bluuebunny/crossref_metadata_2025 huggingface dataset:
Hematopoietic Stem Cells Overexpressing Smad7 Exhibit Increased Self-Renewal and Regeneration Capacity in Vivo.
Authors: Blank Ulrika , Larsson Jonas , Utsugisawa Taiju , Magnusson Mattias , Klintman Jenny , Karlsson Stefan
Published: October 2019

The hematopoietic stem cell (HSC) resides in the bone marrow (BM) and can self-renew to generate more stem cells as well as differentiate into all hematopoietic lineages. The precise molecular mechanisms, which govern HSC fate decisions are poorly understood. The Transforming Growth Factor-β (TGF-β) superfamily of ligands, including the TGF-βs, Activins and Bone Morphogenetic Proteins (BMPs), encompasses an important group of growth factors, many of which have been shown to modulate and regulate hematopoiesis. Smad7 is known to block the phosphorylation event of receptor-activated Smads, thus creating a block in the entire signaling cascade downstream of TGF-β and related factors. To assess the effect of blocking the entire Smad signaling pathway downstream of TGF-β/Activin and BMP in HSCs in vivo, we have overexpressed the inhibitory Smad7 by a retroviral gene transfer approach. Both control and Smad7 vectors were MSCV based and expression was driven by the LTR promoter. The Smad7 vector contained the cDNA sequence for murine Smad7 and an internal ribosomal entry site (IRES) followed by GFP, whereas the control vector contained IRES and GFP only. In these experiments BM from wild type C57/B6 mice could efficiently be transduced with Smad7 or control vectors respectively. Upon transduction, cells (Ly5.2) were transplanted in a competitive fashion into lethally irradiated recipients (Ly5.1) and transduced cells were monitored by GFP fluorescence. Smad7 overexpressing cells were able to long-term reconstitute as well as give rise to both lymphoid and myeloid compartments at normal distributions. When self-renewal was assessed by secondary transplantations, Smad7 overexpressing cells showed significantly increased reconstitution ability compared to control transduced cells (blood samples at 12 weeks post transplant: 37.3 ± 5,9 for Smad7 vs. 5.06 ± 1,7 for control. Data represent % GFP positive cells ± SEM). Furthermore, Western blot analysis showed efficient expression of Smad7 protein in BM cells originating from transduced cells of transplanted mice. In addition, Smad2 and Smad1 phosphorylation was blocked upon TGF-β, Activin or BMP stimulation in BM cells, suggesting that Smad7 was functionally active in BM cells in vivo. However, when cultured under serum-free conditions in vitro, Smad7 overexpressing cells exhibited reduced proliferative capacity as compared to control transduced cells (3.5 times fewer cells by day 12 post transduction), suggesting that the in vivo phenotype was dependent on the BM microenvironment. Taken together, our data indicate that blocking of several TGF-β pathways simultaneously increases the self-renewal ability of HSCs in vivo, but not in vitro.
Mutations of the SHP-2 Proteine Tyrosine Phosphatase in Adult Patients with Acute Myeloid Leukemia.
Authors: Thiede Christian , Steudel Christine , Schäkel Ulrike , Ehninger Gerhard
Published: October 2019

Acute myeloid leukaemia (AML) represents a heterogeneous group of early stem cell malignancies characterized by an uncontrolled expansion of malignant cells blocked at certain stages of myeloid differentiation. Several genetic abnormalities have been described, including translocations involving genes of the CBF family, as well as activating mutations of signal transduction proteins, e.g. N-RAS, K-RAS or the Flt3- receptor tyrosine kinase (RTK). More recently, mutations in the SHP-2 protein, a tyrosine phosphatase involved in RTK signal transduction, have been described in patients with juvenile myelomonocytic leukaemia (JMML) as well as paediatric patients with AM. Only limited data on this aberration are available in older patients. In order to investigate the prevalence and the prognostic relevance of this gene in adults, we analyzed the mutational status of SHP-2 in a cohort of patients with AML.

Patients and Methods: DNA samples prepared from bone marrow or peripheral blood samples taken at diagnosis from 977 patients treated in the AML96 protocol of the SHG Dresden were analyzed. Screening for mutations in the mutational hot spot in exon 3 of the PTPN11 gene coding for SHP-2 was performed on a dHPLC system (Transgenomic). Samples with aberrant dHPLC chromatogram were sequenced.

Results: In the 977 patients investigated so far, 29 PTPN11 exon 3 mutations were found (3%). All mutations consisted of single base pair exchanges in codons 60–76, mostly affecting codons 69 (n=6), 72 (n=7) and 76 (n=6). Patients with PTPN11 mutations were more likely to have FAB M0 (p= .038) or M5b (p=.045) morphology than negative cases and had a significantly lower prevalence of FLT3-ITD mutations (p= .017). PTPN11 mutations were associated with a normal karyotype in 15/28 evaluable patients, whereas 13 had cytogenetic abnormalities, all of which were either high risk (−7/7q-, n=4; inv3, n=2, complex, n=2, −5, n=1) or intermediate risk (n=4) aberrations. No significant differences were found in the complete remission rate as well as the overall and event free survival, but a trend for a shorter disease free survival was observed in patients with PTPN11 mutations (median 7.8 vs 14.4; p= 0.06) associated with a significantly increased rate of relapse (p= .02). Although not significant yet, a similar trend was seen for patients with normal karyotype.

