pfizer Japan Inc.: Honoraria, Other: Scholarship; Kyowa Kirin Co., Ltd.: Honoraria; Eli Lilly Japan K.K.: Honoraria, Other: Scholarship; Takeda Pharmaceutical Company Limited: Honoraria, Other: Scholarship; Sanofi K.K.: Honoraria; Ono Pharmaceutical Co., Ltd: Honoraria, Other: Scholarship; AstraZeneca K.K.: Honoraria; Daiichi Sankyo Company, Limited: Honoraria, Other: Scholarship; Novartis Pharma K.K.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria, Other: Scholarship; Shionogi Co., Ltd.: Other: Scholarship; Bayer Yakuhin, Ltd.: Other: Scholarship. Nakaseko: Novartis Pharma KK.: Honoraria. Takamatsu: Adaptive Biotechnologies, Eisai: Honoraria; SRL: Consultancy; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Sakaida: Bristol Myers Squibb: Research Funding; Chugai: Research Funding; Ono: Research Funding; Kyowa Kirin: Research Funding.

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)...

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.

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.

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

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]....

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.

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.

Myeloproliferative neoplasms (MPN), a group of haematopoietic stem cell (HSC) disorders, are often accompanied by myelofibrosis. We previously identified the fusion of the ETV6 gene to the LYN gene (ETV6-LYN) in idiopathic myelofibrosis with ins(12;8)(p13;q11q21). The introduction of ETV6-LYN into HSCs resulted in fatal MPN with massive myelofibrosis in mice, implicating the rearranged LYN kinase in the pathogenesis of MPN with myelofibrosis. However, the signalling molecules directly downstream from and activated by ETV6-LYN remain unknown. In this study, we demonstrated that the direct activation of STAT5 by ETV6-LYN is crucial for the development of MPN. ETV6-LYN was constitutively active as a kinase through autophosphorylation. ETV6-LYN, but not its kinase-dead mutant, supported cytokine-free proliferation of haematopoietic cells. STAT5 was activated in a JAK2-independent manner in ETV6-LYN-expressing cells. ETV6-LYN interacted with STAT5 and directly activated STAT5 both in vitro and in vivo. Of note, ETV6-LYN did not support the formation of colonies by Stat5-deficient HSCs under cytokine-free conditions and the capacity of ETV6-LYN to induce MPN with myelofibrosis was profoundly attenuated in a Stat5-null background. These findings define STAT5 as a direct target of ETV6-LYN and unveil the LYN-STAT5 axis as a novel pathway to augment proliferative signals in MPN and leukaemia.

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.

Clonal expansion of non-leukemic cells expressing two novel MLL – ELL variants differing in transforming activity

Extramedullary blast crisis of chronic myelogenous leukemia (CML) is defined as the development of extramedullary disease caused by the infiltration of blasts regardless of proliferation of blasts in the bone marrow. The onset of extramedullary blast crisis in the newly diagnosed patients is known to be extremely rare. Here, we present a case of extramedullary blast crisis of CML as an initial presentation in a 17-year-old female presenting with pain in the left femur tumor. This case was treated successfully with dasatinib and allogeneic hematopoietic stem cell transplantation, with achievement of long-term remission.

Acute myeloid leukemia (AML) with mixed lineage leukemia-eleven-nineteen lysine-rich leukemia (MLL-ELL) is a rare subtype of MLL-rearranged AML. The outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for patients with this disease remains unknown. In the present study, we retrospectively investigated the efficacy of allo-HSCT in eight adult MLL-ELL-positive AML patients. Although all eight patients achieved first complete remission (CR1), three (37.5 %) patients experienced relapse after induction therapy. Five (62.5 %) patients underwent allo-HSCT during CR1, whereas two (25.0 %) underwent allo-HSCT during disease relapse, and one (12.5 %) during CR2. All three patients who received allo-HSCT beyond CR1 died due to AML progression after allo-HSCT. Of the five patients who received allo-HSCT during CR1, three (60.0 %) remained alive at study conclusion. The overall survival rate at five years was 50.0 %. Intriguingly, clonally expanded non-leukemic cells expressing MLL-ELL during consolidation therapy were found to be eradicated after allo-HSCT during the monitoring of minimal residual disease in one patient; this indicates that allo-HSCT is efficacious for eliminating pre-leukemic cells resistant to chemotherapy. In conclusion, allo-HSCT soon after CR1 represents a promising therapeutic option for adult AML patients with MLL-ELL, although the outcome of allo-HSCT for patients beyond CR1 was dismal.

