bluuebunny/crossref_metadata_2025 huggingface dataset:
Treatment of Osteopenia/Osteoporosis in Pediatric Hematopoietic Cell Transplantation.
Authors: Sanders Jean E. , Hoffmeister Paul A. , Storer Barry E.
Published: October 2019

The bone mineral density during childhood and the teen years is an important determinate of adult bone health. Decreased bone mineral density has been found to be a relatively common complication following adult hematopoietic cell transplantation (HCT), but has not been well studied after HCT among pediatric patients. Not surprisingly, an effective therapeutic approach to decreased bone mineral density has also not been developed for children.

Patients and Methods: The records of 196 children <18 years of age who received HCT between 1/97-6/03 who survived for 1 year or more after HCT were reviewed. Patients selected for analysis had decreased bone mineral density (Z-score of <-1 standard deviation (SD) corrected for patient sex and age) and 2 or more bone mineral density (DEXA) scans performed after HCT. Change in bone density was calculated as the difference in L2-4 lumbar spine SD score. Variables associated with change in bone density were analyzed.

Results: Among the 143 patients who had a DEXA scan performed, 82 (57%) demonstrated bone loss defined as <-1 SD Z-score. Fifty-six (68%) of these 82 patients had two or more DEXA scans and were analyzed. These 56 patients received an HCT when they were a median 10.8 (1.1–17.9) years of age, 33 were male, and 40 had chronic GVHD treated with corticosteroids and calcinurin inhibitor therapy. Patients were grouped according to treatment received for the bone loss. Nine patients changed therapy and were analyzed twice in separate treatment categories, hence there were 65 patients in 3 groups: 16 received a bisphosphonate or calcitonin (B/C), 37 calcium supplement only, and 12 no therapy. The B/C group included patients receiving pamidronate, zometa, fosamax, or calcitonin. Baseline median Z-scores were −2.6 SD for the B/C group, −2.2 SD for calcium, and −1.4 SD for no therapy. The median length of therapy was 446 days for the B/C group and 365 days for the calcium group. For the no therapy group, the time interval between DEXA scans was 324 days. Treatment with B/C or calcium compared with no therapy was significantly associated with improved bone density (p=0.0004). Corticosteroids, length of corticosteroid therapy (months), presence of chronic GVHD, gender, TBI received in preparative regimen, and patient age at therapy were not associated with change in bone mineral density. The mean change in bone mineral density per year was +1.16 SD for the B/C group, +0.16 for calcium, and +0.52 for no therapy. Although not statistically significant (p=0.44), concomitant steroid use for more than half the time interval between DEXA scans decreased therapeutic gains in the B/C group by −0.59 SD/year and −0.38 SD/year for the calcium group. The only observed side effect was hypocalcemia in one patient receiving pamidronate.

Conclusions: These data demonstrate that the greatest improvement in bone mineral density was observed in patients receiving a bisphosphonate or calcitonin. Minimal to no toxicity was observed.
Adhesion Molecule ICAM-4 Participates in Erythroblastic Island Formation.
Authors: Lee Gloria , Lo Annie , Short Sarah , Mankelow Tosti , Parsons Stephen , Spring Frances , Narla Mohandas , Anstee David , Chasis Joel Anne
Published: October 2019

Erythroblasts proliferate, differentiate and enucleate within erythroblastic islands, three dimensional structures comprised of developing erythroblasts surrounding a central macrophage. Collective evidence suggests that erythroblastic islands are highly specialized bone marrow subcompartments where adhesion events, in concert with cytokines, play critical roles in regulating erythropoiesis and apoptosis. ICAM-4, a recently characterized member of the immunoglobulin superfamily, is expressed early in erythroid differentiation. This adhesion molecule interacts with multiple integrin binding partners, including alpha4beta1 and alphaV integrins (alphaVbeta1, alphaVbeta3 and alphaVbeta5). Since erythroblasts express alpha4beta1 and ICAM-4 and macrophages exhibit alphaV, ICAM-4 is an attractive candidate for mediating erythroblast-erythroblast and erythroblast-macrophage attachments. A molecular model of ICAM-4 derived from the crystal structure of closely related ICAM-2 presents the extracellular region of ICAM-4 as two Ig-like domains comprised of A,B,C,D,E,F, and G strands. Employing targeted mutagenesis of surface-exposed amino acid residues, we earlier identified a patch or footprint that mediates adhesion to alphaV integrins comprised of three A strand residues and five G strand residues on N-terminal domain 1. To explore whether ICAM-4 attachments are active in erythroblastic islands we first developed a quantitative live cell assay for reforming islands from single cell suspensions of freshly harvested mouse bone marrow. Islands and their cellular components were identified and quantitated by three-color immunofluorescent microscopy employing fluoresceinated erythroid-specific TER119 antibody, macrophage-specific F4/80 antibody and a DNA probe. To determine the amount of variation in number of islands reformed from a single cell suspension of 1 x 105 cells, we counted islands at the beginning and conclusion of experiments on five different mice. The island numbers were very reproducible and equaled 1000 +/− 158. We then tested the effects of two synthetic peptides that we have previously shown block ICAM-4/alphaV adhesion: peptides FWV and ATSR, corresponding to sequences of the A and G strands of ICAM-4 domain 1, respectively. Both peptides caused a marked, concentration dependent decrease in the percentage of islands formed. 2mM ATSR inhibited island formation by 75% while 2mM FWV inhibited island formation by 70%. In marked contrast, a strand D control peptide had minimal to no effect on island formation. Our data strongly suggest that erythroblast ICAM-4 binding to macrophage alphaV is critical for erythroblastic island formation. We postulate that this newly identified receptor-counterreceptor interaction may be important not only for adhesive integrity of the island structure but also for initiating intracellular signaling essential for normal erythroid terminal differentiation.
Chimeric Anti-CD20 Monoclonal Antibody (Rituximab; Mabtheraâ) in Remission Induction and Maintenance Treatment of Relapsed /Resistant Follicular Non-Hodgkin’s Lymphoma : A Phase III Randomized Intergroup Clinical Trial.
Authors: Van Oers Marinus H.J. , Van Glabbeke Martine , Teodorovic Ivana , Rozewicz Cynthia , Klasa Richard , Marcus Robert E. , Wolf Max , Kimby Eva , Hagenbeek Anton
Published: October 2019

