bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet Birth.
Authors: Cramer-Bordé Elisabeth , Morison Ian M. , Capron Claude , Dunois-Lardé Claire , Baruch Dominique
Published: October 2019

How and where human platelets get born are two questions still harboring great mystery. In vivo and in vitro approaches have contributed to a better understanding of the phenomenon. The recent study of a family with congenital autosomal-dominant thrombocytopenia showed ectopic platelet release in the bone marrow of the affected individuals. Further, molecular studies identified a new mutation in the gene encoding cytochrome c, which yields a cytochrome c with enhanced apoptotic activity. It is known that platelet birth occurs when megakaryocytes (MKs) undergo compartmentalized apoptotic activity. In this family case, enhanced apoptosis in MKs led to the premature release of platelets in the bone marrow, preventing their natural birth into the circulation after MK migration and causing thrombocytopenia. A similar finding, i.e., platelet birth in the marrow, was made in Wiskott Aldrich syndrome, providing a pathophysiological explanation for the thrombocytopenia, related to impaired migration capacities of MKs upon SDF chemotaxis. Indeed, direct platelet delivery in the human bone marrow space is not currently observed. However, maturing MKs are located close to the marrow sinusoids whose barrier they are able to cross, and entire MKs have been seen in the circulating blood where they become exposed to circulatory shear forces. Since the rate of platelet shedding in culture systems is remarkably low, we hypothesised that a missing element for efficient platelet production in static culture systems would be shear stress. Thus, we exposed human MKs, cultured in vitro until full maturation, to a substrate of matrix protein and high shear rates equivalent to those encountered in capillaries and small arteries. Cells were observed by real-time videomicroscopy, immunofluorescence, and electron microscopy. Shear forces specifically induced a sequence of morphological changes, converting mature MKs into proplatelets, and platelets, which consistently detached from the mother cells into the flow,, at a rate 20 times higher than static culture conditions. Using specific antibody inhibition, we showed the major involvement of GPIb in platelet formation, since its blockade inhibited MK adhesion and subsequent platelet formation. In addition, aIIbb3 was essential for firm MK anchorage, which was required for subsequent platelet formation. These experiments show that MK exposure to high shear rates promotes platelet production via GPIb (and secondarily aIIbb3) interactions with vascular matrix proteins. In conclusion, the above studies converge to present evidence that platelet birth takes place in circulating blood, being regulated by the migratory and apoptotic capacities of MKs and dependent on GPIb and hemodynamic forces.
Live-Animal Tracking of Individual Hematopoietic Stem Cells in Their Niche.
Authors: Scadden David T.
Published: October 2019

Stem cells reside in a specialized, regulatory environment termed “niche” that dictates how they generate, maintain, and repair tissues. We have previously documented in living animals that transplanted stem/progenitor populations (HSPC) localize to sub-domains of marrow microvessels, where CXCL12 is particularly abundant. We have now used high-resolution combined confocal and two-photon video imaging of individual hematopoietic cells in the calvarium bone marrow of living animals over time to examine the relationship of stem and progenitor cells to blood vessels, osteoblasts, and endosteal surfaces as they home and engraft. Osteoblasts were enmeshed in microvessels, and relative positioning of stem/progenitor cells within this complex tissue was non-random and dynamic. Both cell autonomous and non-autonomous factors influenced primitive cell localization. Different hematopoietic cell subsets localized to distinct locations according to the stage of differentiation. In settings of engraftment as well as expansion, marrow stem/progenitor cells were in closer proximity to bone and osteoblasts. The hematopoietic stem/progenitor cell niche is both perivascular and periendosteal in trabecular bone with physiologic demand influencing closer positioning of stem cells to bone.
Notch Signaling Is Required for Mast Cell Development in the Zebrafish.
Authors: Da'as Sahar , Klein Lauren C. , Ferrando Adolfo A. , Berman Jason N.
Published: October 2019

Abstract 3588

Poster Board III-525

The molecular pathways regulating mast cell (MC) development in vertebrates remain to be elucidated. The Notch signaling pathway is highly conserved in all metazoans and has been implicated in regulating hematopoietic stem cell induction and lineage cell fate decisions. Notch receptors and their ligands are expressed in a number of hematopoietic cells, including MCs. We were the first to identify zebrafish MC equivalents (Dobson et al., Blood 2008) and examine vertebrate MC transcriptional regulation in vivo. These studies demonstrated the significance of carboxypeptidase A 5 (cpa5) as a zebrafish MC-specific marker. Co-localization studies reveal zebrafish notch3 (a homologue of human NOTCH3) is expressed in a proportion of cpa5 positive cells in 7 day old embryos. Moreover, the zebrafish Notch signaling mutant, mind bomb, displays profound loss of cpa-5 expression, as do wild type zebrafish embryos treated with Compound E (Cpd E), a gamma-secretase inhibitor that blocks Notch signaling. We previously identified pu.1 and gata2 as essential transcription factors for early MC development. Interestingly, we observed a dose-dependent response, with reduced cpa5 and gata2 but preserved pu.1 expression at 50 μM Cpd E, compared with profound decreased expression of all these factors, as well as gata1 and mpo at 75 μM Cpd E. These data suggest a particular role for Notch signaling in regulating MC development, as well as a potentially broader role in regulating the myeloid and erythroid lineages. These studies are currently being validated through reciprocal experiments overexpressing notch mRNA in wild type embryos and rescue experiments overexpressing the notch intracellular domain and the above-mentioned transcription factors in Notch deficient embryos (mind bomb and Cpd E treated). We have also developed a transgenic zebrafish line expressing the human c-KIT D816V mutation found in systemic mastocytosis, which exhibits increased mast cells at the expense of erythroid cells, features in keeping with the human condition. These transgenic fish provide an opportunity to examine if Notch pathway inhibition alone, or in combination with other therapies, such as those targeting the c-KIT kinase, have a therapeutic impact in this condition. Parallel approaches in a human mastocytosis cell line are also being undertaken. These studies promise key insight into the role of Notch signaling in MC development and the opportunity to use the zebrafish as an in vivo model for identifying novel therapeutic strategies in MC diseases.

