bluuebunny/crossref_metadata_2025 huggingface dataset:
C1q Complement Cascade Protein as a Novel Modulator of the SDF-1-CXCR4 Axis and Hematopoietic Stem/Progenitor Cell Trafficking.
Authors: Jalili Ali , Shirvaikar Neeta , Ratajczak Mariusz Z. , Janowska-Wieczorek Anna
Published: October 2019

The first component of the complement (C) system C1q has multiple immune functions including acting as a chemoattractant for mature blood cells, and it has recently been reported that hematopoietic stem/progenitor cells (HSPC) express the C1q receptor, C1qRp. We have reported that the complement components C3 and C5 play an important role in the mobilization of HSPC and that G-CSF mobilization activates C by the classical Ig-dependent pathway (Blood2004;103:2071; Stem Cells 2007, in press). While C3 plays an important role in the retention of HSPC in BM, C5 is pivotal for their egress into peripheral blood. The aim of this study was to examine a possible role for C1q in the mobilization/homing of HSPC. We found (using RT-PCR and Western blotting) that the C1q gene and protein are strongly expressed in human mobilized peripheral blood (mPB) CD34+ cells, but not in steady-state bone marrow (BM) CD34+ cells. Importantly, we found that G-CSF stimulation of BM cells induces the expression of C1q in CD34+ cells, as detected by flow cytometry. Moreover, we demonstrated the presence of the receptor for C1q, C1qRp, in BM, mPB and cord blood (CB) CD34+ cells as well as in myeloid, megakaryocytic and erythroid progenitors ex vivo-expanded from CD34+ cells. The stromal derived factor (SDF)-1-CXCR4 axis plays a critical role in HSPC homing to BM and we showed using confocal microscopy that pre-incubation (for 1 h) of CB CD34+ cells on C1q-coated slides increased incorporation of the SDF-1 receptor, CXCR4, into membrane lipid rafts while C1q-coated filters increased chemoattraction (compared to bare filters) to an SDF-1 gradient. Although C1q itself is not a chemoattractant for HSPC, it primed the chemotactic response of CD34+ cells to a low SDF-1 gradient (10 ng/mL) to the extent that it approximated response to a high SDF-1 gradient (200 ng/mL), as well as the chemoinvasion of CB CD34+ cells across reconstituted basement membrane (Matrigel) to a low gradient of SDF-1, which was inhibited by the monoclonal anti-C1q antibody. In addition, C1q stimulated matrix-degrading metalloproteinase-9 secretion by CB CD34+ cells. In conclusion, we demonstrated for the first time that G-CSF mobilization strongly upregulates C1q in human HSPC which express the functional C1q receptor and that the first component of the complement cascade is involved in HSPC trafficking. This supports our previous data that C as an important modulator of the SDF-1-CXCR4 axis plays a pivotal role in the mobilization of HSPC.
Autologous Implantation of Bone Marrow for Non-Unions and Avascular Necrosis of the Bone.
Authors: Nedelcu Elena , Vierria Dawn , Taylor Douglas , Dwyre Denis , Jamali Amir
Published: October 2019

Introduction: Autologous implantation of bone marrow (BM) stem cells for avascular necrosis of the bone has been previously demonstrated to be feasible and safe procedure. The benefit of using autologous stem cells for bone regeneration consists of avoidance of graft rejection, graft-versus-host disease, and the toxic and infectious complications associated with immunosuppressive regimens.

Materials and Methods: We developed an efficient protocol to process autologous BM for volume reduction and mononuclear cell concentration that can be immediately used for autologous implantation. The end-product is a concentrated BM cell suspension that was defined by several parameters as follows: volume, mononuclear cell count, CD34-positive cell count. A total of five patients under general anesthesia underwent BM aspiration, followed by immediate intraoperative BM concentration and implantation at the injured site. Four patients had avascular necrosis of the bone and one had tibia non-union. The patients were followed for one year and monitored clinically and by imaging studies.

Results: All five patients treated with autologous bone marrow implantation at the avascular necrosis or non-union site tolerated the procedure well. One year follow-up of these patients reveals improved clinical and functional status. The imaging performed pre and one-year-post procedure revealed no evidence of bone collapse and preserved joint space.

Conclusions: Autologous implantation of concentrated bone marrow is a safe and promising treatment for non-unions and avascular necrosis of the bone. Additional studies for further characterization of the concentrated BM used for implantation are in progress.
GATA-1 Represses PU.1/Sfpi-1 Gene Transcription in Erythro-Megakaryocytic Progenitors.
Authors: Chou Stella T. , Bailey Charles L. , Hardison Ross C. , Blobel Gerd A. , Vakoc Christopher R. , Weiss Mitchell J.
Published: October 2019

Commitment of multipotential hematopoietic progenitors to unique cell fates is determined by the induction and interplay of specific nuclear factors that reinforce unilineage transcriptional programs. Previously, we reported that loss of transcription factor GATA-1 promotes the expansion of bipotential megakaryocyte erythroid progenitors (MEPs) from embryonic stem cell or fetal liver derived hematopoietic progenitors. These mutant cells, termed G1ME (for Gata-1− Megakaryocyte-Erythroid), proliferate in culture and differentiate into committed megakaryocytes and erythroblasts when GATA-1 activity is restored. To evaluate gene regulation at the MEP stage of hematopoiesis, we performed microarray analysis of G1ME cells before and after GATA-1-induced differentiation. Expression of GATA-1 in G1ME cells induced numerous erythroid and megakaryocytic target genes. In addition, undifferentiated G1ME cells expressed numerous granulocyte and macrophage genes, suggesting that loss of GATA-1 derepresses a myeloid program in MEPs. Myeloid genes were rapidly inhibited upon expression of GATA-1. In particular, mRNA encoding the myeloid transcription factor PU.1 and more than thirty of its downstream targets were repressed by GATA-1. Chromatin immunoprecipitation (ChIP) showed that GATA-1 bound directly to the PU.1 gene (Sfpi1) at the promoter and at a −18kb upstream region coincident with repression. At the same regions, GATA-1 triggered release of both GATA-2, a related transcription factor expressed in multipotential progenitors, and PU.1, a positive autoregulator of its own gene. While GATA-1 is known to inhibit PU.1 functions through direct protein interactions, this work shows that antagonism also occurs at the level of Sfpi1 gene transcription. Together, our findings indicate that GATA-1 not only positively regulates an erythro-megakaryocytic program of gene expression, but also actively restrains myelopoiesis by inhibiting Sfpi1 expression directly. More generally, our work reveals a new mechanism through which cross-antagonism between transcription factors reinforces lineage commitment decisions in hematopoiesis.
GATA-1 Interferes with Monopoiesis by Blocking a Positive Regulatory Circuit Involving VDR and PU.1 - Results from a Retroviral Dominant Effector Screen.
Authors: Taschner Sabine , Gobel Florian , Mag Mario Kumerz , Jorgl Almut , Richter Susanne , Strobl Herbert
Published: October 2019

