bluuebunny/crossref_metadata_2025 huggingface dataset:
Gender Counts: Defining the Core Genetic Profile of Polycythemia Vera.
Authors: Spivak Jerry L. , Jie Chunfa , Williams Donna M. , Moliterno Alison R.
Published: October 2019

Intra-abdominal venous thrombosis and exuberant extramedullary hematopoiesis leading to painful splenomegaly and hepatomegaly are two of the most serious consequences of polycythemia vera. Although polycythemia vera is slightly more common in men, both of these complications appear to be more common in women, in whom the disease also presents at an earlier age. We previously reported that polycythemia vera peripheral blood (pb) CD34+ cells could be distinguished from their immunophenotypically similar normal counterparts by gene expression profiling. We, therefore, hypothesized that phenotypic differences in disease behavior between men and women with polycythemia vera were a consequence of gender-based genotypic differences and have employed gene expression profiling to examine this issue. For this purpose, cRNA was prepared from the total RNA of pb CD34+ cells of eleven polycythemia vera patients and six normal controls purified by immunomagnetic bead chromatography. The cRNA was hybridized to an Affymetrix HU133 high-density oligonucleotide microarray chip representing 22,000 genes. Approximately 30–45 % of chip genes were recorded as present in the RNA samples. GC-RMA (Robust Multiarray Analysis) was used for normalization, to adjust for probe effects and for signal estimation. A parametric empirical Bayes statistical modeling method was used for differential gene expression analysis between the patients and controls. A posterior probability of >0.5 was taken to indicate significant differential gene expression. As previously reported, all polycythemia vera patients could be unequivocally distinguished from the controls, indicating that gene expression profiling can be employed as diagnostic test for polycythemia vera. With respect to gender, comparing male patients with male controls, 1106 genes were differentially expressed in the patient group; comparing female patients with female controls, 461 genes were differentially expressed in the patient group. Using unsupervised hierarchical clustering, the control patients segregated as a single group while the polycythemia vera patients segregated into two groups based on exuberant extramedullary hematopoiesis but this segregation was not gender-based despite the marked differences in gender-specific gene expression. Importantly, however, after eliminating gender-specific genes, only 93 genes were concordantly expressed in the male and female patients (59 up regulated and 34 down regulated). This small group appears to represent core genes in polycythemia vera whose behavior may be modified by gender-specific genes but the specific up regulation or repression of which is essential for disease expression. These data indicate that gender-specific effects on gene expression must be addressed in order to determine the basic genetic signature of a hematologic malignancy and they also define a core genetic profile in polycythemia vera.
Efficacy and Safety of Cladribine in Adult Systemic Mastocytosis : A French Multicenter Study of 33 Patients.
Authors: Lortholary Olivier , Vargaftig Jacques , Feger Frederic , Palmerini Fabienne , Delarue Richard , Ferriole Valerie , Launay Jean Marie , Bonte Isabelle , Barete Stephane , Bodemer Christine , Freytag Sylvie , Casassus Philippe , Arock Michel , Dubreuil Patrice , Hermine Olivier
Published: October 2019

Systemic mastocytosis (SM) is a myeloproliferative disabling disorder for which no consensual curative therapy is currently available. Recent preliminary experiences in small groups of patients using cladribine (2-CdA) were encouraging. We thus studied the efficacy and safety of 2-CdA in 33 patients enrolled in a compassionate program in France. Characteristics of patients were as follows: 19 male, 14 female, mean age 55y (17–76y), mean duration of disease 10 y (1m–71y). Treatment consisted in intravenous 2-CdA (1 to 6 cycles of 0.15 mg/kg/d administered in a 2-hour infusion or subcutaneously for 5 d, repeated at 4–12 weeks) for severe SM-related infiltration or symptoms. Patients were classified as having indolent SM (n=6), aggressive SM (n=22) or SM with an associated clonal hematologic non-MC-lineage (AHNMD) (n=4), mast cell leukemia (n=1). C-kit mutation analysis was performed in skin and/or bone marrow in 27 cases (D816V =24; WT=3). All failed previous symptomatic therapy and/or recombinant interferon-a (n=5). Evaluation was based according to consensus criteria (Valent et al. Leuk Research 2003). Major response, partial response and no response were observed in 24, 2, 7 patients, respectively. Mean time to best response was 4 months (1–12m), and mean duration of response was 16m (2–36). In responding patients skin lesions, hepatomegaly/ascitis, splenomegaly, bone involvement, peripheral blood cytopenia, major asthenia, flush, syncope/anaphylaxis, GI tract and pulmonary symptoms improved or disappeared. Treatment was overall well tolerated. Adverse events consisted mainly in peripheral blood cytopenia (n=10) with resolutive opportunistic infections in 2 patients. Although mast cell infiltration persisted in bone marrow, the patient with mast cell leukemia, responded to treatment with disappearance of circulating abnormal mast cells, and resolution of thrombocytopenia. Death was observed in 4 cases related to two disease progression and two acute myeloid leukemia.