Background. In view of a) the efficacy of Rituximab (R) monotherapy in relapsed low grade lymphoma; b) the feasibility to combine R with cytostatic drugs c) the potential of such combinations to clear minimal residual disease, in 1999 we started a phase III clinical trial in patients with relapsed follicular non-Hodgkin’s lymphoma, with 2 main objectives. First: to compare CHOP and R-CHOP as to response rate and response quality. Second: to establish the effect of maintenance treatment with R on progression-free survival (PFS ).

Study design. Patients with stages III or IV follicular lymphoma at initial diagnosis and relapsed after or resistant to a maximum of two non-anthracycline containing systemic chemotherapy regimens, were randomized to remission induction with either 6 cycles of standard CHOP (once every 3 weeks) or CHOP + R (375 mg /m2 at day 1 of each cycle of CHOP). Those with a complete or partial remission after 6 cycles of therapy underwent a second randomization to no further treatment or maintenance treatment with R (375 mg/m2 once every 3 months until relapse or for a maximum period of two years).

Results. In February 2004, a preplanned second interim analysis by the IDMC was performed. At that time 461 patients had been included. Of these, 369 could be evaluated for response (188 CHOP; 181 R-CHOP). The groups did not differ as to age, sex, performance status, time from initial diagnosis, number and type of, and best overall response to prior chemotherapy. Both treatment arms yielded similar PR rates: CHOP 53.7%; R-CHOP 52.5 %. However, there was a highly significant difference in CR rates: CHOP 18.1% versus R-CHOP 30.4 % (p=0.0004). 319 patients have been randomized for maintenance treatment. Of these, 268 were evaluable (132 observation; 136 R maintenance). 1 year and 3 year PFS were 54.9% and 31.2% respectively in the observation arm, and 80.2 % and 67.7% in patients with R maintenance treatment (p< 0.0001). There is as yet no impact on OS: the observed difference (in favor of R maintenance) was not significant for an interim analysis (p=0.03). There were no differences in toxicity between CHOP and R-CHOP induction treatments. Maintenance treatment was associated with minimal toxicity. Because the planned second interim analysis revealed that the primary endpoints for both the induction and maintenance part of the study had been reached, the formal criteria for stopping the trial have now been met.

Conclusion. In patients with relapsed/resistant follicular lymphoma R-CHOP remission induction results in a highly significant increase in CR rate as compared to CHOP. Moreover, this is the first trial to show that in these patients Rituximab maintenance treatment achieves a considerable improvement in PFS.
Id/KLH Vaccine (FavId™) Following Treatment with Rituximab: An Analysis of Response Rate Improvement (RRI) and Time-to-Progression (TTP) in Follicular Lymphoma (FL).
Authors: Koc Omer N. , Redfern Charles , Wiernik Peter H. , Rosenfelt Fred , Winter Jane N. , Guthrie Troy H. , Kaplan Lawrence , Holman Peter , Densmore John , Hainsworth John , Lin Thomas , Castillo Rene A. , Janakiraman Nalini , Bender John F.
Published: October 2019

Background: Id/KLH vaccine (FavId) administered as a single agent has been associated with tumor regressions in patients with relapsed/refractory (RR) FL. B-cell depletion has been demonstrated to augment the T-cell immune response to subsequent vaccine administration in mice (Qin 1998 Nat Med 4:627).

Objective: To evaluate the efficacy and safety of Id-KLH administered during the period of rituximab induced B-cell depletion.

Eligibility: FL pts who were: treatment naïve (TN); RR following chemotherapy; or relapsed following rituximab.

Treatment: Following rituximab (375mg/m2 i.v. weekly x 4) pts received Id-KLH (1 mg s.q. monthly x 6) starting on week 12. GM-CSF, 250 mcg, was administered s.q. at the Id-KLH injection site on days 1–4. Pts could continue Id-KLH until progression.

Results: 103 pts received rituximab. Response to rituximab at month 3 was 35% (3-CR; 33-PR). Eleven (11) pts were PD following rituximab (11%). Id/KLH could not be made for 4 pts (4%). Eighty-eight (88) pts were begun on Id-KLH. Among the 45 RR pts, 32 (72%) have not progressed at a median follow-up of 12 months compared with 40% of historical control pts treated with rituximab alone (Witzig 2002 JCO 20:2453). Among the 43 TN pts, 82% have not progressed after a median follow-up of 9 months. RRI (SD to PR, PR to CR after month 3) was observed in 21 pts (12-SD to PR; 9-PR to CR). Robust T-cell responses to both Id and KLH were observed (3 of 3 pts tested). Anti-KLH antibody responses were generally not seen until B-cell recovery. The most frequent adverse event was an injection site reaction. A flu-like syndrome was also observed consistent with GM-CSF administration.