In multipotent hematopoietic progenitors the co-expression of several lineage affiliated transcription factors occurs prior to commitment. The mechanism underlying the repression of alternative lineage pathways is poorly understood. We screened a complex retroviral fetal liver cDNA library for regulatory proteins that repress monopoiesis in a phenotype-based differentiation assay system. This functional genetic “dominant effector” strategy revealed a positive circuit regulating monopoiesis that is established by PU.1 and vitamin D receptor (VDR) as well as a novel repressor function of GATA-1. Increasing levels of PU.1 sensitize progenitors to VDR-dependent monopoiesis, and VDR/retinoic X receptor (RXR) signalling directly transactivates PU.1 gene expression. This positive regulatory loop is repressed by GATA-1 via the VDR. Specifically, we show that the GATA-1 N-terminal region, which is required for the inhibition of monocytic cell proliferation, represses and physically interacts with VDR. Additionally, PU.1 binding via the GATA-1 C-terminus is sufficient to inhibit monocyte differentiation without impairing myeloid cell proliferation. Thus, loss of GATA-1 N-terminal function allows for the expansion of immature myeloid progenitors. We propose that GATA-1 represses monopoiesis in multipotent myeloid progenitors or precursors by interacting with both VDR and PU.1, ultimately enabling alternative lineage programs.
ZFP143 Activates C/EBPα Transcription in Myeloid Cells.
Authors: Bartholdy Boris , Gonzalez David , Tenen Daniel G.
Published: October 2019

C/EBPα is a key transcription factor involved in myeloid differentiation and frequently mutated or deregulated in human acute myeloid leukemias (AML) as well as in blast crisis of chronic myelogenous leukemia (CML). Disruption of its function contributes to the differentiation block observed in these diseases and thus to leukemogenesis. Here, we have identified a conserved region in the C/EBPα promoter that is important for activation of C/EBPα transcription in myeloid cells and narrowed it down to a conserved site of approximately 25 bp that contains a consensus binding site for ZFP143, a seven-zinc finger transcription factor. In gel retardation assays, this region bound a factor of approximately 100 kDa that we biochemically purified and identified by mass spectrometry as being indeed ZFP143. In vivo binding of ZFP143 to its bona fide binding site in the C/EBPα promoter was also detected by chromatin immunoprecipitation assays. We are now studying the in vivo effect of ZFP143 on C/EBPα transcription in mice that carry an inducible gene trap in the ZFP143 locus. While mice homozygous for the active gene trap - that prevents ZFP143 transcription - die at an early embryonic stage, preliminary data from heterozygous mice shows indeed a reduction of C/EBPα mRNA levels, suggesting a role for ZFP143 in C/EBPα transcription activation. Ongoing experiments aim at selectively inactivating ZFP143 in the hematopoietic system using the Mx1-Cre / loxP system.
Evi-1 Promotes Para-Aortic Splanchnopleural Hematopoiesis through Up-Regulation of GATA-2 and Repression of TGF-β Signaling.
Authors: Sato Tomohiko , Goyama Susumu , Kurokawa Mineo
Published: October 2019

The ecotropic viral integration site-1 (Evi-1) gene was first identified as a common locus of retroviral integration in myeloid tumors in mice. Evi-1 is highly expressed in cases with human acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) as a consequence of chromosomal rearrangements involving 3q26, where Evi-1 is mapped. Mice deficient in Evi-1 die during embryogenesis with widespread hypocellularity, hemorrhaging, and disruption in the development of the heart, somite, and neural crest-derived cells. It was recently reported that Evi-1 is expressed at a high level in the para-aortic splanchnopleural (P-Sp) region, from which definitive hematopoiesis originates. Hematopoietic stem cells (HSCs) in Evi-1-deficient embryos are decreased in number with defective proliferation capacity, and this defective proliferation of Evi-1-deficient P-Sp cells is successfully rescued in vitro by retroviral transfer of GATA-2. However, detailed molecular mechanisms underlying Evi-1-mediated hematopoiesis remain to be elucidated. In the present study, we used a coculture system of cells derived from the P-Sp region with a layer of a stromal cell line OP9, on which hematopoietic cell development is efficiently induced. In this culture system, Evi-1-deficient P-Sp-derived cells showed severely decreased colony forming capacity. This defect was overcome by reactivating Evi-1 retrovirally. Using this assay, we examined a hematopoietic potential of a series of Evi-1 mutants and found that the first zinc finger domain and the acidic domain are required for the hematopoietic rescue of Evi-1-deficient P-Sp cells. These two domains were also related to some extent for GATA-2 up-regulation, suggesting that GATA-2 is one of the key molecules in Evi-1-mediated hematopoiesis. Furthermore, we found that blocking of TGF-β signaling is also able to recover the hematopoietic defect of Evi-1-deficient P-Sp cells. These findings suggest that Evi-1 promotes hematopoietic stem/progenitor expansion during embryogenesis through at least two pathways: up-regulation of GATA-2 and inhibition of TGF-β signaling.
Single-Stranded DNA-Binding Proteins (SSBPs) Regulate the Abundance of the LIM-Homeodomain Protein LHX2 and Augment Its Transcriptional Activity.
Authors: Cai Ying , Xu Zhixiong , Nagarajan Lalitha , Brandt Stephen J.
Published: October 2019