Therefore, as a single agent, cladribine is an effective and safe treatment in symptomatic and agressive SM. In contrast with interferon, cladribine may induce regression of mast cell tumoral burden. However, cladribine is ineffective to improve AHNMD. Further work is warranted to define the optimal regimen with respect to dose and schedule, and the usefulness of maintenance cladribine therapy.
Specialized Bone Marrow Endothelium Defines Microdomains for Tumor and Stem Cell Engraftment.
Authors: Sipkins Dorothy A. , Wei Xunbin , Wu Juwell W. , Means Terry K. , Luster Andrew D. , Lin Charles P. , Scadden David T.
Published: October 2019

The organization of cellular niches has been shown to play a key role in regulating normal stem cell differentiation and regeneration, yet relatively little is known about the architecture of microenvironments that support malignant proliferation. Using dynamic in vivo confocal and multi-photon imaging, we show that the bone marrow contains unique anatomic regions defined by specialized endothelium. This vasculature expresses the adhesion molecule E-selectin and the chemoattractant SDF-1 in discrete, discontinuous areas that localize the homing and early engraftment of both leukemic and normal primitive hematopoietic cells. Real-time imaging of cell-cell interactions in SCID mice bone marrow was performed after injection of fluorescently-labeled leukemic and other malignant cell lines. Progressive scanning and optical sectioning through the marrow revealed the existence of unique, spatially-restricted vascular domains to which the majority of marrow-homing tumor cells rolled and arrested. Serial imaging of mice on days 3 – 14 demonstrated that leukemic (Nalm-6 pre-B ALL) extravasation and early proliferation were restricted to these vascular beds. To define the molecular basis of these homing interactions, in vivo labeling of key vascular cell adhesion molecules and chemokines using fluorescent antibodies was performed. We observed that while ICAM-1, VCAM-1, PECAM-1 and P-selectin were expressed diffusely throughout the marrow vasculature, the expression of E-selectin and the chemokine receptor CXCR4 ligand SDF-1 was distinctly limited to vessels that supported leukemic cell engraftment. In vivo co-localization experiments confirmed Nalm-6 binding was restricted to vascular beds expressing both E-selectin and SDF-1. In functional studies, disruption of E-selection had a modest effect on leukemic homing (<20% diminution), while pharmacologic blockade of CXCR4 decreased Nalm-6 binding to vessels by approximately 80%. To explore the normal function of this vascular niche, we next examined whether benign primitive hematopoietic cells might preferentially home to these same vascular microdomains. Fluorescently-labeled stem and progenitor cells (HSPC) isolated from donor balb/c mice were injected into recipient mice and imaging was performed at multiple time points. HSPC were found to adhere to the BM microvasculature in the same restricted domains. At 70 days post-injection, HSPC had extravasated, were persistent in these perivascular areas and had undergone cell division as assessed by dye dilution. Our findings show that these microdomains serve as vascular portals around which leukemic and hematopoietic stem cells engraft, suggesting that this molecularly distinct vasculature provides both a cancer and normal stem cell niche. Specialized vascular structures therefore appear to delineate a stem cell microenvironment that is exploited by malignancy.
Matrix Glycoprotein Osteopontin Is a Stem Cell Niche Constituent That Constrains the Hematopoietic Stem Cell Pool Size.
Authors: Stier Sebastian , Ko Yon , Forkert Randolf , Lutz Christoph , Neuhaus Thomas , Gruenewald Elisabeth , Dombkowski David , Rittling Susan R. , Scadden David T.
Published: October 2019

Stem cells reside in a physical niche where a balance of signals controls their growth, differentiation and death. Niche components have generally been defined in terms of cells and positive effects on stem cell maintenance or expansion. Here we define a role for a matrix glycoprotein that provides a constraining function in the hematopoietic stem cell niche. Osteopontin (OPN) is an abundant glycoprotein in bone that can function as either cytokine or cell adhesion mediator. It is known to be produced by multiple cells types including osteoblasts, cells recently defined to be a regulatory component of the hematopoietic stem cell niche. Using studies combining OPN deficient mice and exogenous OPN, we demonstrate that OPN modifies primitive hematopoietic cell numbers and function. In OPN deficient mice, increased primitive cell numbers were observed in vivo associated with reduced progenitors and reduced primitive cell apoptotic fraction. To determine whether the effect of OPN deficiency was stroma dependent, we performed in vitro stem cell assays on OPN−/− stroma and observed greater LTC-IC supportive capacity compared with wild type stroma. Furthermore, OPN−/− recipients showed a significantly higher proportion of hematopoietic stem cells after transplantation of OPN+/+ bone marrow in comparison to wild-type recipients, indicating that the OPN null microenvironment was sufficient to increase stem cell number. A reduction in apoptotic fraction was seen in primitive cells in the OPN−/− recipient marrows. A role for OPN in apoptosis was confirmed by exogenous OPN in in-vitro studies. Hypothesizing that OPN may serve as a physiologic constraint on stem cell pool size, we compared OPN−/− with wild type animals following parathyroid hormone activation of the stem cell niche. The expansion of stem cells by PTH was superphysiologic in the absence of OPN. Therefore, OPN is a restricting element of the stem cell niche, limiting the number of stem cells produced by niche activation. Extracellular matrix components such as OPN may serve as modulable, regulatory participants in the stem cell niche.
Defining the Bone Marrow Stem Cell Niche According to a Hoechst Dye Diffusion Gradient and Hypoxia.
Authors: Down Julian D. , Parmar Kalindi , Clyne James , Mauch Peter M. , Sackstein Robert
Published: October 2019