Conclusion: Id/KLH vaccine (FavId), administered to pts with FL during a period of B-cell depletion induced by rituximab, can result in an anti-Id T-cell response, and appears to result in an RRI and an increased TTP compared to historical controls. A randomized, double-blind, placebo-controlled, Phase 3 trial of rituximab + FavId has been initiated.
Salvage Therapy of Progressive and Recurrent Hodgkin’s Disease: Results from a Multicenter Study of the Pediatric DAL/GPOH HD-Study Group.
Authors: Schellong Guenther , Doerffel Wolfgang , Claviez Alexander , Koerholz Dieter , Mann Georg , Scheel-Walter Hans G. , Boekkerink Jos P.M. , Riepenhausen Marianne , Lueders Heike , Poetter Richard , Ruehl Ursula
Published: October 2019

To evaluate the efficacy of a salvage therapy (ST-HD-86) developed for children and adolescents with progressive or relapsed Hodgkin’s disease (HD) who were primarily treated in the pediatric DAL/GPOH studies. The essential chemotherapeutic elements were ifosfamide, etoposide, prednisone (IEP) and doxorubicine, bleomycin, vinblastine, dacarbazine (ABVD). 176 patients with progression (N=51) or first relapse (N=125) were enrolled by 67 centers from six countries between 1986 and 2003. The median time from initial diagnosis to progression/relapse was 1.1 (range, 0.02–17.1) years and the median patients age at the diagnosis of progression/relapse was 14.7 (range, 4.3–24.5) years. Salvage therapy consisted of 3–5 alternating cycles of IEP and ABVD, supplemented in part by 1–2 cycles of cyclophosphamide, vincristine, procarbazine, prednisone (COPP) or CCNU, etoposide, prednimustine (CEP). Radiotherapy was given to involved areas using individualized dosages. In the 1990ies, high-dose high dose chemotherapy with stem cell support was introduced for patients with unfavorable prognostic criteria. Disease-free survival (DFS) and overall survival (OS) at 10 years after initiation of salvage therapy are 63±4% and 74±4%. Second events occurred in 70 of 176 pts (40%). Risk factor analysis revealed the time until progression/relapse as the most discriminating prognostic factor (p=.0001). Patients with progression had an inferior outcome (DFS 41±7%, OS 51±8%), whereas patients with late relapses (>12 months after end of therapy) do very well (DFS 87±4%, OS 90±4%). The result of study ST-HD-86 with a long-term survival of 74% in this large cohort of patients with progressive or relapsed HD can be considered favorable. While the salvage strategy for patients with progression has to be further optimized, a reduction of intensity might be considered for those patients with late relapses following HD in childhood or adolescence.
Chronic Myeloid Leukemia (CML) in Childhood - Results of the Multicenter-Trial CML-paed.
Authors: Suttorp Meinolf , Claviez Alexander , Martiniak Yvonne , Klingebiel Thomas , Niethammer Dietrich , Gadner Helmut , Peters Chris , Kabisch Hartmut , Kremens Bernhard , Zintl Felix , Henze Guenter , Goebel Ulrich
Published: October 2019

CML is a rare disease in childhood and hematopoietic stem cell transplantation (SCT) remains the only proven option for cure of young patients (pts). We here report the results of the GPOH-trial “CML-paed”. From December 1995 to June 2004 pts younger than 19 years (median age: 11.4 yrs) with Philadelphia-chromosome positive CML (n=193; 99 boys, 94 girls) were treated by hydroxyurea ± interferon and scheduled for SCT from an HLA-matched family donor within 6 months after diagnosis (Dx) and from an unrelated donor within 12 months. 85% of the pts were diagnosed in chronic phase (CP). Six pts (3%) died from disease without SCT with a median interval from Dx to death of 6.5 months (range 0.5–12 months) and 25 pts are still searching for a donor. 168 pts underwent SCT (n=50 HLA-matched related; n=69 HLA-matched unrelated (MUD); n=18 HLA-mismatched related; n=31 HLA mismached unrelated) in CP (n=139), in accelerated phase (AP, n=9), in blast crisis (BC, n=9), or in 2. CP (n=10). Probability of overall survival (OS) was 75 % if SCT was performed <7 months after Dx (n=53 pts) and 60 % (n=100 pts) if pts were transplanted later, however, this difference was statistically not significant. Conditioning regimens included either total body irradiation (n=82) or busulfan (n=80) resulting in no statistically different impact on OS. 5-year OS was 82 % for SCT from HLA-matched related and 55% for HLA-MUD-SCT reflecting a higher transplant-related mortality for the latter (p=.0017). After SCT from HLA-mismached unrelated and HLA-mismatched related donors, OS was 56% and 50%, respectively. 12 out of 168 pts (12%) relapsed following SCT after a mean interval of 11 months (range: 1–137 mos) and 9 of them so far have died of CML. Outcome was inferior if SCT was performed in advanced stages of CML (OS of all pts in CP: 67%; in AP: 55%, in BC: 21%, respectively). During the last decade the 3-year OS after SCT from HLA-matched unrelated donors improved gradually from 45 % before the year 1994, to 53 % in the period 1995 to 1999 and to 62 % after 2000, respectively. This large series of pts from a controlled trial shows an excellent OS of 82% for pediatric pts with CML undergoing SCT from matched sibling donors and constantly improving results during the last decade in the setting of MUD-SCT.
Characterization of Platelet Lipid Rafts Leads to the Identification of RGS-19, Which Forms a Phosphorylation-Dependent Complex with 14-3-3ζ.
Authors: Foy Martina , Treumann Achim , Fitzgerald Desmond J. , Maguire Patricia B.
Published: October 2019