Disclosures:

Ferrando: Merck, Pfizer: Research Funding.
Regulation of Hematopoietic Stem Cell Quiescence by Ku70.
Authors: Qing Yulan , Wang Zhengqi , Liu Min , Bunting Kevin D. , Gerson Stanton L.
Published: October 2019

Abstract 3613

Poster Board III-549

Genomic integrity is essential for organism development and longevity, and in large part is mediated by DNA repair proficiency. Non-homologous end-joining (NHEJ) is essential for DNA double-strand break repair in all cells and for VDJ processing in B and T cells. NHEJ is also critical for hematopoietic stem cell (HSC) maintenance and function, but the mechanisms by which the NHEJ pathway regulates HSC function are not known. Ku70 is a key component of NHEJ; Ku70-deficient mice are hypersensitive to radiation and show a leaky SCID phenotype. Previously we have shown that HSC from Ku70-deficient mice are defective in repopulation, self-renewal and competitive repopulation. Also, mice defective in other NHEJ components, Ku80 or ligase 4, display impaired HSC function and reduced HSC numbers during aging. Here we used Ku70-deficient mice to investigate the role of the NHEJ pathway in HSC function.

Ku70-deficient mice show comparable HSC (Sca1+,c-Kit+,Lin-) frequency to Wild-type (WT) mice, and HSCs from Ku70-deficient mice display similar apoptosis rates as HSCs from WT mice. SKL cells from both Ku70-deficient and WT mice proliferate at similar rates in vitro in the presence of IL3, SCF, Flt3-L and Tpo. However, a greater percentage of HSCs from Ku70-deficient mice are actively cycling, and fewer are quiescent than those obtained from WT mice. Further, recipients of Ku70-deficient BM are more sensitive to 5-FU treatment than those receiving WT BM. In addition, WT HSCs efficiently replace endogenous Ku70-deficient HSCs when WT BM cells are transplanted into Ku70-deficient mice in the absence of any conditioning.

Together, our data indicate that loss of quiescence in Ku70-deficient HSCs correlates with dramatic defects observed in repopulation, self-renewal and competitive repopulation. These results suggest that the NHEJ pathway, either through the processes of DNA repair and genome maintenance, or through a stress response signaling pathway, is critical for maintaining HSCs in a quiescent state, avoiding age-related physiological stress, and allowing functional stem cell transplantation.

Disclosures:

No relevant conflicts of interest to declare.
TRAF3 Over-Expression Contributes to the Enhanced Alternative NF-κB Activity in Hodgkin's Lymphoma Cells.
Authors: Guo Feng , Zhou Peng , Ma Liang
Published: October 2019

Abstract 3665

Poster Board III-601

Introduction

Hodgkin and Reed-Sternberg (H-RS) cells are originated from germinal center B cells. Constitutive nuclear factor κB (NF-κB) activation is one of the molecular characteristic futures of H-RS cells. TNFR-associated factors (TRAFs) participate in a wide range of biological processes, such as adaptive and innate immunity, stress response, and bone metabolism, which are mediated by the induction of cell survival, proliferation, and differentiation. Among those, TRAF3 are reported as a negative regulator of the alternative NF-κB signaling pathway in B cells. How TRAF3 functions in H-RS cells is currently unclear.

Methods

Electromobility shift assay (EMSA) was performed to examine the NF-κB activity in B cell-derived Hodgkin's cells (L428 and KM-H2). An ELISA-based NF-κB family transcription factor activity assay was performed to quantify NF-κB DNA-binding in nuclear extracts from L428 cells. p100 processing, the expression of other NF-κB family members in the cytoplasm, and TRAF3 expression were detected by Western blot analysis. The effects of TRAF3 in L428 cells were studied by transient expression of TRAF3 expression vector.

Results

In this study, we found that TRAF3 was minimally detected in B cell-derived Hodgkin's cell lines (L428 and KM-H2) either in mRNA or protein levels. Both the classical (p50-RelA) and the alternative (p52-RelB) NF-kB activity were consistently activated in L428 cells, measured by EMSA and TransAM NF-kB activity assay. The enhanced alternative NF-κB activity, accompanied by increased p100 processing and RelB accumulation in the cytoplasm were detected in L428 cells. Transient transfection of TRAF3-expression vector enforced the expression of TRAF3 and blocked the p100 processing in L428 cells. The alternative NF-kB activity was partially decreased whereas the classical NF-kB activity remained intact. In addition, the increased TRAF3 expression did not affect the anti-apoptotic effects in L428 cells.

Conclusions

Not only the classical NF-κB activity but also the alternative NF-κB activity characterized by p100 processing and p52-RelB nuclear localization is constitutively activated in B cell-derived lymphoma cells. Lack of TRAF3 expression might be one of the reasons for the aberrant expression of alternative NF-κB activity. TRAF3 is indeed an important molecule regulating the activation of the alternative NF-kB activity but not the classical NF-kB activity in H-RS cells.