The spatial organization of hematopoietic cell subsets of differing proliferative potential within the bone marrow microenvironment has come under increasing interest. In particular, it has been suggested that hematopoietic stem cells (HSCs) normally reside in a regulatory niche situated at the endosteal bone surface and in close proximity to osteoblasts. In the present study we have investigated how different hematopoietic cell subsets are distributed along a Hoechst dye perfusion gradient that may reflect the distance from marrow blood vessels and the level of oxygenation. C57BL/6J mice were intravenously injected with two doses of Hoechst 33342 at 10 and 5 min before marrow cell harvesting, a period that we determined was insufficient for active dye exclusion in vitro. Flow cytometric analysis revealed a wide distribution of Hoechst staining over 3 logs fluorescence intensity. The cells were then sorted from 6 different regions of the Hoechst gradient and evaluated for short- and long-term in vitro repopulating cells in the cobblestone area-forming cell (CAFC) assay. The primitive CAFC subset appearing at day 28 in culture was shown to be progressively enriched with decreasing Hoechst fluorescence while the short-term repopulating day 7 CAFC subset frequencies was the highest at an intermediate level of Hoechst staining. We further investigated whether primitive HSCs exist in a very low oxygen tension by administering the reductive 2-nitroimidazole compound pimonidazole in vivo and performed flow cytometric analysis on sorted primitive HSCs residing in high Hoechst dye effluxing side population (SP). In comparison to whole bone marrow or non-SP cells, the SP fraction showed increased intracellular staining with an anti-pimonidazole antibody that recognizes pimonidazole adducts formed only under hypoxic conditions (less than pO2 of 10 mm Hg). Comparison with thymocytes that are already known to be hypoxic in vivo (Hale et al. Am J Physiol Heart Circ Physiol282: H1467–H1477, 2002) showed both low Hoechst dye perfusion and positive anti-pimonidazole antibody staining. These results represent the first direct evidence that the hematopoietic cell hierarchy is spatially organized in relation to blood vessels and that the stem cell niche exists at the lowest end of an oxygen gradient. These findings have important implications in hematopoiesis and stem cell lodgement, and suggest that the location of hematopoietic stem cells in situ may render them more resistant to oxygen-dependent mutagenic and cytotoxic agents.
Mesenchymal Stem Cells (MSC) Display Coordinated Rolling and Adhesion Behaviour on Endothelial Cells under Shear Flow.
Authors: Ruster Brigitte , Bistrian Roxana , Gottig Stephan , Seifried Erhard , Henschler Reinhard
Published: October 2019

Although MSC have been applied in a number of transplantation protocols, the mechanisms which are responsible for their extravasation and tissue specific homing are less well understood. To elucidate the potential of MSC to undergo coordinated interaction with endothelial cells, we investigated MSC under controlled shear flow using a parallel plate flow chamber. MSC were isolated from bone marrow of 6 different donors, depleted of hematopoietic cells by plastic adherence to yield >99% CD45− populations and further expanded up to 106 fold in DMEM/FCS/bFGF over 35 days. Their osteogenic, chondrogenic and adipogenic differentiation potential was maintained during the expansion period. Flow cytometric analysis showed that MSC expressed integrins alpha 1, alpha 4, alpha 5 and beta 1, the chemokine receptor CCR6 and, in a small subpopulation also CXCR4, but that they did not express beta 2 integrin, L-selectin or PSGL-1. During continuous laminar flow on early passage HUVEC at wall shear stresses between 0.1 and 1 dyn/cm2, MSC extended cytoplasmic podia towards endothelium, resulting in capturing and subsequent rolling. This resulted in firm adherence of MSC which resisted shear stresses of 2 dyn/cm2 for >20min. This was, in a similar way, also observed with peripheral blood mononuclear cells or CD34+ primary hematopoietic progenitor cells, whereas erythrocytes and primary endothelial cells did not interact with the HUVEC monolayer. Shear stresses of 2 dyn/cm² and more resulted in a decrease of MSC rolling. Prestimulation of endothelial cells with TNF-alpha increased both MSC rolling (3.7-fold ± 0.6 fold) and firm adhesion (3.3 ± 0.3 fold; means ± SD, n=5; p<0.05). Preincubation of endothelial cells with function-blocking anti-P selectin antibodies reduced the number of rolling MSC by 37.7% ± 5.9% and of firmly adhering MSC by 54.6% ± 11.3% (means ± SD; n=4; p<0.05). Although preincubation with function-blocking antibodies against PSGL-1 did not detectably affect MSC rolling and adhesion, preincubation with O-sialoglycoprotease and with fucosidase but not with neuaminidase significantly reduced MSC rolling and adhesion, indicating that a selectin-type ligand is responsible for the binding. Preincubation of MSC with function-blocking anti-beta 1 integrin antibody also led to a statistically significant reduction of MSC firm adhesion by 21.3% ± 5.7%. Taken together, our results demonstrate that MSC can undergo coordinated rolling and adhesion on endothelium under shear flow involving (i) stimulation in a pro-inflammatory environment, (ii) engagement of P-selectin and a fucosylated ligand on MSC inducing capturing and rolling, and (iii) activation of beta 1 integrins mediating firm adhesion to endothelial cells
Conditional Expression of C/EBPα in Mice Reveals Developmental Plasticity of Lymphoid and Erythroid/ Megakaryocyte Precursors in Vivo.
Authors: Fukuchi Yumi , Shibata Fumi , Ito Miyuki , Goto-Koshino Yuko , Sotomaru Yusuke , Ito Mamoru , Kitamura Toshio , Nakajima Hideaki
Published: October 2019