Lipid rafts are specialised membrane regions, rich in dynamic multi-protein complexes that are implicated in the regulation of platelet activation processes, particularly those mediated by GPVI and GPIb/IX/V. To elucidate the role of rafts in platelet function, we carried out a comprehensive characterization of the detergent-insoluble proteins associated with rafts from control and VWF-activated platelets using liquid chromatography coupled to tandem mass spectrometry. Over 160 proteins were identified including 72 proteins unique to platelet rafts and several novel platelet signaling and vesicle transport proteins e.g., rab 5, rab 8 and syntaxin 11. Indeed, the high level of signaling and trafficking proteins identified implicates rafts as concentrating platforms for the critical platelet functions of activation and secretion. Moreover, activation through GPIb-IX-V resulted in the translocation of many proteins into or out of rafts. Proteins recruited into rafts upon VWF-activation included GPIbαand regulator of G-protein signalling-19 (RGS-19). We confirmed that the novel platelet protein, RGS-19, upon VWF activation resides exclusively within the raft fraction and using confocal microscopy, RGS-19 co-localised with GPIbα and the raft-marker flottilin, to both the plasma and vesicle membranes of VWF-activated platelets. The phosphorylation-dependent interaction of RGS proteins with 14-3-3 leads to the inhibition of their GTPase-activating protein (GAP) activity. Aligning RGS-19 to other members of the RGS family, we identified that it contained a novel 14-3-3 binding motif. Immunoprecipitation with an antibody to 14-3-3ζ demonstrated that RGS-19 and GPIbα co-immunoprecipitated with 14-3-3ζ in VWF-activated rafts. Furthermore, we demonstrated that acid phosphatase treatment could significantly reduce the co-immunoprecipitation of RGS-19 with 14-3-3ζ from activated rafts. Thus, within 15 sec of VWF activation we are suggesting maximal suppression of the GAP activity of RGS-19 in rafts through a phosphorylation-dependent association with 14-3-3ζ. Our results provide the basis for a new hypothesis concerning how RGS proteins might govern signaling within platelet rafts.
AKT Activity Regulates Sensitivity of Multiple Myeloma Cells to mTor Inhibitors.
Authors: Frost Patrick , Hoang Bao , Gera Joseph , Sharma Anushree , Shi Yijiang , Yan Huajun , Lichtenstein Alan
Published: October 2019

Inhibitors of the mammalian target of rapamycin (mTOR), such as rapamycin and CCI-779, have potential as anti-tumor agents against multiple myeloma (MM). In a murine xenograft model, CCI-779 demonstrated efficacy against in vivo growth of OPM-2 and 8226 MM cells. In this model, OPM-2 tumors (ED50=2 mg/kg) were considerably more sensitive than 8226 (ED50=20 mg/kg) tumors. CCI-779-induced anti-tumor responses were associated with significant inhibition of proliferation and angiogenesis and concomitant upregulation of apoptosis. OPM-2 cells were also significantly more sensitive to these CCI-779-mediated effects. Other tumor models have demonstrated that heightened AKT activity induces hypersensitivity to mTOR inhibitors. As OPM-2 cells express high levels of activated AKT (due to PTEN mutations) and 8226 cells contain predominantly quiescent AKT, this regulatory role for AKT may be present in MM cells as well. To further test this, we stably expressed an activated AKT allele in U266 (U266myr-AKT) MM cells. The in vivo growth of U266myr-AKT cells was considerably more sensitive than control U266 cells to the anti-tumor effects of CCI-779. The differential sensitivity induced by AKT activation was mirrored in an enhanced sensitivity to CCI-779-mediated apoptosis and inhibition of angiogenesis. Since previous studies demonstrated the ability of AKT/mTOR to regulate the expression of vascular endothelial growth factor (VEGF), we hypothesized that MM cells with heightened AKT activity may be more sensitive to the CCI-779-mediated inhibition of this critical angiogenic factor. In vitro, mTOR inhibitor, rapamycin, was markedly more effective at inhibiting VEGF secretion from U266myr-AKT than control cells. Our results demonstrate that AKT regulates the sensitivity of MM cells to the anti-tumor effects of mTOR inhibitors and that this may be mediated through the inhibition of AKT-dependent survival and growth factors.
P-21 Activated Kinase 1 (Pak1) Is Critical for Normal Mast Cell Degranulation and Cytokine Production in Response to Antigen Stimulation of the High Affinity IgE Receptor.
Authors: Allen Jayme D. , Hoffman Clemens , Derr-Yellin Ethel , Bessler Waylan , Yang Fen-Chun , Ingram David A. , Jaffer Zahra , Tipton Amanda , Chernoff Johnathan , Clapp D. Wade
Published: October 2019

Mast cells participate in normal and pathogenic inflammatory processes, including innate host defense, allergy, and asthma. Stimulation of the mast cell high-affinity IgE receptor (FcεR) by receptor cross-linking activates multiple downstream signaling pathways resulting in degranulation and de novo synthesis of multiple cytokines. However, the molecular mechanisms underlying these processes are incompletely defined. It is known that Rac2 deficient murine bone marrow derived mast cells (BMMCs) have impaired degranulation, but the downstream effectors that modulate this function are unknown. We hypothesized that p-21 activated kinase 1 (Pak1), a downstream effector of Rac proteins, is important in degranulation and de novo cytokine synthesis. Mature BMMCs from wild-type (WT) and Pak1 KO mice were sensitized with anti-DNP IgE then stimulated with DNP-HSA to stimulate FcεR. Interestingly, Pak1 KO BMMCs showed significant impairment in degranulation, as demonstrated by a 3-fold reduction in the percent of B-hexosaminidase released upon IgE stimulation. IgE stimulation of mast cell results not only in degranulation, but also in the production of TNFα, which is critical in the early recruitment of neutrophils to sites of acute inflammation. TNFαsynthesis is influenced by a number of transcription factors, many which are regulated by Erk. Since Pak1 has been shown, in overexpression systems, to phosphorylate Raf and Mek to activate Erk, we examined Erk activation in WT and Pak1 KO BMMCs in response to IgE stimulation. Pak1 KO BMMCs have a 50% reduction in phospho-Erk as compared to controls. We then tested another MAPK member important in mast cell cytokine synthesis, p38, and found phospho-p38 to be decreased in Pak1 KO BMMCs as well. Further, IgE-stimulated Pak1 KO BMMCs produce only 20–30% as much TNFαas controls. To define the role of Pak1 in cytokine production as specific for TNFαversus a more global defect, we also studied IL-6 synthesis and are able to report a 50% reduction in IL-6 production by Pak1 KO BMMCs. Our results indicate that Pak1 is important in BMMC degranulation, cytokine production, and MAPK activation in response to FcεR stimulation. These studies identify Pak1 as a potential therapeutic target in pathologic inflammation. Mechanisms by which Pak1 may be influencing mast cell degranulation as well as further study of transcription factors important in mast cell cytokine production are under current investigation.
HOXB9 Is Overexpressed in Blast Cells from a Subset of Acute Myeloid Leukemia Patients and Supports Proliferation of AML Cell Lines.
Authors: Heinrichs Stefan , Schoch Claudia , Neuberg Donna S. , Look A. Thomas
Published: October 2019