Conclusion: PTPN11 mutations can be found in a small but considerable number of adult patients with AML. Consistent with the data in children, this mutation seems to more prevalent in patients with monosomy 7 and leukemias with monocytic differentiation. Although very preliminary, the data point to a worse prognosis of patients with SHP-2 mutations. We are currently further extending the number of patients and analyze additional exons to delineate the potential prognostic role of this aberration.
Death Associated Protein Kinase 2 (DAPK2): A Novel Epo-Modulated Apoptotic Regulator of Late Erythropoiesis.
Authors: Menon Madhu , Vincent Bethany , Breggia Anne , Wojchowski Don M.
Published: October 2019

Death-associated kinase-1 (DAPK1) is a broadly expressed pro-apoptotic cytoplasmic S/T protein kinase that regulates cell proliferation and survival, modulates a p53 apoptotic checkpoint, and contributes to ALL, AML, and myelodysplastic syndromes. DAPK2, by comparison, is an under-studied (but likewise pro-apoptotic) DAPK1 orthologue that we have discovered to be expressed (via Affymetrix gene profiling) at high-levels in late-stage erythroid progenitor cells. In particular, and as examined in purified and developmentally-staged proerythroblasts, basophillic erythroblasts and orthrochromatic erythroblasts, DAPK2 gene expression was sharply up-modulated as progenitor cells developed to a Ter119(+), CD71-high stage. Using a DAPK2-specific antibody, this was confirmed at the protein level, and initial analyses of expression in cell lines further indicated lineage-restricted DAPK2 expression (e.g. high level in erythroid GIE2 cells but undetectable in pluripotent EML cells). To advance an initial understanding of DAPK2’s role in erythroid cells, its possible regulation via the Epo receptor (EpoR) also was studied. Here, primary erythroid progenitor cells from mice with knocked-in minimal EpoR alleles were used (PY-null and PY343-only EpoR-HM and EpoR-H forms, respectively). Interestingly, the point-mutation of a PY3434/Stat5 specific binding site (to F343) (EpoR-HM allele) resulted in a clear multi-fold de-repression of DAPK2 that also correlated with a decreased survival potential for EpoR-HM erythroblasts. This repressive effect of EpoR/PY343/Stat5 signaling on DAPK2 expression was highly stage-specific, and was exerted distinctly in advance of an observed EpoR/PY343/Stat5-dependent up-modulation of Bcl-xl expression. In addition, in wild-type EpoR pro-erythroblasts, DAPK2 levels increased with decreasing Epo concentrations. Investigations therefore reveal DAPK2 to be a late-stage erythroid- restricted, (and EpoR/PY343/Stat5- modulated) novel candidate regulator of red cell production.
Repetitive High-Dose Therapy with Dose Escalated CHOP + Etoposide Followed by Autologous Stem Cell Transplantation (Mega-CHOEP) in Younger Patients with Primary Diagnosis of Aggressive NHL and Elevated LDH at Diagnosis: Final Analysis.
Authors: Schmitz Norbert , Kloess Marita , Engert Andreas , Berdel Wolfgang E. , Metzner Bernd , Truemper Lorenz , Loeffler Marcus , Pfreundschuh Michael , Glass Bertram
Published: October 2019

As a novel approach to primary treatment of aggressive lymphoma, we tested the feasibility and efficacy of a four cycle high-dose chemotherapy protocol including autologous stem cell transplantation after cycles 2, 3 and 4. In a multicenter Phase II study patients with newly diagnosed aggressive NHL, age between 18–60 years, and LDH > N at diagnosis were included. At dose level 1, cycle 1 consisted of Cyclophosphamide (CY) 1500 mg/m2, Adriamycin (ADR) 70 mg/m2, Vincristine 2mg, Etoposide (ETO) 450 mg/m2, and Prednisone 500 mg/m2. In subsequent cycles doses of CY and ETO were increased: Cycle II and III : CY 4500 mg/m2 and ETO 600mg/m2, cycle IV: CY 6000 mg/m2 and ETO 1000 mg/m2. At dose level 2 ETO was further increased throughout all cycles to 600, 960, 960 and 1480 mg/m2, respectively. From February 97 to August 99, 124 patients were enrolled 14 patients had to be excluded mostly due to correction of initial diagnosis. 110 patients were evaluable with a median observation time of 55 months. 81.8% of patients completed therapy as per protocol. There were 5 cases of treatment related mortality (4.5%), and one of these deaths was due to secondary leukemia (0.9%). Overall survival at 5 years was 67.2 % and freedom from treatment failure was 62.1 %. The following factors were tested with respect to their impact on time to treatment failure (TTTF): age, sex, extranodal disease, bulky disease, performance status, B-symptoms, stage, age-adjusted IPI and LDH. In univariate analysis, only the risk factors of the age-adjusted IPI and IPI itself showed a significant impact on TTTF: see table 1. In a Cox regression multivariate analysis, LDH remained the only independent risk factor with a relative risk of 2.0 (p=0.046). Mega-CHOEP is feasible and effective treatment in younger pts. with aggressive lymphoma. Elevated LDH has major prognostic significance in patients receiving repetitive high-dose therapy and autologous stem cell transplantation. A phase III study comparing Mega-CHOEP + Rituximab to conventional chemotherapy in younger pts. with aggressive NHL is ongoing.

