Abstract: Recent identification of platelet/megakaryocyte-biased hematopoietic stem/repopulating cells requires revision of the intermediate pathway for megakaryopoiesis. Here, we show a unipotent megakaryopoietic pathway bypassing the bipotent megakaryocyte/erythroid progenitors (biEMPs). Cells purified from mouse bone marrow by CD42b (GPIbα) marking were demonstrated to be unipotent megakaryocytic progenitors (MKPs) by culture and transplantation. A subpopulation of freshly isolated CD41+ cells in the lineage Sca1+cKit+ (LSK) fraction (subCD41+LSK) differentiated only into MKP and mature megakaryocytes in culture. Although CD41+LSK cells as a whole were capable of differentiating into all myeloid and lymphoid cells in vivo, they produced unipotent MKP, mature megakaryocytes, and platelets in vitro and in vivo much more efficiently than Flt3+CD41−LSK cells, especially at the early phase after transplantation. In single cell polymerase chain reaction and thrombopoietin (TPO) signaling analyses, the MKP and a fraction of CD41+LSK, but not the biEMP, showed the similarities in mRNA expression profile and visible TPO-mediated phosphorylation. On increased demand of platelet production after 5-FU treatment, a part of CD41+LSK population expressed CD42b on the surface, and 90% of them showed unipotent megakaryopoietic capacity in single cell culture and predominantly produced platelets in vivo at the early phase after transplantation. These results suggest that the CD41+CD42b+LSK are straightforward progenies of megakaryocytes/platelet-biased stem/repopulating cells, but not progenies of biEMP. Consequently, we show a unipotent/highly biased megakaryopoietic pathway interconnecting stem/repopulating cells and mature megakaryocytes, the one that may play physiologic roles especially in emergency megakaryopoiesis. Stem Cells 2015;33:2196–2207

Abstract: Background: Activities of nonhematopoietic cells (NHCs) reportedly underlie lymphomagenesis. In follicular lymphoma (FL), mesenchymal stromal cells (SCs) including follicular dendritic cells (FDCs) have been shown to facilitate FL expansion. However, comprehensive understanding of lymphoma NHC activities have been hampered by indefinite NHC heterogeneity even in normal human lymph node (LN). Indeed, human LN blood endothelial cells (BECs) and non-endothelial stromal cells (NESCs) have not been analyzed at single-cell resolution. Here, we aimed to construct a single-cell atlas of NHCs in human LN applicable to lymphoma researches. We also sought to reveal the landscape of stromal remodeling in lymphomas, particularly in FL, to advance understanding of stromal contributions in lymphomagenesis. Methods: We prospectively performed single-cell RNA sequencing of NHCs ( & gt;100,000 cells) extracted from 27 human samples including metastasis-free LN (MFLN; n=9), nodal FL (n=10), peripheral T-cell lymphoma (PTCL; n=5), and diffuse large B-cell lymphoma transformed from FL (tDLBCL; n=3). Data from MFLN samples were used for the construction of NHC atlas. Immunofluorescence (IF) staining was performed to investigate the existence and topological localizations of each NHC subcluster in the LN. Using the NHC atlas, we performed comprehensive comparative analysis with FL NHCs by differentially-expressed gene (DEG) and intercellular ligand-receptor analyses. We also investigated the prognostic impact of putative stroma-derived biomarkers using deposited microarray data of FL patients. Finally, we examined the applicability of the atlas to NHCs from other lymphoma subtypes by analyzing PTCL and tDLBCL NHCs. Data analysis was performed through multiple pipelines including Seurat, Monocle3, and CellphoneDB. Results: Graph-based clustering analysis revealed that the transcriptional features of NHC subpopulations in MFLN are detectable in FL NHCs. Unsupervised sub-clustering analysis of BECs, lymphatic endothelial cells (LECs), and NESCs revealed 10, 8, and 12 subclusters, respectively, including some lacking mouse counterpart. IF staining successfully identified each NHC subcluster and its localization in the LN. In FL NHCs, the proportion of arterial BEC subclusters markedly increased relative to MFLN, while the proportion of LECs decreased. In FL NESCs, the proportion of marginal reticular cells (MRCs) as well as FDCs greatly increased. DEG analysis revealed that the greatest changes in gene expression occurs in NESC subclusters, particularly in MRCs, T-zone reticular cells (TRCs), pericytes, and FDCs. Notably, in some NESC subclusters, we observed marked upregulation of genes relevant to solid cancers but previously not described in lymphomas (e.g. POSTN, EGFL6, and FAP). Combined interactome and DEG analysis revealed 60 FL-specific interactions between NHC subclusters and malignant B cells. For example, interactions mediated through stroma-derived CD70 were enhanced at medullary SC subclusters and SCs at LN capsule adventitia. Additionally, the CCR7-CCL19 interaction and interactions via B-cell activating factor (BAFF) were unexpectedly upregulated at non-TRC SC and medullary SC subclusters, respectively. Also, the CXCL13-CXCR5 axis was highly activated in MRCs, collectively indicating that non-FDC SCs vigorously participate in FL cell expansion and/or infiltration into extra-follicular lesions. Some intercellular interactions were functionally validated by in vitro binding assays. Based on this dataset, we identified putative stroma-derived biomarkers linked to unfavorable prognosis in FL patients including TDO2, encoding immune-modulators, and LY6H and LOX, tip cell markers. We finally confirmed that NHC subclusters identified in our atlas were also detectable in NHCs of more aggressive lymphoma subtypes including PTCL and tDLBCL. Notably, we found that extra-follicular SCs had further differentiated into follicular SCs in tDLBCL, likely representing a terminal form of stromal remodeling in FL. Conclusion: We constructed a comprehensive single-cell atlas of NHCs in human LN highly applicable to lymphoma NHC researches and revealed a total of 30 NHC subclusters. Our study largely updates NHC taxonomy in LNs and provides a rich resource and deeper insights into lymphoma biology, a contribution that should advance lymphoma management and therapy. Figure 1 Figure 1. Disclosures Usuki: Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Research Funding, Speakers Bureau; Ono Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Janssen Pharmaceutical K.K.: Research Funding; Celgene K.K.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Nippon-Boehringer-Ingelheim Co., Ltd.: Research Funding; Mundipharma K.K.: Research Funding; Amgen-Astellas Biopharma K.K.: Research Funding; Nippon-Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Kyowa-Kirin Co., Ltd.: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; PharmaEssentia Japan KK: Research Funding, Speakers Bureau; Yakult Honsha Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo-Dainippon Pharma Co., Ltd.: Research Funding; SymBio Pharmaceuticals Ltd.: Research Funding, Speakers Bureau; Gilead Sciences, Inc.: Research Funding; Bristol-Myers-Squibb K.K.: Research Funding, Speakers Bureau; Apellis Pharmaceuticals, Inc.: Research Funding; AbbVie GK: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Incyte Biosciences Japan G.K.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Sanofi K.K.: Speakers Bureau; Amgen K.K.: Research Funding.