CCAAT/ enhancer binding protein α (C/EBP α) is a member of the bZIP family of transcription factors that plays a critical role for early myeloid differentiation. C/EBP α knockout mice showed a complete differentiation block at myeloblast stage in hematopoietic system, and mature neutrophils and eosinophils are absent in the peripheral blood. Lineage specification in developmental tree of hematopoiesis is generally determined by a lineage specific transcription factors such as C/EBP α and GATA-1 that allow commitment to the specific lineage with simultaneous extinction of their capacity to differentiate into the other ones. However, recent evidences shown by the ectopic expression of above transcription factors unveiled the unexpected developmental plasticity of various progenitors such as MEP (erythroid/ megakaryocyte progenitor) and CLP (common lymphoid progenitor). GATA-1 is reported to convert CLP and CMP (common myeloid progenitor) into erythroid/ megakaryocyte lineage, however, the effect of C/EBP α on MEP and CLP is still unlcear. In order to investigate the role of C/EBP α in the various aspect of hematopoietic differentiation, especially its effect on the lineage specification at different stages of differentiation in vivo, we generated transgenic mice expressing inducible form of C/EBP α (C/EBP α-ER) under H-2K promoter (C/EBP α-ER Tg). In these mice, C/EBP α activity can be induced conditionally by 4-hydroxy tamoxifen (4-HT) in all hematopoietic cells. As expected, C/EBP α-ER was expressed in almost all hematopoietic tissues including bone marrow, spleen and thymus in these mice. Gel shift analysis revealed that C/EBP α-ER was activated by 4-HT, and showed specific binding to C/EBP-specific oligonucleotide in these tissues. Next we tested differentiation plasticity of erythroid and lymphoid progenitors by ectopically inducing C/EBP α-ER activity in these cells. We sorted MEP and CLP by FACS from C/EBP α-ER Tg and examined their clonogenic activities in the presence or absence of 4-HT. In the absence of 4-HT, MEP and CLP exclusively formed erythroid/ megakaryocyte and lymphoid colonies, respectively, as previously reported. Surprisingly however, these cells dramatically changed their fate of differentiation and formed significant numbers of granulocyte/ macrophage (GM) colonies in the presence of 4-HT, indicating that ectopic activation of C/EBP α-ER activity skewed their differentiation pathways to myeloid lineage. Cytospin preparation of the colonies and RT-PCR analysis revealed that these were accompanied by the morphological differentiation to granulocytes/ macrophages, and upregulation of myeloid-specific genes at mRNA level. These results indicate that MEP and CLP are not fully committed to either erythroid/ megakaryocyte or lymphoid lineage, and possess differentiation plasticity that can be redirected to myeloid lineage.
High-Risk Myelodysplastic Syndrome (MDS): First Results of International Phase 2 Study with Oral Farnesyltransferase Inhibitor R115777 (ZARNESTRATM).
Authors: Kurzrock Razelle , Fenaux Pierre , Raza Azra , Mufti Ghulam , Aul Carlo , Germing Ulrich , Kerstens Rene , DePorre Peter
Published: October 2019

R115777 is an oral selective inhibitor of farnesyltransferase which disrupts critical signaling pathways resulting in antiangiogenic, antiproliferative and apoptotic effects. Single-agent activity of R115777 in patients (pt) with MDS, with manageable toxicity, was previously reported in single-institution studies. The current multicenter study assesses the response rate (IWG-criteria) and survival of R115777 in pt with high-risk MDS, defined by FAB classification as RAEBt and RAEB or CMML with ≥10% bone marrow (BM) blasts. Starting dose of R115777 was 300 mg bid for the first 21 days of every 28-day cycle. Eighty-two pt were enrolled (median age 67 yrs; range 39–86) with RAEBt (n=23), RAEB (n=40) and CMML (n=19). Thirty-six pt (44%) had IPSS score high, 32 (39%) int-2 and 14 (17%) int-1. Fifty-two pt were therapy-naïve, while 30 (37%) had received prior therapy for MDS. Pt were treated for a median of 3 cycles (range: 6 to 553+ days), with 78% relative dose intensity. Pt discontinued treatment primarily for progressive disease (n=37) or for adverse event (n=20), with 6 pt still on therapy. Responses were seen in 28 pt (33%; 6 CR, 2 CR with incomplete platelet recovery, 2 PR, 18 hematological improvement-HI), both in therapy-naïve and pre-treated pt. Responses were durable, with median duration of 14+ months (mo) for CR-PR (11+ to 20+ mo) and 6.7 mo for HI (3–15 mo). In 68 pt with at least 1 on-study BM assessment, BM response (either normalization or ≥50% decrease in BM blast count) was observed in 35 pt (51%). Median time to leukemic transformation (mTTL) was 14.1 mo, median time to leukemia or death (mTTLorD) was 6.4 mo and median overall survival (mOS) was 11.9 mo. Results (in mo) by risk-group are tabulated.

by FAB class by IPSS score RAEBt RAEB CMML high int-2 int-1 n = 23 40 19 36 32 14 mOS 9.2 12.2 15.0 10.4 15.0 15.1 mTTLorD 3.3 7.4 10.8 3.7 7.4 11.9 mTTL 3.7 median was not reached 7.6 median was not reached

Myelosuppression was the most common side effect, with 24% grade (G) 3–4 neutro-penia and 37% G3–4 thrombocytopenia. Non-hematological toxicity was mostly G1–2, rarely G3–4. Most frequently reported were nausea (35%; 4% G3), fatigue (35%; 2% G3), diarrhea (34%; 1% G3), fever (23%, 10% G3), dyspnea (20%; 2% G3–4), purpura (20%; 4% G3) and rash (19%; 2% G3). Conclusion: In a large cohort of pt with high-risk MDS as defined by FAB and IPSS, clinical activity with durable responses and limited toxicity is confirmed for R115777. Further study is warranted.
Expression of Urokinase-Type Plasminogen Activator in Platelets Decreases Oxygen-Induced Lung Injury in Mice.
Authors: Perkowski Sandra Z. , Kowalska M. Anna , Albelda Steven M. , Bdeir Khalil , Cines Douglas B. , Poncz Mortimer
Published: October 2019