Disclosures:

No relevant conflicts of interest to declare.
Rapid Reconstitution of Human Antibody Secreting Cells After Haploidentical Allogeneic Stem Cell Transplantation.
Authors: Steurer Martina , Malenke Elke , Federmann Birgit , Kanz Lothar , Bethge Wolfgang Andreas , Wirths Stefan
Published: October 2019

Abstract 3682

Poster Board III-618

Innate immunity including granulocytes, monocytes, and NK cells is reported to recover rapidly after allogeneic stem cell transplantation within weeks. In contrast, adaptive immunity, including T- and B-cells, has delayed recovery over months. In murine models innate type marginal zone and B1 B cells, established at fetal age and providing natural antibodies, are distinguished from adaptive B2 or follicular B cells. A crucial maturation and survival factor for adaptive murine B cells was shown to be TNF-family member BAFF (B cell-activating factor), while development of innate B1 B cells is BAFF independent. Kinetics in reconstitution of innate and adaptive immunity after ablation in adults may give insight into hierarchy and attribution to innate and adaptive immunity of defined lymphocyte populations. Reconstitution of lymphopoiesis after CD3 and CD19 depleted haploidentical stem cell transplantation was analyzed in 10 patients, which received monoclonal anti-CD3 antibody OKT3 as immunosuppressant only. This model may enable detailed in vivo evaluation of de novo B cell formation. Weekly samples before and after reduced-intensity conditioning were analyzed by flow cytometry for absolute numbers of T-cell, NK-, and B-cell subsets. Their origin of host or donor hematopoiesis was differentiated by HLA-FACS. Antibody secreting cells (ASC) were enumerated by ELISPOT. Plasma cytokine concentrations were determined by bead based arrays and ELISA. Complete reconstitution of allogeneic NK cells was found at day +21 after transplantation. CD4+ and CD8+ T-cells and their subsets had delayed reconstitution with less then 100 cells/μl at 3 months after transplantation. CD19+ B-lymphocytes of naïve and memory phenotype (>0,5% of all lymphocytes) were detected not before day +60. In contrast, complete reconstitution of antibody-secreting cells after a nadir (<0,05/μl) was observed at day +14. Absolute numbers of ASC were comparable to those of healthy controls (d+14: 72 ASC/μl vs. control: 12 ASC/μl). ASC secreting the isotypes IgM and IgA were more prevalent than IgG compared to controls (time increase: IgM 20; IgA 10; IgG 2,9). These ASC appear CD19low/neg, CD38+, and intracellular Ig+ in flow cytometry and carried donor HLA-haplotype. Reconstitution of ASC occurred without detectable circulating T-cells and before increase of BAFF concentrations were observed. In summary, the rapid and complete reconstitution of peripheral blood ASC after allogeneic transplantation, far proceeding detection of naïve and memory type B-cells, is a novel observation. Incidence before T-cell reconstitution and increase in BAFF concentrations indicates a T-cell and BAFF independent mechanism allocating these early ASC to innate immunity, potentially maintaining natural antibody levels.

Disclosures:

No relevant conflicts of interest to declare.
Stimulation of B-CLL Cells with Interleukin 21 and Toll Like Receptor Agonists Induces a CTL-Like Transcriptional Profile.
Authors: Hagn Magdalena , Ebel Verena , Sontheimer Kai , Beyer Thamara , Blackwell Sue E. , Simmet Thomas , Weiner George J. , Jahrsdoerfer Bernd
Published: October 2019

Abstract 3685

Poster Board III-621

Interleukin 21 (IL-21) and CpG oligodeoxynucleotides (CpG ODN) are two novel and highly promising agents for the treatment of hematological diseases. Recently, we reported that IL-21 and CpG ODN induce granzyme B (GrB) and GrB-dependent apoptosis in malignant B cells from patients with B chronic lymphocytic leukemia (B-CLL), but not in healthy peripheral B cells. Using various techniques including FACS analysis, Western immunoblotting and RT-PCR we further characterized the factors accountable for the different apoptotic response of B-CLL cells versus normal B cells. GrB induction in B-CLL cells after stimulation with IL-21 and CpG ODN was associated with upregulation of transcription factors, which are normally involved in the differentiation of cytotoxic T lymphocytes (CTL) including T-bet, Eomesodermin (EOMES) and nuclear factor of activated T cells (NFAT), a finding not observed in normal healthy B cells. Furthermore, the induction of GrB in B-CLL cells by IL-21 and CpG ODN required signaling via a JAK/STAT-dependent pathway, as suggested by simultaneous upregulation of phosphorylated STAT3 and complete abrogation of GrB expression by the pan-JAK inhibitor pyridone 6. Stimulation of B-CLL cells with IL-21 and CpG ODN upregulated molecules involved in cell adhesion (CD54), antigen presentation (MHC class I), co-stimulation (CD40, CD86), and GrB uptake (CD222), suggesting B-CLL cells activated with IL-21 and CpG ODN are able to contact other immune cells and may be able to reabsorb secreted GrB. Similar findings resulted when the toll-like receptor (TLR)7 agonist imiquimod was used instead of the TLR9 agonist CpG ODN, suggesting that comparable differentiation programs are initiated by TLR7 and TLR9. In summary, B-CLL cells can express transcription factors involved in cytotoxic differentiation of CTL as well as GrB in response to IL-21 and TLR stimulation. The B-CLL cell differentiation program described in this study could explain our recent findings of GrB-dependent apoptosis in B-CLL cells but not in benign B cells. Moreover, our data provide novel insights into the aberrant signaling state of B-CLL cells.