Table 1

Risk factor 2-years TTTF p-value LDH (< 2xONV/> 2xONV) 73.1 vs 48.2% p=0.004 Stage (I, II/III, IV) 76.5 vs 60.9% p=0.044 Performance status (ECOG 0, 1/>1) 73.2 vs 51.2% p=0.047 Age-adjusted IPI (1, 2/3) 72.4 vs 45.4% p=0.007
CHOP Improves Response Rates but Not Overall Survival in Follicular and Mantle Cell Lymphoma (MCL)- Results of a Randomized Trial of the German Low Grade Lymphoma Study Group (GLSG).
Authors: Nickenig Christina , Dreyling Martin H. , Schiegnitz Eva , Pfreundschuh Michael , Truemper Lorenz H. , Reiser Marcel , Wandt Hannes , Lengfelder Eva , Ludwig Wolf-Dieter , Berdel Wolfgang E. , Metzner Bernd , Hess Georg , Forstpointner Roswitha , Parwaresch Reza , Hasford Joerg , Unterhalt Michael , Hiddemann Wolfgang
Published: October 2019

In advanced stage follicular lymphoma conventional chemotherapy is non-curative and no major improvement in overall survival has been achieved by different regimens. Similarly, MCL, a lymphoma subtype with an especially poor clinical outcome, cannot be cured by conventional chemotherapy. In 1996, the German Low Grade Lymphoma Study Group (GLSG) started a randomized trial to evaluate the efficacy of two different anthracycline/anthrachinon containing regimens comparing CHOP (cyclophosphamide 750 mg/m2 day 1, vincristine 1.4 mg/m2 day 2, adriamycine 50 mg/m2 day 1, prednisone 100 mg/m2 days 1–5) and MCP (mitoxantrone 8 mg/m2 days 1–2, chlorambucil 3x3 mg/m2 days 1–5; prednisone 25 mg/m2 days 1–5). 415 previously untreated patients with advanced stage indolent lymphoma were prospectively randomized to receive either 6–8 cycles of CHOP or MCP and are evaluable for induction therapy. 277 patients (67%) had a follicular lymphoma, 86 (21%) had a mantle cell lymphoma and 52 (13%) patients had another indolent histology. Responders up to 60 years were subsequently assigned to either myeloablative radiochemotherapy and autologous stem cell transplantation or to interferon-a maintenance (IFNa ), all other patients received IFNa. As stem cell mobilization was hampered in the MCP arm (only 44% sucessful mobilisations after MCP, 93% after CHOP; p=0.0003), from July 1998 all younger patients were asssigned to the CHOP arm. 86% complete and partial remissions (18% CR) were observed in the CHOP arm, whereas after MCP an overall response rate of 77% was obtained (14% CR, p=0.0094). In subgroup analysis similar improvement of remission rates were detected in follicular lymphoma (91% vs 82%, p=0.026) and mantle cell lymphomas (87% vs 73%, p=0.080). No differences, were observed, however, between both regimens for progression-free survival in both lymphoma subtypes. Overall survival was comparable for FL in both study arms (74% vs 69% at five years, p=0.29). In MCL, overall survival was slightly higher in the CHOP arm (5y OS: 57% vs. 31%; p=0.0578), but this difference was mostly due to unbalanced patient characteristics (IPI). In conclusion, CHOP appears superior to MCP in achieving a higher initial response rate but has no long term impact on response duration or overall survival.
Targeting the Insulin-Like Growth Factor-I Receptor (IGF-IR) in Multiple Myeloma Cells Using Selective IGF-IR Tyrosine Kinase Inhibitors.
Authors: Stromberg Thomas , Ekman Simon , Girnita Leonard , Lennartsson Johan , Hellman Ulf , Carlson Kristina , Osterborg Anders , Vanderkerken Karin , Axelson Magnus , Larsson Olle , Nilsson Kenneth , Jernberg-Wiklund Helena
Published: October 2019

In multiple myeloma (MM) emerging evidence suggest the IGF-1R as an important mediator of tumor cell survival and thus resistance to cytotoxic therapy. Since IGF-1R is not an absolute requirement for maintenance of normal cell homeostasis, interfering with IGF-1R signaling at the receptor tyrosine kinase (RTK) level represents an attractive strategy to improve anti-cancer treatment. However, most IGF-1 RTK inhibitors do not fully discriminate between the IGF-1 RTK and the insulin RTK, i.e. are diabetogenic. Recently, members of the cyclolignan family have been shown to selectively inhibit the phosphorylation of the IGF-1R β-chain without down regulating the RTK activity of the insulin R1. The effect of two of these compounds, picropodophyllin (PPP) and deoxypodophyllotoxin (DPPT), was studied in vitro using a panel of nine MM cell lines and primary tumor cells from MM patients, and in vivo using the 5TMM mouse MM model2. Both IGF-1 RTK inhibitors effectively inhibited growth in all MM cell lines providing increased apoptosis and cell cycle arrest in the G2/M-phase. Notably, the two drug-resistant subclones of the MM cell line RPMI 8226/Dox40 (doxorubicin) and RPMI 8226/LR5 (melphalan), were highly sensitive to the IGF-1 RTK inhibitors. In addition, PPP and DPPT showed inhibitory effects on primary MM cells cultured in vitro with bone marrow stromal cells. Inhibition of the IGF-1 RTK with PPP has been shown to be non-competitive with ATP suggesting interference with the IGF-1R at the substrate level1. To identify tyrosine phosphorylation site(s) on the IGF-1R β-chain potentially regulated by the IGF-1 RTK inhibitors, IGF-1R extracted from RPMI 8226 cells was analyzed by tryptic phosphopeptide mapping following 32P-labeling and treatment with PPP or DPPT. Cleaved phosphopeptides were separated by thin layer chromatography and analyzed by a modified radio-Edman degradation. The results show that the IGF-1 RTK inhibitors do not down regulate any specific tyrosine phosphorylation site of the IGF-1R in MM cells. In vitro kinase assay suggests that PPP/DPPT-induced inhibition of the IGF-1R instead is conducted via a general down regulation of the tyrosine kinase activity of the receptor. Extraction of tyrosine phosphorylated proteins followed by electrophoresis and mass spectrometric analysis revealed additional signaling molecules affected by treatment with the IGF-1 RTK inhibitors e.g. impaired tyrosine phosphorylation of CDK1/cdc2, a cell cycle associated protein known as a potent regulator of apoptosis. Taken together, we show that treatment of MM cells with selective IGF-1 RTK inhibitors decreases survival/proliferation and affects the function of crucial intracellular signaling proteins, thus emphasizing the pivotal role for IGF-1R signaling in MM.
Gene Expression Profiling Defines a Set of New Molecular Markers for Sporadic and Familial Myeloproliferative Disorders.
Authors: Kralovics Robert , Teo Soon-Siong , Passamonti Francesco , Buser Andreas S. , Tichelli Andre , Cazzola Mario , Skoda Radek C.
Published: October 2019