We have previously shown that urokinase (uPA) can be ectopically expressed and stored in platelets in a transgenic mouse model and that such platelets can effectively prevent untoward thrombosis. One major advantage of this uPA delivery system is that there is no systemic fibrinolysis and the uPA is selectively released at sites of vascular injury. We have now tested whether such a delivery model may be of use in the care of patients with lung injury where inhibition of fibrinolytic activity and intra-alveolar fibrin deposition are of pathogenic importance. In this study, we evaluated (O2)-induced lung injury in transgenic mice expressing platelet uPA compared to wildtype (WT) littermate controls. Given that pulmonary sequestration of activated inflammatory cells, including neutrophils (PMN) and platelets, occurs relatively early in response to O2, and given the ability of the transgenic uPA platelets to enhance fibrin lysis, we hypothesized that hyperoxic-induced lung injury would be lessened in the transgenic animals. Lung injury was assessed by changes in bronchoalveolar lavage fluid (BAL) protein concentration. In this model, a significant increase in BAL protein concentration, indicative of injury to the alveolar capillary barrier, occurred in WT mice after 72 hr of >95% O2 exposure compared to controls [6.9 ± 0.9 vs. 0.14 ± 0.01 mg/ml (mean ± SEM)], respectively. BAL protein was significantly decreased in platelet uPA+ mice [1.5 ± 0.4 vs. 6.9 ± 0.9 mg/ml, n=6–8, p<0.05]. Immunohistochemical detection of fibrin in control and platelet uPA+ mice lungs using anti-murine fibrin(ogen) antibody showed that very little fibrin(ogen) was detected in control (0 hr) mice or WT mice exposed to >95% O2 for 24 hr. By 72 hr of O2 in WT mice, however, fibrin(ogen) immunoreactivity was seen within the alveolar space and diffusely throughout the interstitium. Fibrin clots within small blood vessels were also seen. In contrast, fibrin(ogen) immunoreactivity was greatly reduced in uPA+ mice. Moreover survival time of uPA+ mice was significantly longer than WT animals (127 ± 28 hr vs. 97 ± 14 hrs, respectively, n=12, p<0.003). Total white blood cell counts and % PMN in the BAL were significantly higher after 72 hr exposure to O2, suggesting that increased pulmonary inflammation is not necessarily associated with increased lung injury, a finding consistent with other studies showing that neutrophils do not play a significant role in the genesis of this lung injury. These data support the hypothesis that a decrease in lung fibrinolytic activity is a primary factor in the development of O2-induced lung injury and that platelet delivery of uPA may be an especially effective and safe way to prevent the development of such lung injury.
Mantle Cell Lymphoma Genotypes Identified with CGH to BAC Microarrays Define Clinical Subgroups of Disease and Strongly Predict Patient Outcome.
Authors: Martinez-Climent Jose A. , Rubio-Moscardo Fanny , Climent Joan , Terol Maria J. , Sarsotti Elena T. , Benet Isabel , Siebert Reiner , Piris Miguel A. , Martin-Subero Inaki , Dyer Martin J.S. , Pinkel Daniel , Garcia-Conde Javier
Published: October 2019

Mantle cell lymphoma (MCL) is a rare, aggressive subtype of B-cell non-Hodgkin lymphoma (B-NHL), characterized by t(11;14)(q13;q32). To identify recurrent genomic events involved in the pathogenesis of this malignancy, we used genome-wide comparative genomic hybridization (CGH) to 1.4 Mb. resolution BAC microarrays in 68 patients and 9 derived MCL cell lines; the genome profiles were compared to those from other B-NHL subtypes. Array-CGH defined a MCL genomic signature different from other B-cell lymphomas, including deletions of chromosomes 1p21.2-p12.3, 11q22.3 at ATM gene locus, 13q14.2 and 13q34, with coincident 10p12-BMI locus amplification and 10p14 deletion, along with a previously unidentified loss within 9q21-q22. Specific genomic alterations were associated with different subgroups of disease. Notably, 11 patients with non-nodal, leukemic MCL, defined on the basis of the absence at diagnosis of lymph node disease, showed a different genomic profile to nodal cases, including deletion of 8p21.2 at TRAIL receptor gene cluster (55 vs. 19%;p=0,01) and gain of 8q24.1 at MYC locus (46 vs. 14%;p=0,015). Additionally, leukemic MCL exhibited frequent IgVH mutation (64 vs. 21%;p=0,009) with preferential VH-39 use (36 vs. 4%;p=0,005), and followed a more indolent course, being 7 of 11 patients (64%) survivors for more than 3 years whereas only 15 of 55 patients (27%) in the nodal group experienced this long survival (median survial time, 42 vs. 18 months; p=0,02). The median overall survival (OS) for the patients in the series was 39 months (range, 0–115). Blastoid variant of MCL, increased number of genomic gains, and deletions of P16/INK4a and P53 genes correlated with poorer outcomes, whilst 1p21 loss was associated with prolonged survival (median OS, 70 vs. 31 months; p=0,02). In multivariate analysis, deletion of chromosome 9q21-q22, which has not been previously reported as a common change in MCL, was the strongest predictor for inferior survival (HR:6; CI:2,3–15,7). Last, the genotypes of 14 patients with MCL who were alive for more than 5 years since diagnosis showed deletion of 1p21 in seven cases (50%), but none exhibited deletions of 9q21-q22, 9p21.3-P16/INK4a or 17p13.1-P53. Our study illustrates the utility of array-CGH in MCL sub-classification and highlights the array genomic profile as the strongest indicator for predicting clinical outcome. The screening of this reduced BAC clone set either by FISH or custom-made array CGH devices could be reliably applied to the clinical diagnostics of MCL.
Enoxaparin Is Effective and Safe as Bridging Anticoagulation in Patients with a Mechanical Prosthetic Heart Valve who Require Temporary Interruption of Warfarin because of Surgery or an Invasive Procedure.
Authors: Turpie Alexander G. , Douketis James D.
Published: October 2019

Background: Enoxaparin is a low-molecular-weight heparin (LMWH) that is widely used in patients with an acute coronary syndrome and venous thromboembolism. However, there is concern about its use in patients with mechanical prosthetic heart valve because of recent reports of fatal TE occurring in pregnant women with a mechanical heart valve who received long-term enoxaparin, instead of warfarin, during pregnancy.