A small family of proteins with putative single-stranded DNA-binding activity has been shown to augment the biological actions of LIM-homeodomain (LIM-HD) transcription factors through the mediation of the LIM domain-binding protein LDB1. We recently established that two of these SSBPs, Ssbp2 and Ssbp3, were components of an E-box-GATA DNA-binding complex in murine erythroid progenitors containing transcription factors Tal1, E2A, and Gata-1 and LIM-only protein Lmo2 and showed that Ssbp2 stimulated E box-GATA DNA-binding activity by inhibiting Ldb1 ubiquitination and Ldb1 and Lmo2 turnover (Genes & Dev.21:942–955, 2007). Since LIM-HD proteins are substrates of different E3 ubiquitin ligases than LIM-only proteins and have the additional property of binding DNA, we sought to determine the effect of SSBPs on LIM-HD expression and function. Using the prototype LIM-HD protein Lhx2 and one of its best-characterized target genes, Cga, for analysis, we found that an Ssbp3-, Ldb1-, and Lhx2-containing complex associated with an Lhx2 binding element in the Cga promoter in vitro and in mouse pituitary cells (alphaT3-1 cell line) in vivo. We then showed that enforced expression of Ssbp2 and Ssbp3 in alphaT3-1 cells increased Lhx2 and Ldb1 protein abundance, Lhx2 DNA-binding activity, and Cga expression and augmented Lhx2 transcriptional activity in an Ldb1-dependent fashion. While Lhx2-Ldb1-Ssbp3 DNA-binding activity increased in Ssbp3- relative to vector-transfected cells, the affinity of this complex for DNA was unaltered. Similar to the effect of Ssbp2 on Lmo2 in murine erythroleukemia (MEL) cells, overexpressed Ssbp3 reduced Lhx2 protein turnover in cycloheximide-treated alphaT3-1 cells without affecting Lhx2 RNA levels. In contrast, knockdown of endogenous Ssbp3, but not Ssbp2 which is expressed at much lower levels in these cells, reduced Lhx2 and Ldb1 abundance, Lhx2 DNA-binding activity, Lhx2, Ldb1, and Ssbp3 loading onto the Cga promoter, Cga promoter activity, and endogenous Cga gene expression. Significantly, neither overexpression nor knockdown of Ssbp2 in MEL cells, which express both the LIM-only protein Lmo2 and LIM-HD protein Lhx2, affected Lhx2 protein abundance, and Lhx2 DNA-binding activity was undetectable in nuclear extracts from these cells despite the presence of immunoreactive Lhx2. These studies indicate that SSBP augmentation of LIM-HD function results from Ldb1-mediated inhibition of LIM-HD protein turnover and increased assembly of a LIM-HD/LDB1/SSBP DNA-binding complex. The much greater affinity for LDB1 of LIM-only compared to LIM-HD proteins is likely a major determinant of the SSBP effect on LIM-HD protein abundance. Finally, these findings are consistent with cell type-specific contributions of different SSBPs, even for similar LDB1-dependent actions.
Human FLT3/FLK2 Targets Hematopoietic Stem Cells and Granulocyte/Macrophage Progenitors.
Authors: Kikushige Yoshikane , Yoshimoto Goichi , Miyamoto Toshihiro , Ishikawa Fumihiko , Iwasaki Hiromi , Akashi Koichi
Published: October 2019

FLT3/FLK2, a member of the receptor tyrosine kinase family, plays a critical role in maintenance of hematopoietic homeostasis, and the constitutively active form of the FLT3 mutation is one of the most common genetic abnormalities in acute myelogenous leukemia. In murine hematopoiesis, Flt3 is not expressed in self-renewing long-term hematopoietic stem cells (LT-HSCs), but its expression is restricted to the multipotent and the lymphoid progenitor stages at which cells are incapable of self-renewal. In order to test whether Flt3 expression can delineate such a developmental pathway also in human hematopoiesis, we have analyzed the expression of human Flt3 (hFlt3) in prospectively-purified human stem and progenitors (PNAS 2002) by utilizing 7-color FACS and a highly efficient xenograft systems. We have found that Flt3 expression in early hematopoiesis is completely different between human and mice: hCD34+hCD38-hCD90+Lin-LT-HSCs capable of long-term reconstitution in xenogeneic hosts uniformly express hFlt3, and its expression is upregulated through hCD34+hCD38+hCD45RA-hCD123+Lin-common myeloid progenitors (CMPs) to hCD34+hCD38+hCD45RA+hCD123+Lin-granulocyte/macrophage progenitors (GMPs), but hCD34+hCD38+hCD45RA-hCD123-Lin- megakaryocyte/erythrocyte progenitors (MEPs) shut off its expression in human. Furthermore, we have also demonstrated that hFlt3 signaling can prevent stem and progenitors from apoptotic cell death in vitro without any effects on lineage fate decision. Next, we tried to find key molecules for Flt3-Flt3 ligand (FL)-mediating anti-apoptotic effect. First, we tested expression pattern of anti-apoptotic Bcl-2 family genes in HSCs, CMPs, GMPs, MEPs and common lymphoid progenitors (CLPs) in human hematopoiesis. Mcl-1, an indispensable survival factor for murine hematopoiesis (Science, 2005), was also expressed at the highest level in human HSCs, whereas Bcl-2 and Bcl-xL was highly expressed in GMPs and MEPs, respectively. Next, we examined whether FL stimulation can upregulate the expression of Bcl-2 family genes in human purified HSCs and progenitors. FL significantly upregulated the expression of Mcl-1, but not of Bcl-2 or Bcl-xL in HSCs as well as CMPs and GMPs. In conclusion, our data show that the distribution of Flt3 is quite different in mouse and human hematopoeisis. Human Flt3 targets LT-HSCs and myeloid progenitors except for MEPs. Flt3 signaling might support cell survival in early hematopoiesis including the HSC and the myeloid progenitor stages through upregulation of Mcl-1. This is a striking example that the expression pattern of key molecules could be significantly different between human and mouse.
N-cadherin Expression Level Distinguishes Reserved Versus Primed States of Hematopoietic Stem Cells.
Authors: Haug Jeffrey S. , He Xi C. , Grindley Justin C. , Wunderlich Joshua P. , Gaudenz Karin , Ross Jason T. , Paulson Ariel , Wagner Kathryn P. , Xie Yucai , Zhu Ruihong , Yin Tong , Perry John M. , Hembree Mark J. , Redenbaugh Erin P. , Radice Glen L. , Seidel Christopher , Li Linheng
Published: October 2019