The description of new molecular markers, such as PRV-1, has revived the interest in myeloproliferative disorders (MPD). To gain information on gene expression in cells derived from the MPD clone, we performed Affymetrix U133A microarray analysis on granulocyte RNA from MPD patients and healthy controls. Granulocytes are relevant for expression profiling in MPD, because they originate from the MPD stem cell clone in all PV, IMF, and in a proportion of ET patients. By analyzing 10 patients with PV, 9 patients with ET and 6 healthy controls we identified more than 300 genes with a significantly altered expression (P<0.05). Both PV and ET samples were clearly separated from healthy controls in unsupervised cluster analysis. The expression profiles of PV and ET showed partial overlap. In addition, we analyzed 4 affected individuals from 3 unrelated families with hereditary MPD-like phenotypes. Interestingly, 2 affected individuals from the same family with a PV-like phenotype clustered with the sporadic PV group, whereas 2 patients with familial ET-like phenotype had an expression profile closely resembling sporadic ET. Together with our finding that the family with PV displays clonal hematopoiesis, these results support the concept that studying familial MPDs, i.e. positional cloning of the mutated genes, could be relevant to advancing our understanding of sporadic MPD. From the 300 genes which displayed altered expression in MPD patients, we selected 15 for a detailed quantitative PCR analysis in a Swiss cohort of 42 MPD patients, 3 patients with chronic myelogenous leukemia, 7 patients with secondary erythrocytosis and 14 healthy controls. These markers include genes encoding transcription factors (KLF4, ETS2), a growth factor (FGF13) and proteins involved in signaling (SOCS3, FYB). A combination of three markers (HP, HRP, and ETS2) was sufficient to detect all PV patients that displayed endogenous erythroid colonies, including PV patients with normal levels of PRV-1. MPD patients with a history of complications, such as bleeding, secondary fibrosis or leukemic transformation, exhibited significantly higher levels of expression in a subset of these markers. In contrast, there was no difference in expression levels between MPD patients who received hydroxyurea and patients without cytoreductive treatment, suggesting that hydroxyurea does not alter the phenotypic characteristics of the MPD clone. We are now validating the usefulness of these markers on an independent test cohort of 93 individuals from Italy. The conclusions from this comparison will be presented.
Effect of Human Plasma-Derived Protein C and Murine Recombinant Protein C on Severe Neonatal Sepsis in Mice.
Authors: Muchitsch Eva-Maria , Schwarz Hans Peter , Varadi Katalin , Esmon Charles , Mancuso Giuseppe , Teti Giuseppe
Published: October 2019

Neonatal sepsis is a leading cause of infant morbidity and mortality frequently associated with activation of the coagulation system. Reduced levels of protein C are found in the majority of patients with sepsis and are associated with an increased risk of death. Although activated protein C is indicated for the treatment of severe sepsis in adults, the risk of severe bleeding may limit its use in neonates. Because the likelihood of inducing bleeding with the zymogen form of protein C is reduced we assessed both human and murine protein C zymogen in a murine neonatal sepsis model. In this model neonatal mice were challenged with viable group B streptococci (GBS). The effect of this septic condition on endogenous protein C levels was evaluated and the effect of treatment with either recombinant murine protein C or human plasma-derived (non-activated) protein C (Ceprotin) investigated. During severe GBS sepsis murine endogenous protein C levels decreased over time in neonatal mice, resulting in a maximum decrease of −30 % at 16 hours after GBS challenge and returned towards baseline at 30 hours. Concomitantly, there was an increase in endogenous protein C activation up to 59 % at 6 hours after GBS challenge, returning to baseline levels at 16 hours. Blocking endogenous murine protein C with an anti-mouse monoclonal antibody increased the mortality rate significantly from 62 to 91 %. Treatment of neonatal septic mice (n=36) with 300 U/kg murine protein C subcutaneously 4 hours before GBS challenge decreased the mortality rate significantly in severe sepsis (LD90) to 64 % (p=0.002). Similarly pretreatment with human plasma-derived protein C (200 IU/kg) 4 hours before GBS challenge increased survival rate significantly in severe septic mice. Treatment with 200 IU/kg (Ceprotin) was even effective given 18 hours after GBS challenge, leading to a decrease in the mortality rate in severe sepsis from 87.5 to 48 %. Despite this species difference and the septic condition, human protein C zymogen was activated to activated protein C and detectable in the circulation of mice. This is the first preclinical study were a beneficial effect of a non-activated protein C could be shown in an animal model of severe neonatal sepsis.
A Factor VII Deficiency Protects Against Lethal Endotoxemia in Mice.
Authors: Xu Haifeng , Ploplis Victoria A. , Joyce David E. , Castellino Francis J.
Published: October 2019