Patients and Methods: We performed a prospective cohort study to assess the efficacy and safety of enoxaparin as bridging anticoagulation in a consecutive patients with a mechanical prosthetic heart valve who required temporary interruption of warfarin therapy because of an elective surgical or other invasive procedure. Patients had warfarin interruption and bridging anticoagulation based on a standardized peri-operative protocol and underwent clinical follow-up for 3 months after surgery. We determined the incidence and associated 95% confidence interval (CI) during the follow-up period for the following outcomes: major bleeding; arterial thromboembolism (stroke, rtansient ischemic attack, systemic embolism, valve thrombosis); all-cause death.

Results: There were 174 patients included in this cohort (100 males; mean age: 67±10 years; mean weight: 76±16 kg; 115 with a mechanical aortic valve; 59 with a mechanical mitral valve or combined mechanical aortic and mitral valves) who received bridging anticoagulation with subcutaneous enoxaparin, 1 mg/kg twice-daily. No patient was lost to follow-up. Warfarin therapy was interrupted 5.7±2.2 days before the procedure. The mean number of doses of enoxaparin (1 mg/kg) administered before the procedure was 5.7±1.4 doses. After the procedure, warfarin was resumed on the evening or the next day after the procedure, and the mean number of doses of enoxaparin administered was 6.0±2.2 doses. After 3 months of clinical follow-up, 4 patients developed non-fatal major bleeding (2.3%; 95% CI: 0.06–5.7), 4 patients died (2.3%; 95% CI: 0.06–5.7), and 1 patient had a non-fatal stroke (0.56%; 95% CI: 0.01–3.1). There were no episodes of valve thrombosis or systemic embolism.

Conclusions: Enoxaparin appears to be effective and safe as bridging anticoagulation in patients with a mechanical prosthetic heart valve who require temporary interruption of warfarin therapy.
The Safety of Co-Administered Low-Molecular-Weight Heparin and Continuous Epidural Analgesia after Major Orthopedic Surgery: Assessment of a Standardized Management Protocol.
Authors: Wang Jenny , Douketis James , Wagler Meghan , Kinnon Krys , Crowther Mark
Published: October 2019

Background: In patients who have had major orthopedic surgery, low-molecular-weight heparin (LMWH) has established efficacy in preventing venous thromboembolism (VTE) after surgery, and continuous epidural analgesia (CEA) provides better pain control than parenteral analgesia and may reduce the risk of cardiorespiratory complications. However, there is concern about the co-administration of LMWH and CEA after surgery because of an increased risk of epidural hematoma. No study, to our knowledge, has investigated the safety of a standardized treatment regimen that allows co-administered LMWH and CEA after surgery.

Patients and Methods: We assessed the safety of a standardized management protocol in 970 patients who were scheduled to undergo knee or hip replacement surgery and who received post-operative thromboprophylaxis with LMWH and pain control with CEA. All patients received intra-operative spinal (regional) anesthesia, with an indwelling epidural catheter left in place after surgery to administer epidural analgesia with an infusion of fentanyl and bupivacaine. Subcutaneous LMWH (enoxaparin 30 mg twice-daily or dalteparin 5000 IU daily) was started on the morning after surgery and continued for at least 5 days. CEA was administered for 2–3 days after surgery, with the epidural catheter removal occurring during the trough anticoagulant effect of the LMWH, 10 hours and 22 hours after the preceding dose of enoxaparin and dalteparin, respectively. In patients who were to receive warfarin, this treatment was initiated, with a 2.5 mg dose, on the evening prior to the scheduled removal of the epidural catheter. Drugs that are known to affect hemostasis were avoided during CEA, and patients at increased risk for bleeding were not given LMWH. During the period of hospitalization, we calculated the incidence and associated 95% confidence interval (CI) for the following outcomes: epidural hematoma; other bleeding (major and minor); VTE, myocardial infarction, stroke, death.

Results: 970 patients (mean age: 68.2 years; mean weight: 81.4 kg; 63% female) who underwent knee replacement (527 patients) or hip replacement (443 patients) received co-administered LMWH and CEA. The mean duration of LWMH therapy was 7.2±5.3 days. The mean duration (±standard deviation) of CEA was 1.9±0.80 days, respectively. Warfarin was initiated in 758 (78%) patients. There were no episodes of symptomatic epidural hematoma (0%; 95% CI: 0–0.38). During the in-hospital follow-up period, there were 13 episodes of VTE (1.3%; 95% CI: 0.71–2.3), 47 bleeds (4.7%; 95% CI: 3.5–6.3); 4 myocardial infarcts (0.41%; 95% CI: 0.12–1.1), and 4 deaths (0.41%; 95% CI: 0.12–1.1).

Conclusion: Co-administered LMWH and CEA appears safe after major orthopedic surgery when using a standardized management protocol.
Initial Outpatient Treatment of Venous Thromboembolism with Fondaparinux (Arixtra®): The Matisse Trials.
Authors: Buller Harry R. ,   the MATISSE Investigators
Published: October 2019

Background: The MATISSE trials showed that a single dose regimen of fondaparinux, a synthetic selective factor Xa inhibitor, was at least as effective and as safe as standard therapies in the treatment of venous thromboembolism (VTE). In these trials, outpatient treatment of fondaparinux was encouraged but left at the investigator’s discretion. We analyzed the data in patients who received fondaparinux on an outpatient basis.