Within the bone marrow, three hematopoietic stem cell niches have been identified; the osteoblastic niche (Arai et al., 2004; Calvi et al., 2003; Nilsson et al., 2001; Zhang et al., 2003), the vascular niche (Kiel et al., 2005), and the CAR cell niche (Sugiyama et al., 2006). The adhesion molecule N-cadherin has been found associated with the osteoblastic and CAR cell niches, implicating N-cadherin’s function for hematopoietic stem cell (HSC) anchoring and signaling (Arai et al., 2004; Muguruma et al., 2006; Zhang et al., 2003). However, as of yet, a HSC population expressing N-cadherin has not been fully characterized. Therefore, we examined how N-cadherin expression in HSCs relates to their function and lifecycle. Unexpectedly, we found that doses of 5000 bone marrow cells expressing the highest level of N-cadherin (N-cadherinhi) failed to reconstitute hematopoietic lineages in irradiated recipient mice. An explanation for this engraftment failure came with detailed cell surface phenotyping which revealed that these N-cadherinhi cells were primarily Lineage+ and devoid of the characteristic hematopoietic stem pool, Lineage-Sca+cKit+ (LSK) cells. Instead, we found that Flk2-LSK HSCs express a gradient of N-cadherin which could be described as low (N-cadherinlo) to intermediate (N-cadherinint) levels (Figure1).

FACS applications were used to isolate pure populations of these N-cadherinlo and N-cadherinint Flk2-LSK HSCs. Real time RT-PCR (N-cadherin primers crossed the intron between Exon 2 and 3).

(Figure 2), microarray studies, and competitive reconstitution transplantation assays revealed that this N-cadherin division of Flk2- LSK HSCs formed two populations with very distinct properties. In transplantation assays the N-cadherinlo population more robustly reconstituted the hematopoietic system. Principle Component Analysis and gene ontology analysis of microarray data revealed that the N-cadherinlo cells expressed genes that may prime them to respond to signals and to mobilize. This data was confirmed with mobilization studies which showed that HSCs mobilized from bone marrow to spleen were predominantly N-cadherinlo. In contrast, the expression profile of N-cadherinint cells suggests they are more ‘reserved’ and this population was also maintained with HSCs spared by 5-fluouracil (5FU) treatment. Our results suggest that differential N-cadherin expression reflects important functional distinctions between HSC subpopulations. N-cadherinlo HSCs, with their robust reconstitution efficiency and properties similar to mobilized HSCs, may have clinical relevance.

Figure Figure Figure Figure
Junctional Adhesion Molecule-A (JAM-A/JAM-1/F11R) Marks Long-Term Repopulating Hematopoietic Stem Cells.
Authors: Sugano Yasuyoshi , Takeuchi Masaki , Hirata Ayami , Matsushita Hirokazu , Kitamura Toshio , Tanaka Minoru , Miyajima Atsushi
Published: October 2019

Junctional adhesion molecule-A (JAM-A/JAM-1/F11R) is a cell adhesion molecule expressed in epithelial cells, endothelial cells and also hematopoietic cells such as leukocytes, platelets and erythrocytes. However, the expression of JAM-A in hematopoietic stem cell (HSC) had not been known. Here we show that JAM-A is expressed at a high level in the enriched HSC fraction, i.e. CD34+ c-Kit+ cells in embryonic day 11.5 (E11.5) aorta-gonod-mesonephros (AGM) and E11.5 fetal liver (FL) as well as c-Kit+ Sca-1+ Lineage- (KSL) cells in E14.5 FL, E18.5FL and adult bone marrow (BM). While the percentage of JAM-A+ cells in those tissues decreases during the development, the expression in HSC fraction is maintained throughout life. In fact, c-Kit+Sca-1+ cells are enriched approximately 200-fold from whole BM cells by anti-JAM-A antibody alone, i.e. the c-Kit+Sca-1+ cells in whole BM was about 0.15% and that in JAM-A+ cells was about 30%. Colony forming assays reveal that multi-lineage colony forming activity (CFU-mix) in JAM-A+ cells is higher than that in JAM-A- cells in the enriched HSC fraction in all those tissues. HSC transplantation assay revealed that long-term repopulating HSC (LTR-HSC) activity is present in the mice received 100 JAM-A+ KSL (7/9) cells and 300 KSL cells (5/6). Since the ratio of JAM-A+ cells to JAM-A- cells in the KSL fraction is about 6.5:3.5, 100 JAM-A- KSL cells are equivalent to 285 total KSL cells. In contrast to JAM-A+ cells, the mice received 1000 JAM-A- KSL cells, which are equivalent to more than 2850 total KSL cells, failed to engraft for long-term (0/6). These data revealed that long-term repopulating HSC (LTR-HSC) activity is present exclusively in the JAM-A+ cells, but not in JAM-A- cells. Moreover only 100 JAM-A+ cells isolated from whole BM cells by anti-JAM-A antibody alone reconstituted the hematopoietic system for long-term (4/7). Together these results indicate that JAM-A is expressed on hematopoietic precursors in various hematopoietic tissues and is an excellent and convenient marker to enrich LTR-HSC from BM cells.
Addition of Rituximab Improves Progression-Free and Overall Survival in Patients with B-Cell Lymphoma Receiving Doxorubicin-Containing Chemotherapy
Authors: Sakai Tatsunori , Nagai Hirokazu , Watanabe Tomoyuki , Uike Naokuni , Okamura Seiichi , Yano Takahiro , Kawano Fumio , Hanada Shuichi , Sunami Kazutaka , Ikeda Hirokazu , Sawamura Morio , Nishiura Tetsuo , Hotta Tomomitsu , Horibe Keizo
Published: October 2019