Disclosures:

No relevant conflicts of interest to declare.
Necdin Regulates Hematopoietic Stem Cell Quiescence and Sensitivity to Genotoxic Stress.
Authors: Asai Takashi , Liu Yan , Giandomenico Silvana Di , Deblasio Anthony , Menendez Silvia , Antipin Jack , Reva Boris , Wevrick Rachel , Nimer Stephen D.
Published: October 2019

Abstract 379

Necdin, a member of MAGE (melanoma antigen) family proteins, is a growth suppressing protein that was first identified in post mitotic neurons. The gene encoding necdin is one of several deleted in individuals with Prader-Willi syndrome, a neurobehavioural disorder associated with an increased risk of myeloid leukemia. It is reported that necdin interacts with p53 and represses p53-mediated apoptosis in neurons, but its role in hematopoiesis is largely unknown. Recently, we defined a critical role of p53 in regulating hematopoietic stem cell quiescence, and identified necdin as a target gene of p53, that is highly expressed in LT-HSCs (Liu Y et al., Cell Stem Cell, 2009). To define the role of necdin in hematopoiesis, we have analyzed the hematopoietic compartment of necdin-null mice. As necdin-null mice die perinatally, we first investigated fetal hematopoiesis and found no alteration in the frequency of fetal liver HSCs, defined as Lin-Sca1+Mac1+CD48-CD150+ within the fetal liver cells. Although necdin-null fetal liver HSCs increase serial replating capability in methylcellulose and maintain stemness in long-term stromal based cultures better than wild type HSCs, necdin-null fetal liver HSCs repopulate lethally irradiated recipient mice similar to wild type HSCs, in primary, secondary, and tertiary serial bone marrow transplantation assays. In addition, necdin-null HSCs show almost comparable repopulating ability as wild type HSCs, after secondary competitive bone marrow transplantation assays. These imply that necdin is dispensable for HSC self renewal. On the other hand, BM-derived necdin-null HSCs show decreased quiescence 4 months after transplantation, and increased proliferation as indicated by in vivo BrdU incorporation assays. Furthermore, recipient mice repopulated with necdin-null HSCs show enhanced sensitivity both to weekly 5-FU administration and to total body irradiation, as manifested by increased mortality. This suggests that the decreased quiescence of necdin-null HSCs leads to their depletion under conditions of genotoxic stress. Gene expression profiling studies have identified several deregulated signaling pathways in the necdin-null HSCs. Expression of several p53 target genes is altered in irradiated necdin-null HSCs, which may account for their enhanced radiosensitivity. We are now investigating these necdin target genes to clarify how necdin functions to critically regulate HSC quiescence. We are also determining whether targeting necdin could be a therapeutic approach to eliminate quiescent leukemia stem cells, using a murine CML model.

Disclosures:

No relevant conflicts of interest to declare.
Bcl-Xl Mitigates p53 Activity in AML1-ETO-Expressing Human Hematopoietic Cells.
Authors: Chou Fu-Sheng , Wunderlich Mark , Mulloy James C.
Published: October 2019

Abstract 387

A subset of acute myeloid leukemia (AML) is caused by chromosomal translocation (8;21), resulting in the expression of the AML1-ETO (AE) fusion protein. We have recently shown that AE represses DNA damage repair genes, resulting in increased DNA damage and upregulation of p53. These findings suggest that AE contributes to leukemogenesis by promoting additional mutations. However, it remains undefined how AE cells survive in the context of increased p53. In the current study, we showed that Bcl-xL is upregulated upon AE expression. Bcl-xL knockdown resulted in growth disadvantage and increased apoptosis in AE cells, but not in normal CD34+ cells. On the other hand, Bcl-xL overexpression in AE cells leads to an expansion of colony-forming cells and an increase in the frequency of long-term culture-initiating cells (LTC-IC). Interestingly, similar results were obtained when a Bcl-xL mutant (Bcl-xLG138A) that is defective in BH3 binding is overexpressed in AE cells, suggesting that the additional cytoprotection is not contributed by pro-apoptotic protein binding. Instead, published evidence on the p53 sequestering function of Bcl-xL prompted us to speculate that AE expression may lead to the establishment of a new balance between pro-apoptotic signals conveyed by activated p53 and the pro-survival characteristic of Bcl-xL. Indeed, using confocal microscopy, we observed colocalization of Bcl-xL and p53 in CD34+ AE cells. In addition, we also observed an increase in p53 target gene expression in AE cells following Bcl-xL knockdown, implicating interplay between Bcl-xL levels and p53 activity. Strikingly, examination of the two critical apoptosis-inducing p53 downstream targets, Bim and Puma, revealed that, whereas Bim is upregluated in AE cells, Puma is downregulated upon AE expression. AE ablation in an established cell line induces Puma expression. A chromatin immunoprecipitation assay revealed that AE binds to the first intron of the Puma gene. These data suggest that AE directly represses Puma gene expression, thereby preventing Puma-induced dissociation of p53 from Bcl-xL. Taken together, our in vitro findings demonstrated that the interplay among p53, Bcl-xL and Puma in response to DNA damage in normal cellular physiology is disrupted in the presence of AE to ensure cell survival. To recapitulate the critical role of Bcl-xL in AE expressing cells in an in vivo xenotransplantation assay, we transduced scrambled and Bcl-xL shRNA into AE/N-RASG12D cells, followed by transplantation into the bone marrow cavity of immunodeficient mice. We found that Bcl-xL knockdown is associated with a significant reduction in the long-term engraftment capability of AE/N-RASG12D cells. In conclusion, our data indicate that AE expressing human hematopoietic cells depend on Bcl-xL for their survival and growth.