Methods: Fondaparinux was administered at a once-daily subcutaneous dose of 7.5 mg (5.0 mg and 10.0 mg in patients <50 kg and >100 kg, respectively). In the MATISSE-DVT trial, fondaparinux was compared with twice-daily subcutaneous enoxaparin (1 mg/kg) in patients with deep-vein thrombosis (DVT). In the MATISSE-PE trial, it was compared with adjusted-dose intravenous unfractionated heparin (UFH) in patients with pulmonary embolism (PE). Outpatient treatment of DVT with enoxaparin was possible whereas outpatient treatment of PE with UFH was not feasible. All drugs were given for at least 5 days and until anticoagulation with oral anticoagulants was therapeutic. The primary efficacy and safety outcomes were recurrent VTE during 3 months’ follow-up and major bleeding (MB) and death during the initial treatment period.

Results: In MATISSE-DVT, 31.4% and 33.8% of the fondaparinux- and enoxaparin-treated patients, respectively, received therapy on an outpatient basis. In MATISSE-PE, 14.3% of the patients received fondaparinux on an outpatient basis, compared with none in the UFH group. In both MATISSE-DVT and -PE, efficacy and safety data from the patients who received fondaparinux on an outpatient basis were similar to those from the total population (Tables). The rates of recurrent VTE and MB in fondaparinux outpatients were similar to those in enoxaparin outpatients or UFH inpatients.

Conclusion: Outpatient initial treatment of both DVT and PE with once-daily fondaparinux is feasible, effective and safe.

MATISSE DVT Enoxaparin Fondaparinux All patients Outpatients All patients Outpatients *As treated patients n 1107 374 (33.8%) 1098 345 (31.4%) Age, yr (mean±SD) 61±17 60±16 61±17 58±17 Male/female 578/529 201/173 581/517 197/148 Hospital discharge, days (mean±SD) 7.0±6.2 1.8±1.9 7.6±7.7 1.6±1.7 ≥2 VTE risk factors, n (%) 283 (25.6) 122 (32.6) 293 (26.7) 97 (28.1) VTE, n (%) 45 (4.1) 16 (4.3) 43 (3.9) 7 (2.0) MB*, n (%) 13 (1.2) 3 (0.8) 12 (1.1) 5 (1.5) MATISSE-PE UFH Fondaparinux All patients Outpatients *As treated patients n 1110 1103 158 (14.3%) Age, yr (mean±SD) 62±17 63±16 57±16 Male/female 477/633 501/601 82/76 Hospital discharge, days (mean±SD) 10.2±6.8 9.7±7.7 4.4±2.2 ≥2 VTE risk factors, n (%) 260 (23.4) 241 (21.8) 35 (22.2) VTE, n (%) 56 (5.0) 42 (3.8) 5 (3.2) MB*, n (%) 12 (1.1) 14 (1.3) 0 (0)
BCR/ABL Expression Increases the Formation of Chromosomal Translocations after DNA Damage.
Authors: Dierov Jamil , Padilla-Nash Hesed , Ried Thomas , Carroll Martin
Published: October 2019

BCR/ABL is the protein product of the t(9;22) translocation and is the cause of the hyperproliferation associated with chronic phase chronic myeloid leukemia (CML). However, whether BCR/ABL induces genomic instability leading to blast crisis is controversial. We have previously demonstrated that BCR/ABL translocates to the nucleus after genotoxic damage and associates with the DNA damage sensor, ataxia telangiectasia and rad 3 related (ATR) protein, disrupting the sensing and repair of DNA double strand breaks. Here, we have asked if BCR/ABL expression leads to permanent changes in the DNA after genotoxic stress. For these experiments we have studied the hematopoietic cell line, Ba/F3pTetOn p210, which expresses p210 BCR/ABL after incubation in doxycycline. Cells were incubated in low doses of etoposide for two hours and then allowed to recover for 48 hours in the absence of further DNA damage. Induction of apoptosis in these conditions was consistently less than 5% as demonstrated by annexin V staining of cells. Cells were examined for alterations in the chromosomes using Giemsa banding and spectral karyotyping (SKY). Cells growing in IL3 showed low levels of DNA damage with a few broken chromosomes present in metaphase spreads and an average of 0.5 new chromosomal translocations per cell as revealed by SKY analysis. In contrast, when cells expressing BCR/ABL were treated with the same conditions, a marked number of genetic abnormalities were seen. 75% of cells showed abnormal chromosome forms with ring chromosomes, triradial forms and other abnormalitites. Analysis of BCR/ABL expressing cells by SKY analysis showed frequent abnormalities, averaging at least 6 new translocations per cell. Several cells had greater than 10 translocations present including multiple complex translocations involving more than two chromosomes. The majority of abnormalities consisted of unbalanced translocations. This data demonstrates that BCR/ABL alters the cellular response to DNA damage leading to an increase in chromosomal translocations in cells expressing the oncogene and suggests that BCR/ABL itself is directly responsible for the genomic instability leading to CML blast crisis.
Differential Phosphorylation of FANCA and FANCG Determines Response of Fanconi Anemia Core Complex to DNA Damage and S Phase.
Authors: Mi Jun , Tomashevski Andrei , Kupfer Gary M.
Published: October 2019