Background: Many clinical trials have documented the efficacy of rituximab in B-cell lymphomas. However, it remains unclear whether rituximab will improve long-term prognosis of the patients. This multicenter retrospective cohort study was conducted to evaluate the clinical impact of the drug in the treatment of B-cell lymphomas. Study design: We retrospectively analyzed clinical characteristics, 2-year progression-free survival(2y PFS) and 2-year overall survival(2y OS) of all B-cell lymphoma patients who were newly diagnosed and initially treated with either CHOP-like regimen alone (R(−) group) or in combination with rituximab (R(+) group) between 2000 and 2004 at our 20 hospitals belonging to the National Hospital Organization. Since rituximab was approved in 2002 for indolent B-cell lymphomas and in 2003 for aggressive B-cell lymphomas in Japan, the patients were almost automatically divided into the 2 groups dependent on the year they underwent treatment.

Results: Of the 1,072 patients enrolled, 335 were given rituximab, while 737 did not receive it for the initial induction therapy. 2y PFS was 74.3% and 61.2% for R(+) group and R(−) group, respectively(P<.0001). 2y OS also significantly improved with the addition of rituximab, 86.6% vs 65.3%(P<.0001). In 746 patients with diffuse large B-cell lymphoma, R(+) group (183 patients) and R(−) group (563 patients) showed 2y PFS of 71.6% and 62.2% (P=.0017), 2y OS of 78.5% and 62.5% (P<.0001) respectively. In 195 patients with follicular lymphoma, R(+) group (104 patients) and R(−) group (91 patients) exhibited 2y PFS of 80.1% and 64.5% (P<.0001), 2y OS of 94.9% and 81.5% (P<.0001), respectively.

Conclusion: The addition of rituximab to the chemotherapy regimen significantly improves the clinical outcome in patients with previously untreated B-cell lymphoma regardless of the clinical aggressiveness.
Non-Pegylated Lyposomal Doxorubicin, Cyclophosphamide, Vincristine, Prednisone and Rituximab (R-COMP) as Initial Treatment for Patients with Splenic Marginal Zone Lymphoma (SMZL). A GISL Study
Authors: Iannitto Emilio , Luminari Stefano , Mammi Caterina , Minardi Viviana , Merli Francesco , Angrilli Francesco , Stelitano Caterina , Cesaretti Marina , Franco Giovanni , Carella Angelo M. , Federico Massimo
Published: October 2019

Background: SMZL is an indolent lymphoma, presenting with massive splenomegaly generally associated with intrasinusoidal bone marrow infiltration. The encapsulation of doxorubicin into non-pegylated liposomes allows targeting of the drug to affected organs including spleen, lymphnodes and bone marrow.

Methods: In 2005 the GISL started a phase II study for the treatment of patients with histologically confirmed SMZL, investigating safety and clinical profile of 6 courses of a modified R-CHOP regimen in which standard doxorubicin was substituted with non pegylated lyposomal doxorubicin (NPLD) used at the same doses (50mg/m2) (R-COMP). Main inclusion criteria were age > 18 yrs, normal cardiac function and active disease (at least one of the following; Hb <10g/dl; plt <100.000/mmc, symptomatic splenomegaly, elevated LDH, B symptoms, extrasplenic disease, LDT <12 months). Splenectomy was allowed prior to treatment start only in case of symptomatic spleen enlargement. The study was planned according to a two-stage Simon design using overall response rate as primary endpoint. We present the results of the analysis performed after the completion of study stage 1. The access to stage 2 is allowed if at least 12 responses are recorded among among the first 19 evaluable cases.

Results: As of January 2007, 20 patient were enrolled with the following characteristics; median age 63 years (30–80), Hb <10g/dl in 20%, Lymphocytes > 5000/mmc in 65%, elevated LDH in 65%; 2 patients were splenectomized before treatment start. One patient was not available for response assessment. A clinical response was observed in all remaining 19 cases (ORR 100%); 12 (63%) cases obtained a CR. So far only one patient progressed at +1 month from treatment. Treatment was well tolerated with grade III/IV neutropenia in 8 patients (42%), grade III pulmonary toxicity in 1 patient and grade II peripheral neuropathy in 5 cases. In one case a reversible cardiac failure was reported after 1 month from the end of treatment.