Disclosures:

No relevant conflicts of interest to declare.
Assessment of Circulating t(14;18)-Positive Cells in 4152 Individuals without Lymphoma Shows Association with Age and Gender, but Not with Smoking Status: Results From the Population-Based Study of Health in Pomerania (SHIP).
Authors: Hirt Carsten , Weitmann Kerstin , Schueler Frank , Späth Christian , Hoffmann Wolfgang , Doelken Gottfried
Published: October 2019

Abstract 3921

Poster Board III-857

The t(14;18)(BCL2/IgH) translocation is the genetic hallmark of follicular lymphoma (FL). Circulating t(14;18)-positive cells can not only be detected in FL patients but also in healthy subjects without lymphoma. Several epidemiological and molecular studies suggest that these t(14;18)-positive cells might represent lymphoma precursor cells and are associated with known FL risk factors like age and geographical differences in lymphoma incidence. We used epidemiological data and blood samples of a population-based study to verify associations of FL risk factors and t(14;18)-positive cells reported so far and to test for new associations.

The study of health in Pomerania (SHIP) collects epidemiological data, a basic health status and blood samples from 4310 randomly selected inhabitants of the study region in the northeastern part of Germany. We tested buffy coat-DNA samples from 4152 study participants (median age 50 years, range 20-81 years, 2100 women) by real-time PCR for the presence and frequency of t(14;18)-positive cells.

t(14;18)-PCR results were evaluable from 2620 subjects, 1533 subjects were tested positive (58.1%, median number of t(14;18)-positive cells in positive subjects 3.9 per million nucleated cells, range 0.6 – 9299 per million nucleated cells). t(14;18)-prevalence was lowest in the age group 20-29 years (42.2%) and increased up to the group 50-59 years (67.0%) but did not further increase up to the age of 81 years. In accordance with previous reports there was a significant increase of the t(14;18)-frequency with age up to the age of 69 years (linear regression, p< 0.0001) in this study. In the SHIP cohort, t(14;18)-prevalence was lower in females compared to males (53.2% versus 63.5%, p< 0.0001), but there was no significant difference in t(14;18)-frequency between males and females. Smoking status (current, former and never smoker) had no influence on t(14;18)-prevalence or frequency.

This study confirms the association of t(14;18)-prevalence with age and shows for the first time that the t(14;18)-prevalence in females is lower than in males. The later finding parallels the observation of a lower FL incidence in females. In contrast to previous studies, smoking was not associated with detection of t(14;18)-positive cells when the analysis was adjusted for age and sex. In summary, this study confirms that the prevalence of t(14;18)-positive cells in non-lymphoma subjects is associated with established FL risk factors. Thus our report adds to the accumulating evidence that circulating t(14;18)-positive cells in non-lymphoma subjects may represent a biomarker of FL risk.

Disclosures:

No relevant conflicts of interest to declare.
Junctional Adhesion Molecule C (JAM-C) Constitutes a New Marker for the Differential Diagnosis of B Cell Lymphomas.
Authors: Matthes Thomas , Ody Christiane , Imhof Beat , Donate Carmen , Cossali Dominique , Ruault Sylvie , Kern Wolfgang
Published: October 2019

Abstract 3940

Poster Board III-876

Introduction

Differentiation of naïve B cells into plasma cells or memory cells occurs in the germinal centres (GC) of lymph follicles or alternatively in the marginal zone via a GC- and T cell independent pathway. It is currently assumed that B cell lymphomas correspond to normal B cell differentiation stages, but the precise correlation of several B cell lymphomas to these two pathways remains controversial. We have previously shown that junctional adhesion molecule C (JAM-C) originally identified at the cell-cell border of endothelial cells, constitutes also a marker of B lymphocytes with a tightly regulated expression during B cell differentiation: immature B cells, GC-B cells and plasma cells stain negatively, whereas mature, memory and marginal zone derived B cells stain strongly positive. Here we test the expression of JAM-C on a series of patients with B cell lymphomas.

Methods

B lymphocytes from the peripheral blood of 158 untreated patients were analyzed using flow cytometry with standard antibody panels (CD5, CD10, CD11c, CD22, CD23, CD25, CD38, CD103, FMC7, sIg). Diagnosis of a B cell lymphoma was established according to WHO guidelines, using additionally RT-PCR, karyotyping, or FISH, if necessary. Expression of JAM-C was studied by flow cytometry with a polyclonal antibody obtained from a rabbit immunized with the soluble JAM-C molecule.

Results

MCL, HCL and MZBL with a supposed origin in the marginal zone stained mostly positive, whereas CLL and FL with a supposed origin in the germinal centre showed mostly a negative staining. No correlation was found in CLL between JAM-C expression and staining for ZAP70 or CD38. In 12 cases routine work-up was not able to precisely establish a diagnosis of CLL or MZBL, and CLL or MCL. In these cases the presence of JAM-C was considered a strong argument against a GC-origin of the malignant B cells. Addition of JAM-C to antibodies used in the Matutes score increased the sensitivity and specificity of this score for the diagnosis of CLL. Furthermore, it may help differentiating MZBL from LPL which otherwise display overlapping immunophenotypes.