Factors associated with the efficiency of PBSC collection in POEMS syndrome patients undergoing autologous PBSC transplantation

Successful treatment with rituximab and donor lymphocyte infusions for fulminant EBV-associated lymphoproliferative disorder that developed 14 years after unrelated BMT

Intravascular large B-cell lymphoma (IVLBCL) is a rare disease entity of malignant lymphoma, characterized by selective growth of lymphoma cells within the vessel lumina in various organs [1]. The ...

Philadelphia chromosome-positive mixed phenotype acute leukemia (Ph(+)MPAL) is a rare type of acute leukemia having myeloid and lymphoid features. In the present study, we describe the successful treatment of a 71-year-old Japanese female patient with Ph(+)MPAL by the alternation of second-generation tyrosine kinase inhibitors according to BCR-ABL1 mutations. The patient survived in her third complete remission (CR) for over 4 years. In her first CR, the patient was treated with multiple-agent chemotherapy and underwent maintenance therapy with imatinib and monthly vincristine and prednisolone (VP). At the first relapse, an examination of the bone marrow revealed a transformation into acute lymphoblastic leukemia and an F317L mutation in BCR-ABL1 gene, which responded preferentially to nilotinib over dasatinib. She achieved second CR, and nilotinib with VP therapy was selected for maintenance treatment. At second relapse, BCR-ABL1 mutational analysis revealed Y253H mutation instead of F317L mutation, resulting in resistance to nilotinib. The patient achieved third CR with dasatinib and VP therapy, and maintained CR with this treatment. This suggests that appropriate alternation of TKIs may contribute to long-term survival in elderly patients with Ph(+)MPAL.

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic stem cell diseases. It has been reported that several autoimmune diseases are associated with MDS. Recently, the co-occurrence of MDS with trisomy 8 and rare disorders of the immune system, such as Behcet's disease (BD), has been described. Prognosis in the older-onset group of MDS-associated BD is unfavorable. Here, we report a case of MDS-associated intestinal BD treated successfully by azacitidine therapy. A 59-year-old Japanese male suffering from recurrent high fever, melena, and oral and genital ulcerations was diagnosed with MDS with trisomy 8 and intestinal BD by endoscopic and bone marrow examinations. Immunosuppressive therapies, including infliximab, were ineffective. Due to his severe emphysema, the patient was considered ineligible for stem cell transplantation, and azacitidine therapy was initiated. With the exception of fever, the symptoms of intestinal BD improved, and severe malnutrition and anemia were ameliorated. Fluorescence in situ hybridization analyses of the bone marrow before the eighth cycle revealed that the trisomy 8 had not decreased. To our knowledge, this is the first report of azacitidine therapy for MDS-associated BD. We suggest that azacitidine may control intestinal BD by mechanisms other than those responsible for its effect in MDS.