Emerging evidence from studies in mice and humans suggests that HOX genes integrate the myelodysplastic and leukemogenic activities of different oncogenes. Translocations involving the MLL gene, a major upstream regulator of the HOXA cluster, have been identified in AML and define a specific cytogenetic subgroup of this disease. Recently, amplification of MLL was found in AML and MDS cases, revealing a second mechanism of deregulation of MLL and its downstream targets. However, additional leukemogenic upstream pathways have to be postulated, since upregulation of specific HOX genes, particularly HOXA9, is more frequent than MLL deregulation. Given the fact that HOXA9 is targeted by several yet to be identified mechanisms, we asked whether other members of the HOX9 paralog group are also highly expressed in myeloid malignancies. Our analysis of 449 AML patients by gene expression microarray revealed that besides HOXA9 (61% of all cases), HOXB9 is upregulated in 15% of the cases, whereas the overexpression of HOXC9 and HOXD9 expression is a very rare event (>1% of all cases). High levels of HOXB9 expression did not correlate with low levels of HOXA9 expression ruling out the possibility that HOXB9 compensates for a lack of HOXA9 activity in the leukemogenesis of a HOXA9 negative AML cases. Interestingly, within the cytogenetically defined subgroups (inv(16), t(15;17), t(8;21)) that have no HOXA9 expression at all, HOXB9 was also low or absent. To test if HOXB9 has the potential to contribute to the leukemogenic phenotype we analyzed a panel of human AML cell lines. The finding that 6 out of 24 cell lines express high levels of HOXB9 further indicates the significance of HOXB9 in myeloid malignancies. We chose K-562 and HEL as model cell lines since they express high levels of HOXB9, but lack HOXA9 expression. To reveal HOXB9 dependent pathways we designed two highly efficient siRNAs to target HOXB9 and knocked down its expression by retroviral transduction. In comparison to cells expressing a control siRNA, the resulting cell lines showed <5% of the control HOXB9 protein levels and a reduction in the cellular growth rate. Consequently, HOXB9 expression is required for the rapid growth of these AML cells. To gain insight into the molecular mechanisms underlying this phenotype we are performing global gene expression analysis. Preliminary results of our nearest neighbor analysis in K-562 cells revealed the upregulation of genes associated with erythroid differentiation upon HOXB9 knockdown. Our results indicate that HOXB9 expression inhibits the differentiation of leukemic myeloid progenitor cells, consistent with a role for the aberrant expression of this major HOX protein in leukemogenesis, by maintaining cells of the leukemic clone in an undifferentiated and rapidly proliferative state.
The Osteo-Hematon: Modeling the Ontogenic Development of Stem-Cell Niches into Discrete Organotypic Proliferative Units.
Authors: Blazsek Istvan , Oberlin Estelle , Souyri Michele , Peault Bruno
Published: October 2019

Hemogenic stem cells (HSC) emerge at distinct allocation territories during ontogenesis. They irrigate freely the whole body by the blood stream, but the adult HSC expand and differentiate exclusively in the bone marrow (BM). This implies that stem cells are not autonomous units of development; rather, tissue specific niches control their destiny. We investigated the spatio-temporal emergence of stem cells and nest-forming cells, and basic mechanisms by which they shape together the functional-proliferative units in the liver and BM during ontogenesis. Video microscopy in gut/liver primordium cultures provided direct evidence for intrinsic emergence of a primitive vasculo-hemogenic meshwork guided by pulsative, cardiomyogenic cells around nascent hepatic cords in E8.75-E10 mouse embryos. Gain-of-competence and development of hepatic units required firm aggregation of cholangio-hepatic (CK19+/CK18+), hemogenic (CAFC) and vasculogenic stem/progenitor cells and nest-forming cells into compact 3-D structures, similarly to BM hematons. Colonization and expansion of HSC in the mouse liver at E12, then in the BM at postnatal d2, numerically correlated with the increase in isolatable niches/hematons. Time-lapse video microscopy, quantitative colony assays, FACS and immunocytochemistry analyses of the adult human BM showed that the hematon is enriched in mesenchymal stem cells, preadipocytes/adipocytes, contractile myogenic cells, a vascular meshwork, guardian macrophages and hemopoietic stem/progenitor cells (Sca-1/c-kit/CD34/Thy-1/CD31+). A hematon module plays the role of an organizing center that emits growth signalling factors and accumulates morphogen/differenciator factors, retinoids and 1,25-di(OH)-vitaminD3. The physical attachment of many hematons to spongy bone suggests that they form the basic structural-functional units in the ultimate, fully competent BM. We suggest designating these units osteo-hematons. A comparative analysis of BM aspirates from healthy donors (n=75) and patients (n=210) revealed the disorganization of osteo-hematon modules in AML prior to therapy and in CML during all stages of the disease.
High Cyclin D1 Expression Is Associated with Increased Proliferation Rate and Decreased Survival in Mantle Cell Lymphoma (MCL) and Is Caused by Genomic Deletions and Mutations that Enhance Stability of Cyclin D1 mRNA.
Authors: Wiestner Adrian , Chiorazzi Michael , Lai Raymond , Rosenwald Andreas , Muller-Hermelink H. Konrad , Ott German , Chan Wing C. , Greiner Timothy C. , Weisenburger Dennis D. , Vose Julie , Armitage James O. , Gascoyne Randy D. , Connors Joseph M. , Campo Elias , Montserrat Emilio , Bosch Francesc , Smeland Erlend B. , Kvaloy Stein , Holte Harald , Delabie Jan , Fisher Richard I. , Grogan Thomas M. , Miller Thomas P. , Wilson Wyndham H. , Jaffe Elaine S. , Staudt Louis M.
Published: October 2019