Conclusion

JAM-C constitutes a new diagnostic marker for the differential diagnosis of B cell lymphomas, and is particularly useful for the distinction between CLL and LPL (negative staining) on the one hand and mantle cell and marginal zone B cell lymphomas (positive staining) on the other hand.

Disclosures:

No relevant conflicts of interest to declare.
Sezary Syndrome Is a Malignancy of Central Memory T Cells with Skin Homing Properties, While Mycosis Fungoides Is a Malignancy of Skin Homing Effector Memory T Cells.
Authors: Campbell James , Clark Rachael A , Kupper Thomas S.
Published: October 2019

Abstract 3954

Poster Board III-890

The relationship between leukemic CTCL (L-CTCL), which includes Sezary Syndrome (SS), and mycosis fungoides (MF), a CTCL variant characterized by infiltration of skin, is incompletely understood. In the present study, we examined skin and/or peripheral blood from more than 300 patients with diagnoses of CTCL accrued over a five year period. We identified a subset of patients with leukemic CTCL, all of whom had absolute CD4 counts >2000, CD4/CD8 ratios of > 10, and other features of SS (loss of CD7 and/or CD26). Using specific TCR-Vb antibodies, we could identify malignant clonal populations from CD4+/CD8- peripheral blood mononuclear cells of 12 L-CTCL patients, and performed comprehensive analysis of the malignant clones by flow cytometry. With regard to skin-associated trafficking markers, we found consistently high CCR4 expression, but very heterogeneous expression of CCR6, CCR10 and CLA. Lymph-node homing molecules CCR7 and L-selectin were consistently expressed at high levels on the malignant clonal cells. CD45RA-to-CD45RO ratios, which typically distinguish between non-malignant naïve and antigen-experienced CD4 T cells, were unexpectedly variable from patient to patient. However, CD27, whose expression is lost in normal T cells upon effector/memory cell differentiation, was highly expressed by all malignant clonal cells in SS/L-CTCL. We compared this pattern to the expression of homing and maturation markers on malignant cells extracted from lesional skin of patients with MF. These cells reliably expressed CLA and CCR4, but expressed very low levels of CCR7 and L selectin. In addition, they expressed low levels of CD27. Based on this evidence, L-CTCL/SS appears to be a T central-memory cell CD4-lymphocyte malignancy that, with the exception of CCR4, has variable expression of the putative skin-homing markers CLA, CCR6, or CCR10. This is in direct contrast to MF, wherein clonal T cells in lesional skin bear uniform expression of some skin-homing markers but low expression of central memory markers. These data support a growing body of evidence that SS/L-CTCL and MF are fundamentally different malignancies, rather than different clinical stages of a single disease.

Disclosures:

No relevant conflicts of interest to declare.
Simultaneous Detection of BCL2 Translocation and Copy Number Alteration Including Intrachromosomal Amplification in FL and DLBCL.
Authors: Chhabra Harinder , Mizhiritskay Valentina , Barnabe Charles , Montella Lya Josephine , Nelson Viera , Sun Guoxian
Published: October 2019

Abstract 3955

Poster Board III-891

Causal overexpression of BCL2 protein in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) can be deregulated either by chromosome translocation t(14;18)(q32;q21) or by copy number increase (CNI) of the BCL2 gene, the latter of which includes gains of 18q and extra- or intra-chromosomal amplification. To study the utilization and advantages of interphase fluorescence in situ hybridization (FISH) testing, we retrospectively analyzed abnormal results from 226 consecutive cases with FL or DLBCL using a dual-color dual-fusion t(14;18)/IGH-BCL2 probe (Abbott, IL) performed on various types of specimens such as paraffin embedded tissue, lymph nodes and bone marrow aspirates. The t(14;18) was found in 133 cases (58.8%) with 5 of them also having one extra BCL2 signal, whereas the remaining 93 (41.2%) cases had increased copy number of BCL2 with a range of 3 to 60. In the CNI group, there were two patterns observed: scattered signals seen in the vast majority of cases; and large clusters of intrachromosomally amplified signals observed in two cases. In a concurrent metaphase cytogenetic analysis on the same tonsil sample in one patient with DLBCL, the amplified BCL2-containing segments were present as homogeneously staining region (hsr) on chromosome 13q as well as 18q with no double minutes or marker chromosomes detected. The second highly amplified case was incidentally found to have enlarged lymph nodes during vascular surgery and diagnosed as follicular lymphoma grade 1/3. Interestingly, FISH on paraffin embedded lymph nodes showed one large cluster of amplified BCL2 with an IGH signal within each cluster, suggesting a coexisting translocation t(14;18) and an amplification of BCL2 per se. The FISH patterns in these two highly amplified cases seem to represent a tandem duplication of the BCL2 gene. Although t(14;18) and BCL2 amplification are generally considered to be mutually exclusive in lymphomagenesis, our latter case may be the first reported one where t(14;18) and BCL2 amplification occurred concomitantly. Our study demonstrated that FISH testing with its high specificity, sensitivity and easy visibility for individual cells from various tissue types can simultaneously detect translocations and copy number alterations, provide valuable diagnostic/prognostic information in lymphoma stratification, and complement conventional cytogenetics and comparative genomic hybridization.