The formation of FVIIa:TF complex is a main contributor to the coagulopathy associated with acute bacterial infection and sepsis. In this study, the role of FVII has been investigated in a murine model of lethal endotoxemia induced by LPS using genetically altered mice expressing low FVII (FVIItTA/tTA). The results demonstrated that FVIItTA/tTA mice had reduced mortality, coagulation, and inflammatory responses compared to wild-type (WT) mice. The reduced thrombin generation in FVIItTA/tTA mice at early stages of post-LPS challenge correlated with less fibrin formation, which was characterized by diminished fibrin deposition in the liver and lower D-Dimer levels in the plasma. Fibrin deposition at late stages of endotoxemia was, however, similar between WT and FVIItTA/tTA mice. There was an apparent increased activation of the intrinsic coagulation pathway in FVIItTA/tTA mice as exhibited by an increased consumption of FXII and FXI zymogen in blood, which appeared to compensate for the diminished thrombin generation characteristic of the low FVII state. Fibrinolytic potential during endotoxemia remained the same between WT and FVIItTA/tTA mice. Reduced inflammatory responses were prominent in FVIItTA/tTA mice, as demonstrated by lower IL-6, MIP-2, and higher IL-10 plasma protein levels, which correlated with the total RNA expression levels in various tissues as determined by Quantitative RT-PCR. These reduced inflammatory responses could be directly due to less FVIIa:TF:PAR-2 signaling, and/or indirectly by less thrombin:PARs signaling in FVIItTA/tTA mice. Additionally, a slight increase in parenchymal organ bleeding was observed in FVIItTA/tTA mice challenged with LPS. Results from these studies indicate that low FVII benefits survival mainly through attenuation of coagulation and inflammatory responses as the result of potentially less coagulation protease signaling. Protection by mechanisms associated with regulation of fibrin formation appears to be limited to early stages of the disease.
Cytogenetic and FISH Study of 92 Patients with Splenic Marginal Zone B-Cell Lymphoma (SMZBCL).
Authors: Salido Marta , Sole Francesc , Espinet Blanca , Gimeno Eva , Florensa Lourdes , Granada Isabel , Astier Laura , Domingo Alicia , Mollejo Manuela , Martinez-Climent Jose A. , Garcia Juan L. , Benet Isana , Hernandez Jesus M. , Martinez Pedro , Piris Miguel A. , Perez-Vila Encarna , Woessner Soledad , Campo Elias , Serrano Sergio
Published: October 2019

Background. In the World Health Organization classification system (WHO), Splenic Marginal Zone B-cell Lymphoma (SMZBCL) is described as an indolent B-cell lymphoma, which generally presents as splenomegaly with involvement of the bone marrow and peripheral blood. The immunophenotype is usually IgM+, IgD+/−, cytoplasmic-Ig−/+, pan B antigens+, CD5−, CD10−, CD23−, CD43−/+ and cyclin D1−. A minority of cases (< 20%) express CD5 antigen. The most frequent cytogenetic aberrations are deletions at 7q22–q32, gains of chromosome 3/3q and involvement of chromosomes 1 and 8. The objective of this study is the genetic characterization of SMZBCL by conventional cytogenetics and FISH.

Patients and methods. We present 92 patients with SMZBCL from a multicenter study after being diagnosed morphologically and immunologically. Conventional cytogenetic studies were carried out on lymphoid cells from 72 hour-peripheral blood and/or spleen cultures stimulated with TPA. FISH was performed with centromeric probes from chromosome 3 and 12 and locus specific probes from P53 gene and 7q31 loci. Cross-species color banding FISH (RxFISH) probe was applied in 11 patients and SKY in 4 patients to redefine/confirm G-banding karyotype.

Results. A clonal chromosome abnormality was found in 62/92 patients (67%) being identified in 37 (60%) as a complex karyotype. The most frequent recurrent abnormalities were deletions of 7q (30 cases) and gains of chromosome 3 (20 cases). 6/20 cases with gains #3, were gains of 3q arm, in three of them resulting from an unbalanced translocation. Thirteen cases showed rearrangements of 3q without gain of material. The chromosomes most frequently involved were: 7q, 3q, 14q, 8q and 1q (in more than 10 cases). Four cases presented gain of chromosome 3/3q and 7q deletion in the same karyotype. Four novel cytogenetic aberrations involving 14q32 were found in this series: t(6;14)(p12;q32) in two cases, t(14;19)(q32;q13) in two cases and t(10;14)(q24;q32), t(1;14)(q21;q32) and t(9;14)(pter-p22::14q32-14q11::9q22) in one case respectively.

Conclusions.

-In our series, the most frequent cytogenetic aberration in SMZBCL is a deletion at 7q (48%) and could be considered as a cytogenetic marker of these entity. -Gains of chromosome 3 or partial gains affecting only 3q arm are frequently observed in SMZBCL (32%) being chromosome 3 commonly rearranged. -RxFISH ans SKY techniques help us to define new aberrations not detected by conventional cytogenetics. -We describe translocations involving 14q32 that could help us to identify novel oncogenes related to SMZBCL.
Identification of Genetic and Expression Markers in DLBCL Cell Lines That Influence the In Vivo Response of DLBCL to Doxorubicin.
Authors: Houldsworth Jane , Petlakh Marina , Chaganti R. S.
Published: October 2019