Introduction: Polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome is a rare paraneoplastic disease due to an underlying monoclonal plasma cell (PC) dyscrasia. Despite of dynamic symptoms associated with highly elevated VEGF, monoclonal PCs are thought to be quite small, and pathogenic significance of these PCs remains undetermined. In this study, we performed whole exome sequencing (WES), target sequencing, and RNA sequencing of PCs in patients with POEMS syndrome in order to define its genetic profiles. Methods: Patients diagnosed with POEMS syndrome at Chiba University Hospital from July 2014 to June 2016 were enrolled. DNA was extracted from either PCs which were isolated from patients' bone marrow (BM) using CD138 MACS (Miltenyi) or buccal cells as controls. WES and target sequencing were performed using HiSeq2500 (Illumina) and MiSeq (Illumina), respectively. The data of WES and target sequencing were analyzed by Empirical Bayesian mutation Calling (EBCall). Copy number was analyzed using the data of WES. RNA sequencing of PCs isolated by MACS and FACS sorting was conducted using HiSeq 1500 (Illumina). PCs from some patients diagnosed with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) were also collected as controls for RNA sequencing. Results: Twenty POEMS patients (M:F 12:8, mean age 42.6, range 16-78; 15 newly diagnosed, 5 refractory or relapsed cases) were included in this study. Regarding the types of M protein, 55% (11/20) were IgA-λ, 25% (5/20) were IgG-λ, and each individual case of the following; IgA-λ+IgG-λ, BJP-λ, IgG-κ, and Castleman's variant with no M protein. The mean serum VEGF was 6,471 pg/ml (range 1,190-13,800), and the mean PCs percentage in the BM was 4.4% (range 0.8-10.5). WES was performed in 15 cases; a total of 359 somatic mutations in 334 genes were revealed in 93.3% of cases (14/15) with a mean number of 23.9 (range 0-119) in each. All these mutated genes were significantly enriched in several pathways related to cell adhesion. Importantly, frequently mutated genes in MM such as NRAS, KRAS, and TP53 were not identified. Among all mutations, 1.7% were frameshift insertions, 2.0% were frameshift deletions, 4.2% were stop gains, 0.8% were non-frameshift deletions, 60.2% were other non-synonymous single nucleotide variants (SNVs), 29.5% were synonymous SNVs, and 1.7% were splicing mutations which were within 2-bp of a splicing junction. Copy-number variations were detected in 33.3% of cases (5/10) including -13 (2 cases), +1q (2 cases), and hyperdiploidy (2 cases). To carry out target sequencing in all 20 cases, we defined 51 target genes which included recurrently mutated genes from our WES data, frequently mutated genes in hematopoietic and lymphoid tissues according to the database (COSMIC), and 15 frequently mutated genes in MM (NRAS, KRAS, TP53, BRAF, CDKN2C, FGFR3, BIRC3, DIS3, CYLD, KDM6A, LRP1B, FAM46C, COL6A3, DNAH5, and KRT6A). A total of 60 somatic mutations were revealed in 65% of cases (13/20), and 9 new somatic mutations were found in the cases in which WES was also performed. Ten recurrently mutated genes were identified; KLHL6 in 20% of cases (4/20), each of LTB, RYR1 in 15% of cases (3/20), and each of EHD1, EML4, HEPHL1, HIPK1, PCDH10, USH2A, and ZNF645 in 10% of cases (2/20). Among frequently mutated genes in MM, only 3 genes (FAM46C, LRP1B, and DNAH5) were mutated, each in a single case. We finally conducted RNA sequencing of the FACS-sorted PCs in 5 POEMS patients compared to 5 MGUS and 4 MM patients. Upregulated genes were significantly enriched in some gene sets, gene ontology terms, and pathways related to immune response and cell adhesion, whereas downregulated genes were related to tumorigenesis. Of note, VEGF was not significantly upregulated in POEMS patients. Principal component analysis distinguished the 3 disease groups of patients with marginal overlaps between POEMS and MGUS, and also MGUS and MM. Conclusions: Our data clearly demonstrate that the genetic profiles of PCs in POEMS syndrome are distinct from those in MM and MGUS. Notably, PCs may not be the main source of extremely elevated VEGF in POMES syndrome. On-going further investigation will help clarify the molecular pathogenesis of POEMS syndrome. Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