Conclusions: In conclusion 6 cycles of R-COMP combination represent a safe and promising treatment option for patients with clinically active SMZL.
A Variant Allele of the Gene Growth Factor Independence 1 (GFI1) Is Associated with Acute Myeloid Leukemia.
Authors: Khandanpour Cyrus , Thiede Christian , Nückel Holger , Lohmann Dietmar , Horsthemke Bernhard , Siffert Winfried , Neubauer Andreas , Grzeschik Karl-Heinz , Ehninger Gerhard , Dührsen Ulrich , Möröy Tarik
Published: October 2019

Acute myeloid leukemia (AML) is characterized by an impaired differentiation and abnormal growth of myeloid-progenitor cells, resulting in an accumulation of malignant blast cells in the bone marrow. It represents 1% of all malignancies with overall incidence of 4.5 per 100 000 adults. About 10% of AML patients carry a chromosomal translocation t(8;21) that leads to the production of an AML1/ETO fusion protein. We have previously shown that mice lacking the transcription factor Gfi1 (Growth factor independent 1) show an accumulation of aberrant myelo-monocytic cells reminiscent of myeloproliferative disorders in patients and that Gfi1 is required to maintain the self-renewal and differentiation capability of hematopoietic stem cells. We show here that a variant allele of the gene encoding the transcriptional repressor GFI1, (GFI136N), is associated with AML in three independent Caucasians patient cohorts with an odds-ratio of 2.89 (p=2 x 10–5). The GFI136N variant occurred in 469 AML patients with an allele frequency of 0.064 compared to 0.023 in 503 healthy controls. Patients homozygous for the common form of GFI1 (GFI136S) and patients either heterozygous or homozygous for GFI136N did not differ with regard to the occurrence of AML with specific FAB subtypes, cytogenetic findings or Nucleophosmin 1 mutations. The variant GFI136N did not alter disease progression of AML overall, but correlated with a worse prognosis for patients carrying the t(8;21) chromosomal translocation. Here, the 5 year survival rate was 64% for GFI136S homozygous patients compared to 40% for patients carrying at least one allele of GFI136N. The sub-nuclear localization of GFI136S or GFI136N was different when transiently expressed in 3T3 cells. GFI136S was located mainly at the nuclear border, whereas GFI136S was distributed in a dotted pattern in the nucleus. In addition, we found that GFI136S, but not the variant GFI136N protein co-localized with AML1/ETO in the nucleus. Both GFI136S and GFI136N were able to repress transcription in established reporter gene assays. However, whereas AML/ETO was able to inhibit this activity of GFI136S, the variant GFI136N form was refractory to this inhibition by AML1/ETO, suggesting that the AML1/ETO-GFI1 interaction may be one of the factors responsible for a favorable outcome of t(8;21) positive AML. Our study indicates that the GFI136N variant allele may serve as a novel genetic marker to measure genetic predisposition for the development of AML and provides a first indication that this specific genetic variation at the Gfi1 locus affects the clinical outcome of t(8;21) positive AML patients.
No Difference in Risk of Recurrent Venous Thrombosis between Men and Women. Results from a Family Cohort Study in 3356 Subjects.
Authors: Lijfering Willem M. , Coppens Michiel , Veeger Nic J.G.M. , Middeldorp Saskia , Hamulyak Karly , Prins Martin H. , Buller Harry R. , van der Meer Jan
Published: October 2019

Recent studies have suggested that men are at higher risk of recurrent venous thrombosis than women. It has been proposed to continue anticoagulant treatment after the first episode of thrombosis for a longer period in men, and to search for unidentified prothrombotic abnormalities in men that could explain these findings. However, men and women differ with respect to hormonal risk factors (pregnancy, oral contraceptives), and known thrombophilic defects did not show to increase the risk of recurrence. Moreover, follow-up in previous studies was relatively short (maximum 8 years). We analyzed sex differences in risk of recurrence in a family cohort of 3356 subjects (877 probands, 2479 relatives), who enrolled 5 studies to assess the absolute risk of venous thrombosis associated with currently known thrombophilic defects. Life-time risk of recurrence was calculated, related to sex, follow-up time, type of first and recurrent event (provoked, idiopathic), and thrombophilic defects. Of 816 subjects with a first episode of venous thrombosis, 337 (41%) had a recurrence after discontinued anticoagulant treatment. Overall relative risk of recurrence was 1.8 (95% CI, 1.5–2.3) in men compared to women. Women were younger at time of their first event (mean 34 years vs. 44 years, P< 0.001) and at time of recurrence (40 years vs. 48 years, P< 0.001). Excluding provoked (recurrent) venous thrombosis, the relative risk was 1.2 (95% CI, 0.8–1.7), while mean age at recurrence was comparable in men and women (50 years vs. 49 years, P= 0.595). Women revealed recurrence after a longer period than men (P=0.003), but this difference was not demonstrated if only idiopathic events were considered (P=0.938) (Figure). Recurrences were observed in 111/187 subjects (59%) with antithrombin, protein C or protein S deficiency, compared to 159/479 subjects (33%) with factor V Leiden, prothrombin G20210A or high factor VIII levels (P< 0.001). Within these subgroups, the risk of recurrence after an idiopathic event was comparable in men and women. We conclude that life-time risk of recurrent venous thrombosis is not different between men and women. A difference can be observed when the follow-up period is relatively short and is explained by a younger age of women at time of the first episode of venous thrombosis due to pregancy/puerperium and oral contraception and a longer interval between provoked first episode and recurrence in women.

Figure Figure
Efficacy and Safety of a New Intravenous Immunoglobulin Product in Patients with Chronic Immune Thrombocytopenic Purpura.
Authors: Robak Tadeusz , Salama Abdulgabar , Kovaleva Lidia , Vyhovska Yaroslava I. , Davies Simon , Mazzucconi Maria G. , Zenker Othmar
Published: October 2019

Background Intravenous immunoglobulin (IVIG) is an accepted treatment for immune thrombocytopenic purpura (ITP). A new liquid 10% human IgG preparation stabilized with proline at a pH of 4.8 (trade name: Privigen) was recently developed. It can be stored at room temperature and is therefore always ready to use. Here we report its efficacy and safety in patients with chronic ITP.

Patients and Methods Fifty-seven patients with chronic ITP and a platelet count below 20 x 109/L were included in this open-label, single arm, multi-center Phase III trial. IVIG was given at a dose of 1 g/kg on 2 consecutive days at a maximum infusion rate of 4 mg/kg/min. A subset (56.1%) of the patients received premedication (acetaminophen or diphenhydramine) to avoid adverse events. The primary endpoint was the platelet response rate, defined as the percentage of patients showing an increase in platelet count to ≥50 x 109/L within 7 days of the first infusion. Secondary endpoints included platelet counts at specified time points, time to platelet response, duration of platelet response, and regression of hemorrhages at different bleeding sites. Safety was evaluated by the frequency and severity of adverse events.