Abstract: Abstract 3413 Background: Disseminated intravascular coagulation (DIC) is a lethal complication in patients with hematological malignancies. Although standard therapy against DIC remains to be established, soluble recombinant thrombomodulin (rTM), which serves as a receptor for thrombin, has been developed and its effectiveness for DIC was recently reported (Saito et al, J Thromb Haemost 2006). We retrospectively analyzed 55 DIC episodes treated with rTM in patients with hematological malignancies. Patients and Methods: 55 consecutive DIC episodes in 47 patients with hematological malignancies (AML except for APL, 21; APL, 8; ALL, 8; lymphoma, 8; myeloma, 2) hospitalized between November 2009 and July 2012 in University of Tsukuba Hospital were retrospectively analyzed. Diagnosis of DIC was based on DIC score of Japanese Ministry of Health and Labor Welfare criteria (Kobayashi et al, Bibl Haematol 1983). DIC was induced by hematological malignancy itself and severe infection secondary to hematological malignancy in 39 and 16 episodes, respectively. In every episode, 380 units/kg/day of rTM was administered intravenously from the onset of DIC for median of 7 (range, 2–22) days. The fibrin degradation products (FDP) level, DIC score, recovery time from DIC (recovery, the day when DIC score was decreased to 5 or less), and overall survival were analyzed. Results: Median DIC score at the onset was 7 (range, 6–11). 15 episodes were accompanied by bleeding tendency. Average of FDP level at the onset was 64.6 ƒÊg/dl (range, 20.6–202.4) in malignancy-induced DIC and 30.1 ƒÊg/dl (range, 13.2–72.0) in infection-induced DIC (P=0.03). FDP level 14 days after rTM administration was 10.1 ƒÊg/dl (SD: 3.7–27.9) and 20.3 ƒÊg/dl (SD: 9.3–44.3), respectively (P=0.04). Recovery rates from DIC 7 days after rTM administration were 72% in malignancy-induced DIC and 39% in infection-induced DIC (Fig. 1, P=0.02), and 100-day overall survival after the onset of DIC were 89% and 15% (Fig. 2, P 〈 0.01), respectively. In multivariate analysis, infection-induced DIC was an only significant risk factor and presence of bleeding tendency, FDP level at the onset, DIC score at the onset, period of rTM administration, and number of rTM administration did not influence the recovery from DIC and overall survival. There were no severe hemorrhagic events after rTM administration or deterioration of bleeding tendency that led to discontinuation of rTM. Discussion and Conclusion: The recovery rate from hematological malignancy-induced DIC in the current cohort was comparable to that of rTM-treated DIC group (66%) and can be superior to that of heparin-treated DIC group (50%) in a previously reported phase III trial (Saito et al, J Thromb Haemost 2006). Although the use of heparin has fostered bleeding tendency in a number of previous DIC reports, bleeding tendency was reduced after rTM administration in all the DIC episodes analyzed with the current cohort. Therefore, this analysis traced the core conclusion of the previous phase III trial, emphasizing that rTM can be an effective anti-DIC agent without causing adverse hemorrhagic event even in DIC cases with preexisting bleeding tendency. However, the result was still significantly worse in infection-induced DIC secondary to hematological malignancies. Disclosures: Chiba: Asahi Kasei Pharma: Research Funding.