Most MCLs harbor the t(11;14) translocation resulting in overexpression of cyclin D1 that promotes tumor proliferation. Gene expression profiling of 92 cyclin D1 positive MCL biopsy samples defined a gene expression signature of tumor proliferation that provided strong prognostic information: the quartile of patients with the highest proliferation gene expression signature had a 0.8 year median survival whereas the quartile with the lowest expression had a 6.7 year median survival (Cancer Cell 2003, 3:185). Sixteen of the 23 MCL samples in the highest proliferation quartile displayed exceptionally high cyclin D1 levels and preferentially expressed short cyclin D1 mRNA forms that lacked parts of the 3′UTR known to harbor RNA-destabilizing elements. To test whether expression of short cyclin D1 mRNA is due to genomic deletions, we screened DNA from all samples in the highest proliferation quartile by quantitative genomic PCR. We found genomic deletions of the 3′UTR region in 6 of the 10 samples that expressed short mRNAs. We next sequenced the proximal 400bp region of the 3′UTR in those samples with high expression of short mRNAs that did not have genomic deletions. In 2 samples, we found 2 distinct mutations: a 3bp deletion and an A insertion. Both of these mutations created a premature polyadenylation signal (AATAAA) about 300bp 3′ of the stop codon, giving rise to a predicted mRNA of 1.5kb that lacks the destabilizing elements present in the full-length form. We identified two MCL cell lines, Jeko-1 and Z138, that also showed high-level expression of the short cyclin D1 form. In Jeko-1 we found a deletion of the 3′UTR similar to that observed in the MCL patient samples, whereas the mechanism responsible for expression of the short cyclin D1 mRNA in Z138 remains unclear. In both lines, we confirmed that the half-life of the short cyclin D1 mRNA is longer than that of the full length mRNA. Our results demonstrate that MCLs increase cyclin D1 expression not only through the t(11;14), but also by the additional mechanisms of genomic deletions and mutations that create alternative polyadenylation sites that yield more stable mRNAs. Higher cyclin D1 expression increases proliferation and decreases survival, suggesting that strategies aimed at reducing cyclin D1 expression in MCL could have therapeutic promise.
Incidence of Recurrent Venous Thromboembolism According to Duration of Vitamin K Antagonists: Results of a Meta-Analysis on Individual Data.
Authors: Boutitie Florent , Pinede Laurent , Schulman Sam , Agnelli Giancarlo , Rascob Gary , Jullian Jim , Hirsh Jack , Kearon Clive
Published: October 2019

Objective: 1) to describe the incidence of recurrent venous thromboembolism (VTE) after stopping treatment with vitamin K antagonists (VKA); 2) to determine if the incidence of recurrent VTE is influenced by the duration of VKA therapy.

Methods: We performed a meta-analysis on the individual data of patients included in five randomized controlled trials (ODAC, DURAC 1, LAFIT, WODIT DVT, DOTAVK studies) that assessed several durations of oral treatment with VKA after an episode of VTE. A Poisson linear regression model was used to estimate the rate of recurrent VTE over time in patients off treatment with VKA, according to the duration of treatment (1.5, 3, 6 and ≥12 months).

Results: 2365 patients were followed for an average of 24.5 months after stopping therapy, and 330 recurrent VTE were observed. Estimation of the hazard functions in patients off treatment revealed that the rate of recurrent VTE decreased rapidly over the first year following the index event, independently of the duration of treatment with VKA, and stabilized afterwards. In addition, a peak of recurrences was observed in the first 1 to 3 months after stopping therapy, in all treatment duration groups. The rate of recurrent VTE after the short-term peak did not seem to be modified by the duration of therapy, after accounting for the time elapsed since the index event.

Discussion: After a treated episode of VTE, the rate of recurrence declined exponentially over time. This result may be explained by 1) the natural evolution of recurrence rate after an acute VTE; 2) the heterogeneity of recurrence rate among patients (with higher risk patients experiencing recurrences sooner than lower risk patients). The peak of recurrent VTE observed in the first 1 to 3 months after stopping treatment regardless of the duration of anticoagulant therapy may suggest that some patients are exposed to a very high risk of recurrence as soon as the protective effect of treatment is released.
siRNA-Mediated Silencing of αII Spectrin Gene Expression in Cells Containing DNA Interstrand Cross-Links and Its Relationship to Fanconi Anemia.
Authors: Lambert Muriel W. , McMahon Laura W. , Lefferts Joel A. , Sridharan Deepa M.
Published: October 2019