[Backgrounds] POEMS syndrome is a rare plasma cell dyscrasia and its pathogenesis including the significance of monoclonal plasma cells in disease progression is poorly understood. Monoclonal plasma cells only produce λ immunoglobulins, and genes encoding immunoglobulin λ light chain (IGL) V regions are derived from IGLV1-44 or IGLV1-40 germline sequences. Here we analyzed the clonality in IGLV gene rearrangements using next generation sequencing (NGS) to evaluate the significance of monoclonal plasma cell clone size and follow its changes in clinical course to understand the pathogenesis of POEMS syndrome. [Methods] Patients who were diagnosed with POEMS syndrome between November 2006 and October 2015 at Chiba University Hospital were included in the study. As positive controls, 3 multiple myeloma (MM) patients with λ-type monoclonal light chain, and 9 negative control patients were also analyzed. NGS libraries were constructed from genomic DNA samples, extracted from bone marrow mononuclear cells. The IGLV1 and IGLV2 genes were amplified by polymerase chain reaction (PCR) using a 5' primer for the IGLV1/2 framework 3 (FR3) region and 3ʹ consensus primers for the IGLJ1/2/3 joining regions. Multiple samples were pooled, and paired-end 2 × 250 base pair sequencing reactions were carried out using an Illumina MiSeq sequencer. The closest matched germline sequences were determined using the ImMunoGeneTics database. Subsequently, frequencies of each clonotype that were characterized by a unique V-J rearrangement, conserved complementarity determining region 3 (CDR3) anchors and a unique CDR3 amino acid sequence, were calculated. [Results] Twenty-eight patients with POEMS syndrome were enrolled. All the patients had λ-type M protein. The median follow up time of the patients was 24.4 months (range, 3.7 - 113.9). Firstly we analyzed the usage of IGLV germline genes in each case. In 8 cases, the POEMS syndrome-specific germline sequences, IGLV1-40 or IGLV1-44, were dominant; accounting for more than 40% of all germline sequence usage. However, other samples showed minimal or no differences from controls, indicating that the clonal expansion of monoclonal plasma cells is generally low in patients with POEMS syndrome. Analyzing frequencies of the most dominant rearrangement in each germline, the clonal IGLV gene rearrangements of POEMS syndrome-specific germline sequences were significantly increased in 10 POEMS patients (35.7%; IGLV1-44: n = 8, IGLV1-40: n = 2). Significant increase of clone sizes were not directly linked to the initial disease status (vascular endothelial growth factor [VEGF] level and percentage of plasma cells in the bone marrow), overall survival and progression-free survival of POEMS patients. In 12 patients that we were able to follow their clinical courses, the clone size of IGLVgene rearrangements correlated with disease course assessed with serum VEGF level in most cases (n = 8), as they decreased with serum VEGF levels in disease remission and increased with re-elevation of serum VEGF in relapse cases. Clone sizes without significant increase at diagnosis were constantly flat even after achieving disease remission (n = 3). In one case, clone size with significant increase at diagnosis was unchanged even after serum VEGF level decreased (n = 1). Further observation is needed in this case. [Discussion] Considering the cases with significant increase of IGLV rearrangement clones, it was confirmed that clonal light chain gene expression is restricted to the IGLV1-44 and IGLV1-40 germline sequences, as previously reported. IGLV gene rearrangement clone was not detected as significant increase in some cases. Therefore, we speculated that there are certain numbers of patients with POEMS syndrome with extremely low frequency of clone cells. On the other hand, significant increase of clone size did not reflect disease status, suggesting that disease status is not regulated by tumor burden alone. By contrast, in cases with significantly increased clones, clone sizes changed dependiing on the disease status. These data do demonstrate that monoclonal plasma cells are related to the pathogenesis of POEMS syndrome. [Conclusions] Our analysis of IGLV gene rearrangements has demonstrated the association between the size of IGLV gene rearrangement clones and the clinical courses in POEMS syndrome. Disclosures No relevant conflicts of interest to declare.

Low-dose trimethoprim–sulfamethoxazole for Pneumocystis jiroveci pneumonia prophylaxis after allogeneic hematopoietic SCT

POEMS syndrome is a rare plasma cell disorder characterized by peripheral neuropathy, monoclonal gammopathy, and high levels of serum vascular endothelial growth factor, the pathogenesis of which remains unclear. A unique feature of this syndrome is that the proliferating monoclonal plasma cells are essentially λ-restricted. Here we determined complete nucleotide sequences of monoclonal immunoglobulin λ light chain (IGL) variable regions in 11 patients with POEMS syndrome. The V-region of the Igλ gene of all 11 patients was restricted to the Vλ1 subfamily. Searching for homologies with IGL germlines revealed that 2 germlines, IGLV1-44*01 (9/11) and IGLV1-40*01 (2/10), were identified, with an average homology of 91.1%. The IGLJ3*02 gene was used in 11 of 11 re-arrangements with an average homology of 92.2%. These data suggest that the highly restricted use of IGL Vλ1 germlines plays an important role in the pathogenesis of POEMS syndrome.