Fanconi anemia (FA) is a genetic disease marked by bone marrow failure, congenital defects, and cancer. In spite of the identification of at least 8 genes, the biochemistry of the disease and its normal pathway in the cell remains elusive. The FA core complex is composed of at least 5 proteins, 2 of which, FANCA and FANCG, we have shown to be phosphorylated. In these studies, we show that both FANCA and FANCG are phosphorylated in response to DNA damage. In the case of FANCG, we have mapped the site of this phosphorylation to serine 7, using a phosphoserine 7 FANCG antiserum. Because of the link of FA function and the FA core complex-dependent monoubiquitination that occurs both as a result of DNA damage as well as at S phase, we also examined if phosphorylation occurred at S phase as well. While FANCG serine 7 phosphorylation occurs both at S phase and after DNA damage (similar to FANCD2 monoubiquitination), FANCA phosphorylation occurs only after DNA damage. Recent data have implicated the kinase ATR as important in the pathway. In order to assess whether a downstream target of ATR is differentially phosphorylated in FA cells, we tested the phosphorylation status of chk1 in FA-A mutant and corrected cells. Chk1 kinase is phosphorylated at serine 318 in response to DNA damage only in corrected cells but not mutant FA cells, while signaling through chk2 kinase is unaffected. These data suggest the importance of phosphorylation in the FA pathway in the regulation of both cellular responses to DNA damage as well as engagement of the cell cycle.
Primary Treatment of Waldenstrom’s Macroglobulinemia (WM) with Dexamethasone, Rituximab and Cyclophosphamide.
Authors: Dimopoulos Meletios A. , Kyrtsonis Maria Christina , Tsatalas Constantinos , Kokkini Garyfallia , Repoussis Panagiotis , Zomas Athanasios , Symeonidis Argiris , Michali Evridiki , Anagnostopoulos Athanasios , Anagnostopoulos Nikolaos , Economopoulos Theophanis , Pangalis Gerasimos
Published: October 2019

Introduction: The three main choices for the primary treatment of WM are alkylating agents, nucleoside analogues and rituximab. There is evidence that combinations of nucleoside analogues and rituximab are highly active. However, when such regimens are applied, there are concerns regarding myelosuppression, immunosuppression and impact on stem cell collection. We designed a phase II study which included previously untreated WM patients (with clearly defined criteria for initiation of treatment) who were treated with a combination of dexamethasone, rituximab and cyclophosphamide.

Patients and methods: Patients with WM received primary treatment with dexamethasone 20 mg i.v. followed by rituximab 375mg/m2 i.v. on day 1 and cyclophosphamide 100mg/m2 p.o. bid days 1–5 (total dose 1000 mg/m2). This regimen was repeated every 21 days for 6 courses.

Results: Since July 2002, 34 patients have been enrolled in this multicenter study. Patients characteristics include: median age of 75 years (range 53 to 86 years), 59% male, 25% B symptoms, 25% splenomegaly, 35% lymphadenopathy, 25%hyperviscosity syndrome, 59% hemoglobin < 10 g/dl, 37% serum monoclonal protein ≥ 4 g/dl, 39% serum albumin < 3.5 g/dl, 54% serum β2 microglobulin > 4 mg/dl. So far, on an intent-to-treat basis, 78% of patients achieved at least 50% reduction of serum monoclonal protein concentration. The median time to response was 3.6 months (range 0.7 months to 5.4 months). With a mean follow-up of 18 months, 90% of patients are progression-free. With the exception of one patient who died of interstitial pneumonia, the DRC regimen was well tolerated. 15% of patients developed grade 3, 4 neutropenia. Mild or moderate effects related to rituximab infusion such as fever, rigors and headache were seen in 20% of patients.

Conclusions: We administered a regimen which combined rituximab with cyclophosphamide, a non-stem cell toxic alkylating agent. The DRC regimen is an active and well tolerated primary treatment for symptomatic patients with WM. Patient accrual and evaluation is ongoing.
Immunotherapy of Chronic Lymphocytic Leukemia using CD40L and IL2 Expressing Autologous Tumor Cells.
Authors: Biagi Ettore , Popat Uday , Raphael Rousseau , Eric Yvon , Giampietro Dotti , Lawrence Rice , Andreef Michael , Brenner Malcolm
Published: October 2019

Transgenic human CD40 ligand (hCD40L) activates B-Chronic Lymphocytic Leukemia (B-CLL) cells by CD40 stimulation and may thereby enhance their capacity to present tumor antigens. Pre-clinical models show that co-expression of IL-2 further potentiates the immunogenicity of CD40L-expressing tumor cells. To discover whether these promising pre-clinical data could usefully be applied to patients with B-CLL, we used adenovectors to prepare hIL2- and hCD40L-expressing autologous tumor vaccines. Within these vaccines, a mean of 92% B-CLL cells were CD40L positive (versus <1% pre-treatment), and costimulatory molecules were also upregulated on the tumor cells (CD80 from 6% to 74% and CD86 from 16% to 73% positive cells). The mean secretion of IL-2 (measured by ELISA at 72 hours after transduction) was 1,780 pg/ml/10E6 cells (range = 175–400). To date, 8 patients have received between 5 and 8 subcutaneous injections in a modified Phase I design, in which the dose of IL-2 secreting cells was fixed at 2x10E7 per injection, while the dose of CD40L-expressing leukemia cells was escalated from 2x10E5 (level 1) to 2x10E7 (level 3) per injection. 12 additional patients will receive this final dose combination. Of those treated, two patients were in Rai stage 4; 2 in stage 3; 2 in stage 2; and 2 in stage 1. There were no adverse events from any injection in the patients. Injection-site biopsies revealed a modest infiltration of CD3+ cells, and the vaccine also had systemic immunomodulatory effects. Total T-cell numbers were significantly increased in only 2 patients, but an anti-B-CLL immune response was detected in 5/6 individuals currently analyzed. Using ELISPOT assays with autologous B-CLL cells as stimulators, and allogeneic B-CLL cells as controls, we found a rise in IFN-gamma spot-forming T-cells (<1/100,000 pre to >1/1500 post vaccination) and of Granzyme-B expressing T-cells (<1/100,000 pre to >1/2500 post). This reactivity is mediated at least in part by CD8-positive T cells with specificity to survivin, a known B-CLL associated tumor antigen. Although these immunologic effects have as yet been accompanied by only transient disease responses, our continuing accrual of additional patients who will receive immunomodulatory doses of vaccine should indicate whether the approach could contribute to the management of early or advanced B-CLL.