Fanconi anemia (FA) is characterized by bone marrow failure, a predisposition to cancer and, at the cellular level, a hypersensitivity to DNA interstrand cross-linking agents that correlates with a defect in ability to repair cross-links produced by these agents. We have previously shown that the structural protein, nonerythroid a spectrin (αSpII∑*), binds to DNA containing interstrand cross-links and plays an important role in the repair of this type of damage and that there is a deficiency in this protein in cells from FA-A, FA-B, FA-C, FA-D1, FA-F and FA-G cells. We have also shown that after damage to normal cells, αSpII∑* co-localizes in nuclear foci with FANCA, FANCF and the DNA interstrand cross-link repair protein XPF. In FA-A cells, where there is a deficiency in αSpII∑*, damage-induced nuclear foci formation is significantly reduced. In order to further assess the functional importance of αSpII∑* in the repair process and the DNA repair defect in FA cells, studies were carried out on siRNA-mediated silencing of αII spectrin gene expression. Three siRNA oligonucleotide templates were synthesized, which were targeted for different αII spectrin gene sequences. The 21-mer siRNAs produced were purified and normal human lymphoblastoid cells were transfected with these siRNAs. αII spectrin mRNA and protein levels in transfected cells were determined by RT-PCR and Western blot analysis, respectively. By 48 hours after transfection, levels of αII spectrin mRNA and protein were approximately 34% and 30% of mock transfected cells, respectively. Cells were then damaged with 8-MOP plus UVA light so as to produce DNA interstrand cross-links and localization of αII spectrin and FANCA in the nuclei was examined by immunofluorescence microscopy. The results showed that, in cells transfected with αII spectrin siRNA, there was a marked reduction in the number of αII spectrin and FANCA foci in the nuclei of the siRNA transfected cells compared to the mock transfected cells. There was also markedly reduced survival of the siRNA transfected cells after damage compared to mock transfected cells. These results show that there is a correlation between a reduction of αII spectrin levels in these cells and decreased formation of FANCA as well as αII spectrin nuclear foci after damage with a DNA interstrand cross-linking agent and that this in turn correlates with decreased survival and DNA repair in these cells after DNA damage. These results support our model that αII spectrin is needed in the DNA repair process where it acts as a scaffold in the recruitment and alignment of FANC and repair proteins at sites of DNA damage and that, in FA cells, where there is a deficiency in αII spectrin, this recruitment and repair are defective.

α∑
Prospective Evaluation of a GvHD-Specific Proteom Pattern.
Authors: Weissinger Eva M. , Kaiser Thorsten , Kamal Haytham , Kolb Hans-Jochem , Holler Ernst , Ganser Arnold , Hertenstein Bernd
Published: October 2019

We have recently described and published a proteom pattern specific for the early diagnosis of acute GvHD, based on the application of capillary electrophoresis (CE) and mass spectrometry (MS). Here we report the application of the previously described GvHD-pattern, consisting of 29 polypeptides discriminatory for differential diagnosis of GvHD, towards the analysis of urine of a new group of 35 patients in the follow up phase after allogeneic HSCT. The samples were collected prospectively and analysed in a blinded fashion.

Fifteen patients were transplanted from matched unrelated donors, while 20 received stem cells from family donors. GvHD prophylaxis was metotrexat and cyclosporin A. Urine samples were collected prior to conditioning and once a week during the follow up phase. Screening of the patients’ urine with CE-MS yielded between 500 and 2500 polypeptides defined via their mass, charge, and retention time in the CE-MS. These polypeptides were depicted as a three dimensional picture (contour plot, Diapat) and the data for each individual patient were stored in a Microsoft Access data base. The evaluation of urine of 40 patients after HSCT (5 autologous, 35 allogeneic) led to the establishment of a GvHD-specific pattern, consisting of 16-GvHD-specific and 13- sepsis-specific polypeptides (Kaiser et al., Blood 2004). Sixteen differentially excreted polypeptides formed a pattern of early GvHD markers, allowing discrimination of GvHD from patients without complications with 82% specificity and 100% sensitivity, cross validated. Inclusion of 13 sepsis-specific polypeptides allowed to distinguish sepsis from GvHD with a specificity of 97% and a sensitivity of 100%. Sequencing two prominent GvHD-indicative polypeptides led to the identification of a peptide from leukotriene A4 hydrolase and a peptide from serum albumin. The comparison of the newly collected data to the GvHD-specific patterns will be presented.

Figure Figure
Dysregulation of MMSET Is Tumorigenic In-Vivo.
Authors: Chesi Marta , Naik Kruti , Robbiani Davide F. , Affer Maurizio , Nickerson Helen D. , Bergsagel Peter L.
Published: October 2019

Approximately 15% of multiple myeloma (MM) is characterized by a t(4;14) translocation that causes the simultaneous dysregulation of MMSET on der(4) and fibroblast growth factor receptor 3 gene (FGFR3) on der(14). We reported several lines of evidence indicating a role for FGFR3 in myeloma tumorigenesis. First, activated FGFR3 is an oncogene capable of transforming fibroblasts. Second, FGFR3 activating mutations are acquired by MM cells during tumor progression. Third, targeted inhibition of FGFR3 leads to terminal differentiation and apoptosis in two t(4;14) MM cell lines. However, expression of FGFR3, but never of MMSET, is lost in about 25% of t(4;14) MM. Therefore, the overexpression of MMSET in all MM tumors with a t(4;14) translocation, and its homology to MLL, the oncogene on 11q23 translocated in acute leukemia suggest a critical role for MMSET in MM.