Results The primary endpoint, an increase in platelet counts to ≥50 x 109/L, was achieved by 81% of the subjects (95% CI: 69–89%). The highest median platelet count (149 x 109/L) was observed on day 8. Median time to response was 2.5 days, with 43% of subjects responding within 1 day. Median duration of platelet response (days with platelet count ≥ 50 x 109/L) was 15.4 days. Regression rates for various bleeding sites ranged from 78% to 100%. Regression of bleeding correlated with increases in platelet counts. Adverse events were reported in 52 (91%) subjects. The most common adverse event was headache (67%), the incidence and severity of which was attenuated by premedication with acetaminophen/diphenhydramine. There were 3 serious adverse events, one of which (aseptic meningitis) was considered related to the study medication.

Conclusions The present study has shown the safety and significant efficacy of a novel, 10% liquid, ready for use IVIG preparation, in patients with chronic ITP. A rapid increase in platelet counts to a level where severe bleeding episodes become more unlikely was seen in the majority of patients, as expected with IVIG given at 1 g/kg on 2 consecutive days. The increase in platelet counts was associated with a regression of bleeding. Adverse events were generally mild to moderate in severity, corresponded to those expected with IVIG, and could be prevented with premedication.
A Phase 2 Open-Label, Sequential-Cohort, Dose-Escalation Study of AMG 531 in Japanese Patients with Chronic Immune Thrombocytopenic Purpura (ITP).
Authors: Shirasugi Yukari , Ando Kiyoshi , Hashino Satoshi , Nagasawa Toshiro , Kurata Yoshiyuki , Kishimoto Yuji , Iwato Koji , Sonehara Yusuke , Ohkura Masayuki , Ohtsu Tomoko , Nichol Janet L.
Published: October 2019

AMG 531 is a novel thrombopoiesis-stimulating peptibody that is being studied for its ability to increase platelet production by stimulating the thrombopoietin (TPO) receptor. This phase 2 study was conducted to identify the appropriate starting dose of AMG 531 for treatment of chronic ITP in adult Japanese patients. The study consisted of 2 phases: a 2-week cohort dose-escalation phase to determine the starting dose for subsequent phase 3 evaluation, and a treatment continuation phase (that lasted until completion of the dose-escalation phase) which provided continuation of treatment for those with a platelet response. Patients in the continuation phase received the dose of AMG 531 they had received in the dose-escalation phase with the option for subsequent dose adjustments. Twelve patients were enrolled with a mean platelet count of 11.8x109/L and a median age of 60.5 years (range 32 to 63); 8 were female. Four patients enrolled into each of 1μg/kg, 3μg/kg, or 6μg/kg dose cohorts and received AMG 531 by subcutaneous injection on days 1 and 8 with no dose adjustments. Cohort dose escalation was to be stopped in the event of an observed platelet count >1000x109/L. Comparison of dose cohorts showed that the proportion of patients achieving a platelet response (doubling of baseline counts and ≥50x109/L) was greater in cohorts receiving higher doses of AMG 531 (see Table). A dose response was also observed in the mean peak platelet counts, the mean fold changes from baseline in peak platelet counts, and the maximum platelet count. Because one patient in the 6μg/kg cohort had an excessively high platelet count (980x109/L), this dose was eliminated from consideration as the starting dose in phase 3 and further dose escalation to 10μg/kg dose cohort was stopped. Five of 7 eligible patients (3μg/kg, 1/4; 6μg/kg, 4/4) elected to enter the treatment continuation phase. There were no study withdrawals, and no serious adverse events (AEs) were reported during the study in either phase. The most common treatment-related AE in the cohort phase was headache (25%), and in the treatment continuation phase were arthralgia, contact dermatitis, and malaise (each 20%). No patients received rescue medications during the entire treatment period. No antibodies against either AMG 531 or endogenous TPO were detected. AMG 531 was well-tolerated and produced a dose-responsive increase in platelet counts. The phase 3 study in Japanese patients with ITP will be initiated with a starting dose of 3μg/kg based on the outcome of this study.

Key Measures of Platelet Response
Methylen Blue Photoinactivated Plasma vs Fresh Frozen Plasma in Thrombotic Thrombocytopenic Purpura Treatment: A Multicentric Prospective Cohort Study.
Authors: Alvarez-Larrán Alberto , Del Río-Garma Julio , de la Rubia Javier , Zamora Concepción , Galmés Antonio , Ramírez Consuelo , Rodríguez-Vicente Pilar , Martínez Clara , Alegre Adrian , Viejo Aurora , Arbona Cristina , Serrano Alfons , Castellá María Dolors , Corral Mercedes , Peña Francisco , Pereira Arturo
Published: October 2019