Disclosures:

No relevant conflicts of interest to declare.
Kinase Activity of RTKs Is Dispensable for Factor Independent Growth and Transformation of a Myeloid Cell Line 32D in the Presence of Cbl Mutants.
Authors:   Bülent Sargin on behalf of the Study Alliance Leukemias (SAL), Bandi Srinivasa Rao , Rensinghoff Marion , Grundler Rebekka , Tickenbrock Lara , Duyster Justus , Berdel Wolfgang E , Brandts Christian , Serve Hubert , Müller-Tidow Carsten
Published: October 2019

Abstract 3970

Poster Board III-906

Purpose

The Cbl proto-oncogene products have emerged as important components of the signal transduction cascades downstream of both non-receptor and receptor tyrosine kinases (RTKs). By regulation of receptor trafficking and degradation, they have been shown to tightly regulate the intensity and amplitude of RTK activation. c-Kit belongs to the family of the class-III RTKs and plays an important role in the pathogenesis of acute myeloid leukemia (AML). So far, very little is known about the role of c-Cbl mutants in the role of c-Kit signaling.

Results

We analyzed the interaction of c-Cbl with c-Kit and the functional relevance of this interaction in the IL-3-dependent murine myeloid progenitor cell line 32Dcl3. We recently identified the first c-Cbl mutation in human disease in an AML patient, named Cbl-R420Q. Co-expression of two different dominant negative mutants of c-Cbl (Cbl-R420Q or Cbl-70Z) with Kit induced cytokine-independent proliferation, survival and clonogenic growth. Importantly, transformation was observed also with kinase-dead forms of Kit and Flt3 in the presence of Cbl-70Z, but not in the absence of Kit or Flt3, suggesting a mechanism dependent on RTKs, but independent of their kinase activity. Instead, transformation appeared to depend on Src family kinases (SFKs), as c-Cbl co-immunoprecipitated with SFKs and SFK inhibition abolished transformation.

Conclusion

Our results indicate that c-Cbl has an important role in c-Kit signal mitigation. They demonstrate that disturbed mechanisms of c-Kit internalization have important implications for its transforming potential, possibly in the development of AML. Furthermore, these findings may explain primary resistance to tyrosine kinase inhibitors targeted at RTKs.

Disclosures:

No relevant conflicts of interest to declare.
Loss of Tnk1/Kos1 in Mice Is Associated with B-Lymphomas Specifically DLBCL.
Authors: Hoare Kishalay , Reinhard Mary K , Hoare Sarasija , Flagg Tammy , May W. Stratford
Published: October 2019

Abstract 3972

Poster Board III-908

The ubiquitously expressed nonreceptor tyrosine kinase (NRPTK) Tnk1/Kos1 (Thirty-eight negative kinase/Kinase of the embryonic stem cell) functions as a negative regulator of growth in both murine and human cells by suppressing the Ras-Raf1-MapK growth pathway. Since Tnk1 requires its intrinsic protein tyrosine kinase activity to suppress Ras activity and cell growth, the kinase domain is critical for its function and deletion by targeted homologous recombination leads to spontaneous tumor development in mice. To date, Tnk1/Kos1 is the only reported NRPTK that functions as a tumor suppressor in vivo, while other tyrosine kinases may be oncogenic when mutated or activated. While Tnk1 knockout mice may develop primary tumors in different tissues/organs, mainly B-cell lymphomas develop in Tnk1-/- (80%, 47 of 60) and Tnk1+/- (57%, 31 of 54) mice with similar characteristics of Diffuse Large B-Cell Lymphoma (DLBCL) and Burkitt Lymphoma types. Typically in lymphomas from Tnk1+/- mice the intact wild type allele is epigenetically modified and silenced by promoter methylation. Importantly, the absence of Tnk1 occurs only in the tumor tissue but not in the adjacent uninvolved tissue. Now we find allelic loss with associated reduced expression of Tnk1 transcripts and protein in a cohort of human DLBCL patients. These data underscore the potential clinical relevance of Tnk1 in human hematological malignancies. Furthermore, the B-cell lymphomas that develop in the Tnk1 knockout mice express aberrantly high Ras activity indicating that unmutated Ras is a likely necessary effector of B-cell lymphoma development and survival. We also recently determined that the aberrantly high levels Ras activity in lymphoma (but not paired uninvolved lymphoid tissue) from mice results from a novel mechanism involving stabilization of the Grb2-Sos1 complex to maintain activated Ras in these tissues. Therefore, the Tnk1 knockout mouse provides a unique opportunity to test whether and how Tnk1 is involved in the development and/or maintenance of the B-cell lymphomas that develop in the absence of Ras mutation which may have clinical significance for patients with lymphoma.

Disclosures:

No relevant conflicts of interest to declare.
DECREASED ACTIVITY of ADAMTS13 IN PATIENTS with DEEP Vein Thrombosis.
Authors: de Moraes Mazetto Bruna , Orsi Fernanda A. , Montalvao Silmara Aparecida Lima , Mello Tayana B , de Paula Erich Vinicius , Annichino-Bizzacchi Joyce Maria
Published: October 2019

Abstract 3991

Poster Board III-927

Introduction

Several risk factors, including increased levels of factor VIII (FVIII) and von Willebrand factor (VWF), have already been identified in patients with deep vein thrombosis (DVT). Recently we published a study showing that in a Brazilian population, plasma levels of FVIII over 180U/dl and VWF over 165U/dl were associated with the occurrence of venous thrombosis (odds ratio = 4.1 and 3.8 respectively) (Mello TB et al, 2009). The level of VWF in plasma and consequently FVIII is the result of genetic and acquired factors. ADAMTS13 (ADisintegrin And Metalloprotease with Thrombospondin type 1 repeats) is an enzyme responsible for cleavage of VWF, and its activity could contribute to VWF and FVIII plasmatic levels in patients with DVT.