The expression of NR4A3, which is a member of the gene encoding NR4A orphan nuclear receptor subfamily, has been reported to be commonly silenced in blasts of patients with acute myeloid leukemia (AML), irrespective of karyotype. In line with this finding, Nr4a1-/-/Nr4a3-/- mice rapidly develop AML within one month following birth (Mullican et al., 2007). In addition, Nr4a1+/-/Nr4a3-/- and Nr4a1-/-/Nr4a3+/- mice show myelodysplastic/myeloproliferative neoplasms (Ramirez-Herrick et al., 2011), suggesting that NR4A3 functions as a tumor suppressor gene in myeloid malignancies. The extremely short latency of AML development in Nr4a1-/-/Nr4a3+/- mice indicates that silencing these tumor suppressors is sufficient to induce AML and that NR4A3 has a crucial role in the pathogenesis of AML. Thus, unveiling the molecular mechanism that regulates NR4A3 expression in AML would facilitate the development of novel therapies, including transcriptional reactivation of the gene. However, the therapeutic modalities targeting NR4A3 have been hindered by our minimal understanding of the mechanism underlying reduced NR4A3 expression, particularly in human AML cells. Abnormal epigenetic regulation is a common mechanism in the pathogenesis of several types of cancers. For instance, the expression of several tumor suppressor genes, such as p16 and MLH1, is repressed due to DNA hypermethylation at their promoter regions. Given that loss-of-function mutations in NR4A3 have not been reported in AML to date, we hypothesized that DNA hypermethylation contributes to a reduction in NR4A3 expression in AML. To test our hypothesis, we analyzed DNA methylation status of NR4A3 in human AML cells. We first compared the level of NR4A3 expression in eight human AML cell lines and two human primary AML samples, with that in CD34+ mononuclear bone marrow (BM) cells from healthy human controls. As expected, the expression of NR4A3 was markedly reduced in all of the AML cell lines and primary AML cells compared with that in the cells of the healthy controls. To evaluate the function of NR4A3 in human AML cells, we ectopically overexpressed NR4A3 in a human AML cell line (NB4 cells). The growth of NR4A3 -overexpressing NB4 cells was remarkably compromised compared with that of the controls, suggesting a tumor suppressive function of NR4A3 in both human AML and murine cells. To investigate the DNA methylation status of NR4A3, we performed bisulfite sequencing assays using eight human AML cell lines (HL60, NB4, Kasumi, TF1, U937, K562, MOLM13, and THP1) as well as CD34+ BM cells from healthy controls. Unexpectedly, a hypermethylated CpG site in the promoter region was not detected in any of the cell lines. However, the drastically or mildly methylated region including twenty eight CpGs was identified approximately 3 kb downstream of the transcription start site in six AML cell lines (97.5%, 78.3%, 77.1%, 89.9%, 95.2%, and 86.9% in HL60, NB4, Kasumi, TF1, U937, and K562, respectively) and two mixed lineage leukemia-related cell lines (31.0% and 53.6% in MOLM13 and THP1, respectively), whereas this site was hypomethylated in the controls (n = 2; mean, 12.7%; range, 7.1%-18.2%). To evaluate the contribution of this hypermethylated region to reduced NR4A3 expression, the six AML cell lines with heavily hypermethylated CpGs at NR4A3 and two human primary AML cell samples were treated with a DNA methyltransferase inhibitor (decitabine; DAC) for three or five days. DAC exposure inhibited cell growth and restored the expression of NR4A3 in all AML cell lines and primary cells in a dose- and time-dependent manner. Next, we examined the status of DNA methylation at the CpG site following DAC treatment with bisulfite sequencing assays. The frequencies of methylated CpG in HL60, NB4, and K562 cells was reduced from 97.5% to 53.6%, 78.3% to 68.5%, and 86.9% to 67.5% after DAC treatment, respectively. In contrast, the methylation status in Kasumi, TF1, and U937 cells did not significantly changed after DAC treatment. Our findings in the present study suggest that DNA hypermethylation may partially account for the transcriptional inactivation of NR4A3 in AML. However, the mechanism of reduced NR4A3 expression is complex and variable depending on the genetic background. We are currently working on a more detailed analysis of DNA methylation using human primary cells, by extending the regions for investigation, such as enhancer regions. Disclosures Nakaseko: Novartis: Honoraria, Research Funding, Speakers Bureau; Otsuka: Honoraria, Research Funding; BMS: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau.

Blesa, Zaragoza; Hospital Universitario de Salamanca, Salamanca; Hospital Universitario La Fe, Valencia; Hospital Universitario de Canarias, Santa Cruz de Tenerife; Hospital Clinico San Carlos, Madrid; Hospital General de Segovia, Segovia; Hospital Universitario 12 de Octubre, Madrid; Clinica Universitaria de Navarra

Auto-SCT has been established as an effective treatment for patients with hematological malignancies such as lymphoma or myeloma. Unfortunately, some patients fail to mobilize a sufficient number of PBSCs for transplantation. Plerixafor is the first molecule to reversibly inhibit the binding of chemokine stromal cell-derived factor-1α (SDF-1α) to its cognate receptor, CXCR4.1 Many recent reports have shown that there is a potential clinical application for plerixafor in PBSC harvesting.2, 3, 4, 5, 6