Diffuse large B-cell lymphoma (DLBCL) is the largest aggressive group of non-Hodgkin’s lymphoma (~40%) and exhibits great clinical and pathological heterogeneity. While DLBCL represents a chemosensitive disease, ultimately, only approximately 50% of patients are cured with current treatment options. Standard front-line therapy comprises CHOP and more recently Rituximab-CHOP. CHOP commonly contains doxorubicin as the curative anthracycline. Controversy currently exists in the use of genetic and expression markers in predicting response to treatment and outcome of DLBCL, thus, we examined the cellular responses of a panel of eight DLBCL cell lines to doxorubicin in order to define molecular markers that influence the response. Expression profiling and subsequent classification of the cell lines revealed that six of the lines were of a germinal center B-cell-like (GCBL) subtype and two (Ly3 and Ly10) were of a non-GCBL subtype. Ly3 and Ly10 exhibited a rapid apoptotic response within 24 hours after exposure to doxorubicin as evaluated by Hoescht staining. Expression profiling of the response of Ly10 cells (in triplicate) revealed the up-regulation of transcripts consistent with mediation of the cellular response via a p53-dependent pathway. Mutation analysis and Western blotting revealed wild type TP53 in Ly3 and Ly10 with an induction of p53 levels after doxorubicin. The remaining cell lines exhibited mutated TP53 sequences and no induction of p53 by doxorubicin. Therefore, in DLBCL cell lines, wild type p53 effects an early rapid apoptotic response to doxorubicin. In the six non-GCBL cell lines, a decrease in viability after doxorubicin was observed at much later times (up to 48 hours after treatment) by both apoptotic (exemplified by Ly2 and Ly8) and non-apoptotic mechanisms (exemplified by Ly1). Expression profiling of these three cell lines revealed alterations in expression of transcripts consistent with a G2/M cell cycle delay induced by doxorubicin, which was confirmed by follow-up flow cytometric analysis. An induction of transcripts consistent with NF-κB and AP-1 activation was observed in Ly1, but not in Ly2 and Ly8, which was confirmed by electrophoretic mobility shift assays. In order to determine if this cellular response plays a role in determining the response of DLBCL to doxorubicin in vivo, the expression profiles of untreated Ly1 and Ly2 were compared, and the resulting 160 differentially expressed transcripts were used to cluster 41 newly-diagnosed DLBCL specimens which had previously been profiled and had a known response to doxorubicin-containing therapy. This analysis revealed a marked correlation between capacity to induce NF-κB and AP-1 with in vivo treatment failure. Overall, these experimental studies have begun to outline genetic and biologic features that together contribute to the response of DLBCL to doxorubicin in vivo.
Efficacy and Safety of Fondaparinux (ARIXTRA®) in the Initial Treatment of Venous Thromboembolism in Obese Patients.
Authors: Buller Harry R. ,   the MATISSE Investigators
Published: October 2019

Background: The MATISSE trials demonstrated that once-daily subcutaneous fondaparinux, a synthetic selective factor Xa inhibitor, was at least as effective and as safe as standard therapies in the initial treatment of deep-vein thrombosis (DVT) or pulmonary embolism (PE). Since obesity is a risk factor for venous thromboembolism (VTE), we analyzed the MATISSE data from the subpopulation of obese patients (body-mass index, BMI ≥30 kg/m2).

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 DVT. In the MATISSE-PE trial, it was compared with adjusted-dose intravenous unfractionated heparin (UFH) in patients with PE. 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: The percentage of obese patients was 26.3% in MATISSE-DVT and 28.7% in MATISSE-PE (Tables). In both MATISSE-DVT and -PE, efficacy and safety data from obese patients were similar to those from non-obese patients (BMI <30kg/m2). The rates of recurrent VTE and MB in obese patients were not significantly different between fondaparinux and either enoxaparin (MATISSE-DVT) or UFH (MATISSE-PE).

Conclusion: Once-daily fondaparinux is at least as effective and as safe as standard therapies in the initial treatment of DVT or PE in obese patients.

MATISSE DVT BMI<30 kg/m2 BMI≥30 kg/m2 Enoxaparin Fondaparinux Enoxaparin Fondaparinux *As treated patients n 791 803 300 280 VTE, n/N 35/791 (4.4%) 31/803 (3.9%) 10/300 (3.3%) 10/280 (3.6%) MB*, n/N 10/788 (1.3%) 12/798 (1.5%) 3/297 (1.0%) 0/279 (0%) MATISSE PE BMI<30 kg/m2 BMI≥30 kg/m2 UFH Fondaparinux UFH Fondaparinux *As Treated Patients n 760 757 322 314 VTE, n/N 35/760 (4.6%) 30/757 (4.0%) 20/322 (6.2%) 12/314 (3.8%) MB*, n/N 8/752 (1.1%) 11/751 (1.5%) 4/314 (1.3%) 2/311 (0.6%)
BCR/ABL Oncogenic Kinase Promotes Unfaithful Repair of the Reactive Oxygen Species - Dependent DNA Double-Strand Breaks.
Authors: Skorski Tomasz , Nowicki Michal O. , Falinski Rafal , Koptyra Mateusz , Slupianek Artur , Stoklosa Tomasz , Gloc Ewa , Nieborowska-Skorska Margaret , Blasiak Janusz
Published: October 2019