To determine whether MMSET is an oncogene in vivo, we have generated transgenic mice in which MMSET expression in driven in lymphocytes by the lck proximal promoter juxtaposed to the Emu enhancer. Using the same expression vector we and others have obtained specific, high levels of transgene expression in B and T cells from spleen, bone marrow and thymus. Four transgenic lines were generated and although we detected MMSET expression in T cells in each of them, unexpectedly no expression in B cells was seen. This is consistent with our inability to ectopically express MMSET in B cell lines. Nevertheless B lymphoid tumors expressing MMSET developed at 23 month of age in each line (18/51 mice). Only 1/19 wild type matching control mice developed a splenomegaly. By Southern blot, monoclonal rearrangements of IgH, IgL and TCR β were detected within the same tumor population.

In conclusion, this is the first report that MMSET is an oncogene capable of transforming lymphoid cells in an animal model. We are currently crossing these mice with FGFR3 transgenic mice to assess cooperation between these two oncogenes in tumorigenesis. Obviously a more restricted expression of MMSET in germinal center cells is required to investigate the role of MMSET in MM. Therefore, as we have done for c-myc, we are generating new transgenic mice in which MMSET expression will be activated sporadically in germinal center B cells by somatic hypermutation.
Bexarotene-Induced T-Cell Immunomodulation and Response in CTCL.
Authors: Porcu Pierluigi , Baiocchi Robert , Buckner Maureen , Byrd John C. , Magro Cynthia M.
Published: October 2019

Cutaneous T-cell lymphoma (CTCL) is a group of chronic lymphoproliferative disorders mostly of skin-homing CD4+ T-cells associated with profound suppression of cell-mediated immunity and loss of T-cell reportoire. The immunological effects of current CTCL therapies and their impact on response have not been studied in large samples of patients. Bexarotene is a synthetic retinoic X receptor (RXR) agonist that induces apoptosis in malignant T-cells and has significant clinical activity in CTCL. Bexarotene also exerts multiple effects on normal T-cells. We investigated the in-vivo immunomodulatory effects of bexarotene in patients with CTCL and correlated them with response. 37 patients (pts) with stage IB-III CTCL (33 Mycosis Fungoides, 1 ALCL, 3 pleomorphic small cell) received oral bexarotene (150–300 mg/m2/day) for a median duration of 13 months (range 4–18). Peripheral blood (PB) T-cell subpopulations were measured by multicolor flow cytometry at baseline and during therapy. Circulating CTCL cells were defined as CD4+ CD7− T-cells. 32/37 patients had an elevated PB CD4/CD8 ratio at diagnosis, regardless of the presence of circulating CTCL cells (3/37 pts) and 33/37 pts had a low absolute CD8+ T-cell count (median 98 cells/mm3, normal 150–1000/mm3). After a median time of 6.5 weeks on bexarotene (range 3.5–12) the CD8+ T-cell count had returned within normal range in 26/33 pts and the CD4/CD8 ratio had decreased in 27/32 pts. Responses (defined as Pysician Global Assessment [PGA] of clinical condition) were observed in 24/37 pts (64.8%). Responders had significantly higher peak CD8+ T-cell counts compared to non-responders (median 975/mm3 vs 221/mm3, P=0.002) and lower CD4/CD8 ratios (median 0.8 vs 2.4, P=0.005). At this time 21 pts have relapsed, with median duration of response 9.5 months. A ≥50% decrease in the PB CD8+ T-cell count preceded cutaneous relapse in 17/21 pts (81%) by a median time of 4.5 weeks (range 3–6.5 weeks). Functional analysis (mitogenic response, cytokine secretion, antigenic repertoire) of PB T-cell subpopulations from these pts at baseline and during therapy with bexarotene is in progress. Bexarotene appears to have a profound in vivo T-cell immunomodulatory effect in CTCL pts. The importance of these immune effects for clinical response vis-a-vis direct induction of apoptosis in CTCL needs to be further studied. If these results are confirmed in larger samples, monitoring of PB T-cell subpopulations may provide clinically valuable information in predicting response and relapse.
Mutations in the Gene of Neutrophil Elastase Associated with Congenital Neutropenia Lead to Alteration of Intracellular Trafficking and Proteolytic Function of NE Protein.
Authors: Beger Carmela , Koellner Inga , Zeidler Cornelia , Germeshausen Manuela , von Neuhoff Nils , Schlegelberger Brigitte , Welte Karl
Published: October 2019

Severe congenital (SCN) and cyclic neutropenia (CyN) are characterized by early-stage maturation arrest of myelopoiesis and often are found associated with different heterozygous mutations in the gene encoding neutrophil elastase (ELA2). Neutrophil elastase (NE) is a serin protease stored mainly in azurophilic granules. However, the consequences of the mutations of this protein up to date are not clear. Recent studies suggest a correlation with incorrect intracellular transport leading to mis-localization of the mutant protein. In the present study, we have generated an ELA2-inducible expression system based on the monoblast-like cell line U937. This system allows controlled expression of the transgene to study functional correlations with specific mutations of ELA2. Applying this system, we have studied intracellular processing and proteolytic activities of ten different mutations of ELA2 that are typically detected in patients suffering from SCN or CyN. We detected two clusters within the neutrophil elastase protein, in which mutations caused alteration of protein glycosylation, associated with prevention of membrane localization of the mutant protein. Instead, the mutant protein was incorrectly directed to the nuclear region. Proteolytic activities of NE protein were measured using different substrates. NE activities were suppressed in most but not all mutations tested. To extend our localization studies, we analyzed the intracellular distribution of NE in granulocytes of patients suffering from CyN or SCN. Again, NE protein of patients was mis-localized to the nucleus correlated with loss of detection in granules and membranes as compared with healthy donors without or with rh-G-CSF treatment. In summary, our data suggest that intracellular processing and localization is altered due to specific mutations of ELA2, which may be crucial for the molecular mechanisms resulting in the neutropenic phenotype.