Introduction: Chronic myeloid leukemia (CML) is characterized by the myeloproliferative clonal hyperplasia caused by the protein product of the BCR-ABL fusion gene. However, the mechanism underlying the release of increased numbers of myeloid cells from bone marrow (BM) to circulation which causes the accompanied remarkably high count of peripheral white blood cells (WBC) has not been fully elucidated. We have recently reported that G-CSF treatment induced the shedding of an LDL receptor family member LR11 (also called SorLA or SORL1) from myeloid cells, and the released soluble receptor accelerate the trans-endothelial migration of myeloid cells in cooperation with an endothelial activator, tumor necrosis factor-a (TNF-a) (Shimizu et al Ann Hematol 2014). Another group showed that the levels of TNF- a and G-CSF production by CML stem/progenitor cells were increased in the BM of CML. Thus, we hypothesized that the expanded mobilization of peripheral cells in CML is caused by the abnormally induced shedding activity which releases the two soluble key molecules for the migration of clonally expanded cells from BM to peripheral blood. Based on these backgrounds, we examined soluble LR11 levels in the BM of CML patients. Materials and methods: We analyzed 11 patients (comprising three CML, six acute myeloid leukemia (AML) and two immune thrombocytopenia (ITP)), who underwent bone marrow aspiration at the time of diagnosis in Chiba University Hospital between 2011 and 2012. We measured the levels of sLR11 in supernatants of BM by an ELISA method. We also examined the levels of TNF-a and MMP9 in some patients. Then we compared with clinical data including WBC and total nuclear cell count (TNC) of BM. Results: sLR11 levels of supernatant of BM in CML patients were significantly higher than AML patients (134.5+-215.7 ng/ml vs 15.0+-46.1 ng/ml, p=0.0389, Mann-Whitney test, Figure 1). The levels of two ITP patients were 14.8, 14.2 ng/ml respectively. There was no significant correlation between TNC and the levels of sLR11 (p=0.4386) in all patients. However, the WBC counts in CML patients (53,400+-60,253/ml) were slightly higher than AML patients (2970+-34,624/ml) (p=0.1967, Mann-Whitney test, Figure 2). We next evaluated myeloid TNF-a and MMP9 levels in patients with CML or other hematological malignancy). A CML patient revealed remarkably higher levels of TNF- a (16.8 pg/ml), and also MMP9 (22,000 pg/ml), compared with other hematological diseases. Conclusions: soluble LR11 levels were largely increased in the BM of patients with CML. Considering that the myeloid TNF-a level was increased in a case among them, the increased soluble LR11 in BM may induce myeloid cell release from BM into peripheral blood through the cooperative activation of trans-endothelial migration in cooperation with TNF-a. Figure 1. The levels of sLR11 in BM in patients with CML and AML. (p=0.0389) Figure 1. The levels of sLR11 in BM in patients with CML and AML. (p=0.0389) Figure 2. The WBC count in the patients, CML and AML. (p=0.1967) Figure 2. The WBC count in the patients, CML and AML. (p=0.1967) Disclosures No relevant conflicts of interest to declare.

Recently, novel agents such as bortezomib and lenalidomide have been introduced for multiple myeloma (MM) treatment and have improved patients' survival drastically. However, dexamethasone remains a mainstay in the treatment of MM. Dexamethasone effectively induces tumor cell death when used for the initial treatment of MM. In addition, dexamethasone has a synergistic effect with novel agents and is hence used in combination with such agents. However, prolonged dexamethasone exposure may lead to drug resistance. To elucidate the mechanism of dexamethasone resistance, we generated a dexamethasone-resistant subline of the MM cell line RPMI8226. We cultured RPMI8226 cells with 1 µM dexamethasone for 7 weeks and established the dexamethasone-resistant cell line Dex-R. This cell line showed no difference in survival in the presence or absence of 1 µM dexamethasone. We then examined differences in gene expression between RPMI8226 and Dex-R cells using cDNA microarray. Expression of the FARP1 gene, which is a transforming growth factor beta (TGF-b) target gene in myeloma cells, was increased approximately 50-fold in Dex-R cells compared to that in RPMI8226 cells. In some myeloma patients who become chemoresistant, myeloma cells show high levels of FARP1 expression at the initial stage. FARP1 has a Rho-GEF domain and can associate with proteins on the cell membrane through the FERM domain. In the nervous system, FARP1 is involved in synaptogenesis via the activation of Rac1. Based on these observations, we hypothesize that Dex-R cells acquires dexamethasone resistance with an increase in the level of FARP1 expression via the activation of Rac1. To verify this hypothesis, we established inducible FARP1 knockdown Dex-R cells using the TET-ON lentiviral system. We cultivated these cells for 24 h with doxycycline and added 1 µM dexamethasone. A total of 48 h after adding dexamethasone, we measured cell viability using the MTS assay. We cultured Dex-R cells with a Rac1 inhibitor (NSC23766) and added dexamethasone 12 h later. FARP1 expression decreased to approximately 10% in FARP1 knockdown cells 24 h after the addition of doxycycline. Without dexamethasone, there was no difference in survival in the presence or absence of doxycycline. However, when cells were cultured with dexamethasone, the growth of FARP1 knockdown Dex-R cells was significantly inhibited compared with that of the control (Fig 1). Next, we examined the change in dexamethasone resistance on the addition of the Rac1 inhibitor. The number of cells increased after 96 h without dexamethasone. On the other hand, the number of cells significantly decreased when cultured with dexamethasone (Fig 2). These data suggest that resistance to dexamethasone in Dex-R cells was mitigated by the inhibition of Rac1. We conclude that the activation of Rac1 through FARP1 is one mechanism of dexamethasone resistance in MM. Disclosures No relevant conflicts of interest to declare.