Abstract: Actin polymerization is crucial in throm-bopoiesis, platelet adhesion, and mega-karyocyte (MK) and platelet spreading. The Wiskott-Aldrich syndrome protein (WASp) homolog WAVE functions downstream of Rac and plays a pivotal role in lamellipodia formation. While MKs and platelets principally express WAVE1 and WAVE2, which are associated with Abi1, the physiologic significance of WAVE isoforms remains undefined. We generated WAVE2−/− embryonic stem (ES) cells because WAVE2-null mice die by embryonic day (E) 12.5. We found that while WAVE2−/− ES cells differentiated into immature MKs on OP9 stroma, they were severely impaired in terminal differentiation and in platelet production. WAVE2−/− MKs exhibited a defect in peripheral lamellipodia on fibrinogen even with phorbol 12-myristate 13-acetate (PMA) costimulation, indicating a requirement of WAVE2 for integrin αIIbβ3-mediated full spreading. MKs in which expression of Abi1 was reduced by small interfering RNA (siRNA) exhibited striking similarity to WAVE2−/− MKs in maturation and spreading. Interestingly, the knockdown of IRSp53, a Rac effector that preferentially binds to WAVE2, impaired the development of lamellipodia without affecting proplatelet production. In contrast, thrombopoiesis in vivo and platelet spreading on fibrinogen in vitro were intact in WAVE1-null mice. These observations clarify indispensable roles for the WAVE2/Abi1 complex in αIIbβ3-mediated lamellipodia by MKs and platelets through Rac and IRSp53, and additionally in thrombopoiesis independent of Rac and IRSp53.

Abstract: Primary central nervous system lymphoma ( PCNSL ) is a rare subtype of lymphoma that arises within the brain or the eyes. PCNSL recurs within the central nervous system ( CNS ) in most relapsed cases, whereas extra‐ CNS relapse is experienced in rare cases. The present study aimed at identifying the presence of common precursor cells ( CPC ) for primary intra‐ and relapsed extra‐ CNS tumors, and further assessing the initiating events in bone marrow ( BM ). Targeted deep sequencing was carried out for five paired primary intra‐ and relapsed extra‐ CNS tumors of PCNSL . Two to five mutations were shared by each pair of intra‐ and extra‐ CNS tumors. In particular, MYD 88 mutations, L265P in three and P258L in one, were shared by four pairs. Unique somatic mutations were observed in all five intra‐ CNS tumors and in four out of five extra‐ CNS tumors. Remarkably, IgH clones in the intra‐ and the extra‐ CNS tumors in two pairs were distinct from each other, whereas one pair of tumors shared identical monoclonal IgH rearrangement. In a cohort of 23 PCNSL patients, L265P MYD 88 mutations were examined in tumor‐free BM mononuclear cells ( MNC ) in which the PCNSL tumors had L265P MYD 88 mutations. L265P MYD 88 mutations were detected by a droplet digital PCR method in nine out of 23 bone marrow mononuclear cells. These results suggest that intra‐ and extra‐tumors are derived from CPC with MYD 88 mutations in most PCNSL , arising either before or after IgH rearrangement. The initiating MYD 88 mutations may occur during B‐cell differentiation in BM .

Abstract: We report the case of a 76-year-old man who was diagnosed as having chronic myeloid leukemia (CML) with p190 BCR-ABL while receiving treatment for symptomatic multiple myeloma (MM). The diagnosis of MM was based on the presence of serum M-protein, abnormal plasma cells in the bone marrow, and lytic bone lesions. The patient achieved a partial response to lenalidomide and dexamethasone treatment. However, 2 years after the diagnosis of MM, the patient developed leukocytosis with granulocytosis, anemia, and thrombocytopenia. Bone marrow examination revealed Philadelphia chromosomes and chimeric p190 BCR-ABL mRNA. Fluorescence in situ hybridization also revealed BCR-ABL -positive neutrophils in the peripheral blood, which suggested the emergence of CML with p190 BCR-ABL . The codevelopment of MM and CML is very rare, and this is the first report describing p190 BCR-ABL -type CML coexisting with MM. Moreover, we have reviewed the literature regarding the coexistence of these diseases.

Abstract: A 38-year-old woman with aggressive clinical course of chronic lymphocytic leukemia (CLL) was treated with 8 courses of R-CHOP. Clinical remission was achieved, while B-cell clonality remained. Allogeneic hematopoietic stem cell transplantation was performed with reduced intensity conditioning (fludarabine and 2-Gy total body irradiation). However, autologous hematopoietic recovery occurred within a month after the transplant. Nevertheless, B-cell clonality became undetectable at 14 days after transplant, which has been kept so for over 10 years with clinical remission. Cytogenetic analyses were repeatedly performed and demonstrated nonclonal chromosomal aberrations, although the patient did not develop any secondary malignancies. One possible explanation for the clinical course is a very short-term allogeneic immune reaction helping eradication of residual CLL cells.