The oncogenic BCR/ABL tyrosine kinase induces constitutive DNA damage in Philadelphia chromosome (Ph1)-positive leukemia cells. We find that BCR/ABL kinase - induced reactive oxygen species (ROS) cause chronic oxidative DNA damage as indicated by an enzymatic assay detecting oxidized bases. These DNA lesions result in DNA double-strand breaks (DSBs) detected by comet assay, immunofluorescent gamma-H2AX nuclear foci and linker-ligation PCR (LL-PCR). Combined analysis of the length of LL-PCR products and the sequences of two reference genes DR-GFP and Na+/K+ ATPase revealed that ROS dependent DSBs occurred in the regions containing multiple, 5–9bp long stretches of G/C, in concordance with the notion that oxidative DNA damage is predominantly detected in G/C-rich sequences. Elevated numbers of DSBs were detected in BCR/ABL cell lines, murine bone marrow cells transformed with BCR/ABL and in CML patient samples, in comparison to normal counterparts. Inhibition of the BCR/ABL kinase by STI571 and diminishion of ROS activity by the ROS scavenger PDTC reduced DSBs formation. Cell cycle analysis revealed that most of these DSBs occur during S and G2/M phases, and are probably associated with the stalled replication forks. Homologous recombination repair (HRR) and non-homologous end-joining (NHEJ) represent two major mechanisms of DSBs repair in S and G2/M cell cycle phase. Using the in vivo recombination assay consisting of the DSB-dependent reconstitution of the green fluorescent protein (GFP) gene we found that HRR is stimulated in BCR/ABL-positive cells. In addition, in vitro assay measuring the activity of NHEJ revealed that this repair process is also activated by the BCR/ABL kinase. RAD51 and Ku70 play a key role in HRR and NHEJ, respectively. The reaction sites of HRR and NHEJ in the nuclei could be visualized by double-immunofluorescence detecting co-localization of gamma-H2AX foci (DSBs sites) with RAD51 (HRR sites) or Ku70 (NHEJ sites). Equal co-localization frequency of gamma-H2AX foci with RAD51 and Ku70 was detected, suggesting that both HRR and NHEJ play an important role in reparation of ROS-dependent DSBs in BCR/ABL-transformed cells. Analysis of the DSBs repair products in the reporter DR-GFP gene in BCR/ABL cells identified ~40% of HRR and ~60% of NHEJ events. Sequencing revealed point-mutations in HRR products and large deletions in NHEJ products in BCR/ABL-positive cells, but not in non-transformed cells. We propose that the following series of events may contribute to genomic instability of Ph1-positive leukemias: BCR/ABL → ROS → oxidative DNA damage → DSBs in proliferating cells → unfaithful HRR and NHEJ repair. Since BCR/ABL share many similarities with other members of the fusion tyrosine kinases (FTKs) family, these events may contribute to genomic instability of hematological malignancies caused by FTKs.
Rhogtpase RAC2 Is Required for In Vivo Transformation Activity by P210 Bcr-Abl Fusion Oncogene.
Authors: Krishnan Nithya , Bailey Jeff R. , Summey-Harner Victoria , Brunstein Claudio , Verfaillie Catherine M. , Williams David A.
Published: October 2019

Bcr-Abl, the translocation product of the Philadelphia chromosome implicated in human chronic myelogenous leukemia (CML), is a kinase affecting hematopoietic stem cell (HSC) behavior with respect to proliferation, apoptosis, adhesion and migration. Rho GTPases, particularly the Rac subfamily, have been shown to regulate these same cell functions in normal HSC and also regulate gene expression in many mammalian cells. BCR contains a “GTPase-activating protein” domain and a guanine nucleotide exchange domain, the latter or which is preserved in p210 Bcr-Abl. Since HSC functions regulated by Bcr-Abl and Rac are similar, we studied the potential involvement of Rac activation in

Bcr-Abl signaling cascade. Human CML samples demonstrate baseline activation of Rac proteins that is reversed by in vitro treatment with STI571. To study the specific involvement of Rac2, we used a gene targeted mouse model with Rac2 null bone marrow. Using retovirus-mediated gene transfer, we introduced p210 Bcr-Abl in the MSCV vector into wild-type or Rac2−/− HSC/P and studied the behavior of these cells in vitro and in vivo.

Irradiated recipient mice injected with LDBM cells transduced with p210 developed a uniformly fatal myeloproliferative syndrome (Median survival: 45 days, N=12), while mice injected with p210 transduced Rac2−/− LDBM cells (N=12, 2 independent exp.) had 100% survival and no development of leukocytosis, splenomegaly or organ infiltration of hematopoietic cells. These data suggest that Rac GTPases are critical for the transformation of HSC by Bcr-Abl and provide an additional therapeutic target for intervention in CML.

WILD TYPE Rac 2 −/− Empty Vector MSCV-p210 Empty vector MSCV-p210 *p < 0.01 vs WT-MIEG3, **p< 0.01 vs WT-p210 bcr-abl. Proliferation (CPM) Medium 562 ± 278 16,207± 1605* 819.7 ± 363 3,135.5 ± 498** SCF (100ng/ml) 856 ± 187 23,226 ± 2203* 853.7 ± 524 3,756.8 ± 207** Cytokines (SCF, GCSF, MGDF) 8011± 1412 42,711± 13393* 4833 ±1019 3,614.5 ± 1982** Migration (%) Fibronectin 7 ± 0.4 38 ± 1.9* 0.4 ± 0.0 0.8 ± 0.1** SDF-1α 30 ±2.8 13 ±1.1* 0.5 ± 0.0 0.6 ± 0.0** Adhesion (% ) Fibronectin 76± 2.9 40 ±3* 4 ±0.4 10 ±0.1 **
Transplantation with Higher Dose of Natural Killer Cells Associated with Better Outcomes in Terms of Non-Relapse Mortality and Infectious Events after Allogeneic Peripheral Blood Stem Cell Transplantation from HLA-Matched Sibling Donors.
Authors: Sohn Sang Kyun , Kim Dong Hwan , Lee Nan Young , Baek Jin Ho , Kim Jong Gwang , Suh Jang Soo , Lee Kun Soo , Lee Kyu Bo , Shin Ho Jin , Chung Joo Seop
Published: October 2019

Background: Little is known about the role of the CD56+ natural killer (NK) cell dose on the outcome of allogeneic peripheral blood stem cell transplantation (PBSCT). Recently, a higher dose of NK cells has been associated with a lower incidence of severe GVHD in a PBSCT setting. Therefore, the current study attempted to evaluate the effect of the NK cell dose on transplant outcomes, including non-relapse mortality (NRM) and infectious events, in an allogeneic PBSCT setting.