Successful combination treatment with bevacizumab, thalidomide and autologous PBSC for severe POEMS syndrome

Successful engraftment by second cord blood transplantation with reduced-intensity conditioning after graft rejection due to hemophagocytic syndrome following initial CBT

Introduction LR11, which is also known as SorLA or SORL1, is a type-I membrane protein from which a large extracellular part, soluble LR11 (sLR11), is released by proteolytic shedding. We have demonstrated that serum sLR11 levels are significantly elevated in patients with acute leukemia and that sLR11 levels are associated with the peripheral blast percentage (Sakai et al. Clin Chim Acta. 2012). In addition, we found that high sLR11 levels have a significant negative prognostic impact on progression-free survival in patients with follicular lymphoma (FL; Kawaguchi et al. Br J Haematol. 2013). In this FL analysis, the immunohistological intensity of LR11 in lymph nodes of FL patients did not show a significant association with serum sLR11 levels. Therefore, a shedding mechanism is presumed to play a key role in the functions of LR11. A disintegrin and metalloproteinase 17 [ADAM17, also known as tumor necrosis factor (TNF)-α converting enzyme (TACE)] has been identified as the enzyme that cleaves TNF-α from its transmembrane precursor form, and it has been found to cleave the ectodomains of other cell surface proteins. LR11 is also shed by ADAM17. Tetraspanin CD9 has been recently shown to inhibit the shedding activity of ADAM17. Therefore, we investigated the role of CD9 in sLR11 release. Materials and methods First, we examined the gene expression levels of LR11 by quantitative real-time PCR and the cell surface expression of LR11 and CD9 by flow cytometric analysis in normal human peripheral-blood mononuclear cells (PBMCs). Second, we investigated the cellular expression and the released levels of LR11 and cellular CD9 in THP-1 monocytes, phorbol 12-myristate-13-acetate (PMA)-induced THP-1 macrophages (PMA/THP-1), and the B lymphoblastoid cell line CCRF-SB by immunoblot analysis. Furthermore, to assess the relationship between LR11 and CD9 in PMA/THP-1, we performed double immunofluorescence staining of these molecules followed by confocal microscopy analysis. Third, we examined the effects of ectopic neoexpression of CD9, anti-CD9 mAbs, and CD9 silencing. THP-1 cells stably interfered with CD9 were generated by the transfection of the shRNA expression vector that is specific for CD9. Results The gene expression levels of LR11 were significantly higher in CD3+ T cells than in CD14+ monocytes, and there was no difference between monocytes and CD19+ B cells. However, LR11 was only expressed on the cell surface of monocytes in normal PBMCs. CD9 was broadly expressed on the cell surface of every PBMCs. Because it has been reported that CD9 associates with ADAM-17 on the cell surface, we hypothesized that CD9 associates with the shedding of LR11 on the cell surface. To verify this hypothesis, we chose the monocytic cell line THP-1 because monocytes have both LR11 and CD9 on their surface. In addition, because the gene expression levels of LR11 were significantly higher in human monocyte-derived macrophages than that in monocytes, we investigated the levels in PMA/THP-1. LR11 was not expressed or released in undifferentiated THP-1 cells, but it was expressed or released in differentiated PMA/THP-1. CD9 was poorly expressed in THP-1 cells, and the CD9 expression levels were increased in PMA/THP-1. The confocal microscopy analysis showed colocalization of LR11 and CD9 proteins in PMA/THP-1. We next investigated the effects of ectopic neoexpression of CD9 in CD9-negative cells, neutralizing anti-CD9 mAbs (clone ALB-6) in CD9-positive cells, and CD9 silencing in THP-1 on the shedding of LR11. When CCRF-SB, LR11-positive and CD9-negative cells were transiently overexpressed with CD9, the amount of sLR11 released from CD9-overexpressing cells was decreased compared with that from control cells. The amount of sLR11 released from LR11-transfected THP-1 cells that were cultured with ALB-6 was significantly increased compared with that cultured with an isotypic control antibody. The PMA-stimulated release of sLR11 was significantly increased in CD9 shRNA-interfered THP-1 cells compared with cells that were transduced with the control shRNA. The increase in CD9 shRNA-interfered THP-1 was nullified by treatment with the metalloproteinase inhibitor GM6001 (Figure 1). Conclusion These results suggested that the tetraspanin CD9 inhibits ADAM17-mediated LR11 shedding in leukemia cells and that it may have a role in controlling the function of LR11. Disclosures: No relevant conflicts of interest to declare.