Objective

To evaluate the activity of ADAMTS13 in patients with DVT associated with an increase of VWF and FVIII. Patients and methods: Fifty-six patients with FVIII > 180U/dl or FVW>165U/dl were selected from a cohort of 175 patients with DVT from the study mentioned above. Fifty-four normal subjects were selected as controls. The activity of ADAMTS 13 was performed by binding of residual VWF to collagen; VWF activity was measured by collagen binding, VWF antigen was determined by ELISA and FVIII was measured by a one-stage coagulation assay. Continuous variables were analyzed by Mann-whitney test and categorical variables by the Chi-square test.

Results

The demographic distribution of patients and controls were similar. Among the 56 patients the median age was 37.5 years, 39 were women, 46 had blood typing “non-O”, 34 had DVT caused by transient risk factor, especially the use of oral contraceptives, 10 patients had a hereditary thrombophilia and 3 were carriers of antiphospholipid antibodies. No patient had renal, hepatic or malignant disease. The median ADAMTS 13 activity was significantly lower in patients (112.9%, 44.4 - 327.6%) when compared to controls (142.9%, 76.7 - 323.6%), P = 0.001. The VWF activity was also higher in patients (109.7%, 26.8 - 422.6%) when compared to controls, (79.1%, 45.5 - 203.8%), P=0.038. The median level of VWF antigen was significantly higher in the group of patients when compared to the control group (178.1U/dl versus 111.9 U/dl respectively, P <0.0001). There was an inverse correlation between ADAMTS13 activity and VWF activity. Conclusion: This study suggests that the increased VWF and FVIII activity in patients with DVT can be a result of decreased ADAMTS13 activity. The decreased activity of ADAMTS13 may be influenced by the action of cytokines in inflammatory processes, even after acute period. Future studies will be important to determine the correlation between activity of ADAMTS 13, VWF, and inflammatory markers in the pathogenesis of DVT.

Disclosures:

No relevant conflicts of interest to declare.
Expression of Apoptosis Repressor with Caspase Recruitment Domain (ARC), An Antiapoptotic Protein, Is Strongly Prognostic in Acute Myeloid Leukemia (AML).
Authors: Carter Bing Z. , Zhang Nianxiang , Coombes Kevin R. , Mak Duncan H. , Qiu YiHua , Thomas Deborah A. , Ravandi Farhad , Kantarjian Hagop , Andreeff Michael , Kornblau Steven M.
Published: October 2019

Abstract 400

The identification of relevant targets to induce apoptosis and sensitize to chemotherapy is a focus in AML research. Reverse-phase protein array (RPPA) is a validated and robust proteomic system which can quantitatively determine protein expression and facilitate the selection of molecular-targeted therapies. ARC, located on chromosome 16q21-q23, is an antiapoptotic protein. It inhibits both the intrinsic and extrinsic apoptosis pathways by suppressing the activity of caspase-2 and -8 and by blocking Bax activation. ARC also negatively regulates p53 by inhibiting its tetramerization and triggering its cytoplasmic localization and degradation of ARC depends on the p53-induced ubiquitin E3 ligase, MDM2. ARC has only recently been demonstrated to be expressed in various cancer cells and to confer both chemo- and radio-resistance. To determine its expression and assess its importance in AML, we profiled ARC expression in 511 newly diagnosed patients with AML using RPPA. Similar levels of ARC expression were found in 140 paired blood and bone marrow samples and no significant difference was detected between diagnosis and relapse in 49 paired samples from the same patients. ARC levels were inversely correlated with the protein levels of Bad (p<0.00001), cleaved caspase-9 (p<0.00001), cIAP1 (p<0.00001), caspase-9 (p<0.00001), XIAP (p<0.00001), and Bcl-2 (p=0.00001). Further, ARC levels are significantly higher in FAB M4 (p<0.00001) and M3 (p=0.012) and lower in FAB M2 (p=0.002) AML, but it is neither associated with favorable, intermediate, or unfavorable cytogenetics nor with FLT-3 mutation status. ARC levels do not correlate with bone marrow blast counts, but shows an inverse correlation with blood blast counts (p<0.00001). Importantly, AML patients with low or mid range ARC protein levels had significantly longer overall survival (Median 53 or 61 vs. 38 weeks, p=0.004, A) and remission duration (Median 75 vs 45 vs 29 weeks, p=0.0008, B) (see the figure below) than patients with high levels of ARC. Multivariate analysis indicated that ARC is a statistically significant independent predictor of survival in AML. The final model included: Gender (p=0.008), age at diagnosis (p<0.00001), cytogenetic groups (p<0.00001), FLT-3ITD (p=0.001), WBC (p<0.00001), platelet (p=0.009), albumin (p=0.00006), and ARC (p=0.00006). Results suggest that the expression levels of ARC are highly prognostic in AML and that ARC could be a potential therapeutic target in AML.

Disclosures:

No relevant conflicts of interest to declare.