Plasma exchange (PE) with plasma infusion is the treatment of choice for Thrombotic Thrombocytopenic Purpura (TTP) but doubts remain as to whether all kinds of plasma are equally effective. Since October 2004, a multicentric prospective cohort study is being conducted in Spain to compare Methylene Blue Photoinactivated Plasma (MBPIP) with Fresh Frozen Plasma (FFP) in the treatment of TTP. Sixty-three first episodes of idiopathic TTP were included. MBPIP was used in 38 and FFP in 25. The treatment schedule consisted of daily PE (> 40 mL/kg of body weight) and costicosteroids (1.5 mg/kg/d). Response was defined as the achievement of a platelet count >= 150 x 109/L for at least three consecutive days, normal LDH level and absence of TTP-related symptoms and signs. Recurrence was defined as a fall in platelet count below 50 x 109/L or below 50% of the highest count achieved after response. Response lasting for more than 15 days was considered as remission. The prognostic significance of the kind of plasma used was investigated by logistic regression analysis after adjustment for other variables that had previously been found to influence on response to PE (gender; the Rock score; days from first medical attendance to PE; volume of plasma infused in the first 7 days of treatment). Both groups were comparable with regard to clinical and biological parameters at diagnosis. A severe deficit in ADAMTS13 activity was found in 9 out 12 (75%) patients treated with MBPIP and in 12 out of 16 (75%) patients treated with FFP. When compared to FFP, patients treated with MBPIP required a higher number of PEs (16±13 vs 9±7, p=0.004) and a larger volume of plasma (763±678 ml/kg vs 413±326 ml/kg, p=0.02) to achieve a remission and presented more recurrences while on PE treatment (21 out of 38 vs 6 out of 25, p=0.02). Splenectomy or rituximab was required in eight patients (21%) treated with MBPIP to achieve a remission vs in none out of the 25 patients treated with FFP (p= 0.02). After adjustment for other prognostic variables, patients in the MBPIP group had a lower likelihood of remission by the 8th treatment day (OR: 5.1; 95% CI: 1.6 – 15.9) and a higher risk of recurrence while on PE treatment (OR: 4.2; 95% CI: 1.3 – 13.5). In conclusion, MBPIP is less effective than FFP in the treatment of TTP.
Initial and Long-Term Outcomes of Splenectomy in Adult Immune Thrombocytopenic Purpura (ITP) Patients: A Systematic Review and Meta-Analysis.
Authors: Mikhael Joseph , Kessler Craig M. , Danese Mark , Deuson Robert
Published: October 2019

Splenectomy serves as a common therapy in treating adults with chronic ITP. Laparoscopic splenectomy was introduced in 1991 with the goal of reducing complication rates. This study was designed to estimate both the initial surgical non-response rate and the long-term relapse rate of splenectomy in the post-laparoscopic era. A systematic literature review was conducted by searching PubMed for articles published between January 1, 1991 and October 3, 2006. Selection criteria included: chronic ITP, study enrollment in 1990 or later, ≥12 months of follow-up, ≥15 patients with ITP, ≥75% of patients at least 14 years of age, not HIV positive, and not undergoing a second splenectomy. Response rates for surgery were collected according to an author-defined platelet response criterion (platelet counts <50x109/L or <30x109/L in 75% of studies). Relapse rates post-splenectomy (using the same platelet response criteria) were also collected. A meta-analysis was conducted to estimate a pooled surgical non-response rate as well as a pooled relapse rate in responders by weighting the study-specific estimates by the inverse of their variances, using Stata 9™. The pooled relapse rate was converted to a probability using the exponential formula, e(-rate x time). We identified 161 articles for detailed review. Of these, 24 met our inclusion criteria, all of which were observational studies. These studies represent 1,138 laparoscopic splenectomies (66 or 5.8% were converted to open splenectomy during surgery), and 317 open or undefined splenectomies. The average surgical non-response rate across 21 studies reporting data was 12.2% (95% CI: 10.3–14.3%). The average long-term relapse rate per person year across all studies was 53 per 1,000 patient years (95% CI: 45.0–62.0%). This corresponds to a 5% per year failure rate, or 32% failure rate after 5 years, for all patients initiating surgery. Initial non-response rate to planned splenectomy is 12% in adults with ITP. For patients undergoing laparoscopic splenectomy, 6% required conversion to open splenectomy. The 5 year relapse rate post-splenectomy is 32%. Splenectomy, although the current standard of care, is not always initially successful and does not provide a durable response in all patients. The benefits and risks of splenectomy must be weighed in light of newer options of therapy currently available.

Relapse Rate per 1,000 Patient-years of Follow-up for Each Study Relapse Rate per 1,000 Patient-years of Follow-up for Each Study
Health-Related Quality of Life (HRQoL) in Patients with Chronic Immune Thrombocytopenic Purpura (ITP): Interim Results from an Open-Label Study of AMG 531.
Authors: Tarantino Michael D. , Henry David H. , Go Ronald S. , Lefrere Francois , Lichtin Alan E. , Terebelo Howard R. , Viallard Jean-Francois , Mathias Susan D. , Deeter Robert , Nie Kun , Nichol Janet L.
Published: October 2019

The ITP Patient Assessment Questionnaire (ITP-PAQ) was developed to complement clinical data by assessing multiple facets of disease-specific HRQoL in adult patients with chronic ITP (Mathias et al., Health Qual Life Outcomes, 2007). Here we present interim results from an ongoing, open-label extension study that will evaluate the ability of AMG 531 to improve their HRQoL in the long-term. AMG 531 is a novel thrombopoiesis-stimulating peptibody that is being studied for its ability to increase platelet production by stimulating the thrombopoietin receptor. Adult chronic ITP patients receiving AMG 531 completed the ITP- PAQ at baseline, and weeks 4, 12, 24, and every 12 weeks thereafter. The ITP-PAQ consists of 44 items and 10 scales. Data have been assessed for this interim analysis through week 48, a time-point with reasonable patient numbers at the time of the data cut-off. Mean scores at each assessment, and changes in mean scores from baseline to week 48 were calculated, with positive changes in mean score indicating improvement in HRQoL. Compliance rates for the ITP-PAQ were 95% at baseline, 93% at week 4, and 83% at week 48. Over the 48-week period, results demonstrated an upward trend for the scale scores (see Table). The greatest improvements were reported in the Symptoms, Bother, Activity, Reproductive Health, and Overall Quality of Life scales, particularly during the first 4 weeks of the study. Changes in mean score from baseline to week 48 were significantly increased for Symptoms, Bother, Fatigue, Activity, Fear, Psychological Health, Work, Social Activity, and Overall Quality of Life. ITP patients receiving AMG 531 had an initial, yet sustained, improvement in ITP-PAQ scores. Subsequent analyses of results collected in this ongoing study will be used to substantiate our findings, and the therapeutic benefit of AMG 531 on HRQoL in patients with chronic ITP will be provided by correlations with clinical data.

ITP-PAQ Scale Scores a by Assessment and Change in Mean Scores from Baseline