Introduction Although thrombopoietin-receptor agonists have recently been introduced for the treatment of refractory immune thrombocytopenia (ITP), splenectomy is still the standard treatment option for patients with steroid-refractory ITP. However, open and laparoscopic splenectomy is associated with a mortality rate of 1.0% and 0.2%, respectively, and the major known long-term risk of splenectomy is severe bacterial sepsis. Partial splenic embolization (PSE) is an alternative to splenectomy for patients with steroid-refractory (Kimura et al., AJR 2002). PSE does not require general anesthesia and has not been associated with severe complications. In this study, we analyzed the efficacy and long-term outcomes of PSE in steroid-refractory ITP patients. Patients and Methods Between 1988 and 2009, 77 patients with chronic ITP [30 men and 47 women, 16.5–80.9 years old (median age, 46.5 years)] underwent PSE at Chiba University Hospital. Almost all patients did not respond to steroid treatment or other therapies including Helicobacter pylori eradication and immunosuppressive drugs. A 5-French catheter was introduced via the femoral artery to the middle splenic artery and a 0.089-cm guidewire was inserted coaxially. Embolization of intrasplenic arterial branches was performed by the injection of 2 x 2 mm fragments of Gelfoam and an antibiotic solution. Splenic embolization was monitored angiographically during the procedure, and embolization was stopped after 70% of the splenic parenchyma had been ablated. The therapeutic response to PSE was evaluated on the basis of the platelet (PLT) count at 1 year after PSE: a complete response (CR) was defined as PLT count of more than 10 x 1010/L without any medication and a partial response (PR) was defined as PLT count = 5–10 x 1010/L without any medication. Patients who required medication to maintain a PLT count of more than 5 x 1010/L or patients with a PLT count of less than 5 x1010/L were considered to have no response (NR). Results The average PLT count prior to PSE was 2.7 ± 2.4 x 1010/L. The mean time to achieve the peak PLT count was 10.2 ± 5.2 days after PSE. The PLT count was 11.1 ±10.3 x 1010/L (n = 74), 9.8 ± 8.3 x 1010/L (n = 70), 9.1 ± 8.2 x 1010/L (n = 65), and 9.1 ± 7.5 x 1010/L (n = 69) at 1 month, 3 months, 6 months, and 1 year, respectively. One year post PSE, CR or PR was obtained in 33 (58%) of the evaluable 57 patients and 24 patients (42%) had NR. The group in which the peak PLT count after PSE was over 30.0 x 1010/L showed a significantly higher PLT count than the group in which the peak count was under 30.0 x 1010/L after PSE treatment (Figure 1). The peak PLT count was significantly higher in CR and PR groups (37.4 x 1010/L) than in the NR group (15.0 x 1010/L) (p = 0.041). In addition, 52% patients did not require additional treatment for 10 years after PSE (Figure 2); in 16 patients, PSE was performed for the second time after recurrence; and CR or PR was obtained in 5 patients (31%). There were no severe PSE-related complications such as splenic abscess and no PSE-related deaths. Conclusions PSE might be a safe and effective alternative therapy to splenectomy for patients with steroid-refractory ITP as it generates long-term durable responses. Disclosures: No relevant conflicts of interest to declare.