Methods and Materials: Sixty-one cytokine mobilized PBSC recipients from HLA-matched sibling donors were analyzed according to the infused dose of CD34+ cells and NK cells in relation to overall survival (OS), NRM, GVHD, and infectious events.

Results: The group of patients that received a higher dose of NK cells (≥ 5x107/Kg) showed a lower incidence of NRM (p=0.0186) and infectious events (p=0.0107). When confining the analysis to the group that received a CD34+ cell dose of ≥ 6x106/Kg, those patients that received a higher dose of NK cells exhibited a lower incidence of extensive chronic GVHD (p=0.0704). In a multivariate analysis using Cox’s regression model, a higher dose of NK cells was significantly associated with better transplant outcomes (for NRM, NK cell dose p=0.042, for CD34+ cell dose p=0.018; for infectious events, NK cell dose p=0.013, CD34+ cell dose 0.016; for bacterial infection, NK cell dose p=0.049). The group that received a higher NK cell dose also showed a faster immune recovery (p=0.046 for NK cell recovery, p=0.034 for helper T-cell recovery) in serial measurements of peripheral lymphocyte subsets at D+90, +180, and +365.

Conclusions: The present data suggests that a high dose of NK cells may play an important role in improving transplant outcomes, in terms of reducing NRM and infectious events together with CD34+ cells. The protective role of NK cells against infections may also be associated with a faster immune recovery after allogeneic PBSCT.

Figure Figure

Figure Figure
The C-Mpl Receptor Expressed on Endothelial Cells Does Not Contribute Significantly to the Regulation of Circulating Thrombopoietin (TPO) Levels.
Authors: Geddis Amy E. , Fox Norma E. , Kaushansky Kenneth
Published: October 2019

Blood levels of TPO, the primary regulator of platelet production, are thought to be regulated primarily by cellular degradation following its binding to, and internalization by its cell surface receptor, c-Mpl. This physiology is supported by in vitro studies, in which TPO can be removed from solution by incubation with platelets, and accounts for the very high levels of circulating TPO in individuals with congenital amegakaryocytic thrombocytopenia (CAMT), as in this disorder there are no c-Mpl receptor-bearing cells to bind and remove the hormone. Previous reports have demonstrated that in addition to hematopoietic cells, c-Mpl is expressed on several types of endothelial cells. We hypothesized that the c-Mpl expressed on endothelial cells would contribute to the regulation of circulating TPO levels. As a model to test this hypothesis, we transplanted 10 Mpl knock-out mice and 10 wild-type (WT) controls with WT marrow cells and analyzed platelet counts and plasma TPO levels over the course of 6 months. The resulting two groups of chimeric mice both express c-Mpl on megakaryocytes and platelets, but only the WT recipients express the receptor on endothelial cells. If c-Mpl receptors normally expressed on endothelial cells take up circulating TPO and degrade it, we expected that the Mpl-null mice reconstituted with WT cells would display increased TPO levels and an increased steady state platelet count compared to the WT recipients. As expected, pre-transplant the platelet counts in the Mpl-null mice were low (average value 398x109/L) and TPO levels were high (average value 8040 pg/ml), as compared to WT controls (platelet counts average 883x109/L, TPO level 745 pg/ml). However, following transplant with WT cells, we found that TPO levels in the chimeric mice were virtually identical (954 pg/ml for the Mpl-null recipients vs. 829 pg/ml for the WT recipients) as were the steady state platelet counts up to 6 months following transplantation (891x109 /L vs. 883x109/Lx109 /L, respectively). These results indicate that the c-Mpl receptor expressed on endothelial cells does not contribute significantly to the regulation of circulating TPO levels or to steady state platelet counts. The apparent lack of TPO adsorbtion by the endothelial cells is surprising, as prior published studies indicate that Mpl receptors expressed on human umbilical vein endothelial cells bind TPO and that c-Mpl is functionally active on hepatic endothelial cells; in addition, the composite surface area of the endothelium is large. These results also imply that patients with CAMT that have successfully engrafted with normal hematopoietic stem cells should have normal (not elevated) TPO levels. Similarly, gene replacement strategies designed to restore c-Mpl in CAMT do not need to be expressed on endothelial cells to establish the normal regulation of TPO.
TLR9 Agonist Immunomodulator Treatment of Cutaneous T-Cell Lymphoma (CTCL) with CPG7909.
Authors: Kim Youn , Girardi Michael , Duvic Madeline , Kuzel Timothy , Rook Alain , Link Brian , Pinter-Brown Lauren , Comerci Carol , McAuley Sonja , Schmalbach Tess
Published: October 2019

CPG 7909 belongs to a new class of chemically defined CpG immunomodulators that target dendritic cell TLR9 receptors inducing IL-12, IFN-gamma, and NK cell function. The rate and durability of response to CPG 7909 was investigated in refractory patients with recurrent or advanced CTCL, who had failed one or more systemic therapies. Dose escalation with weekly sc dosing of patients at 0.08, 0.16, 0.24, or 0.28 mg/kg (3 patients/cohort) for 24 weeks is nearing completion. Additional patients continue to receive treatment at 0.32 (4 patients) or 0.36 mg/kg (12 patients). Clinical response, monitored by Composite Assessment of Index Lesion Disease Severity (CA) and Physician’s Global Assessment of Clinical Condition, has been documented. Of 28 patients enrolled, 7 (25%) have achieved objective clinical response, 5 with partial response (PR) and 2 with complete response (CR). Eleven patients have maintained stable disease (SD), while 10 have had progressive disease (PD). Eight patients have completed 24 weeks of treatment (5 SD, 2 PR, 1 CR) with 12–16 weeks of response while on study. Six patients (3 SD, 2 PR,1 CR) are currently ongoing in the study. Three patients (2 PR, 1 SD) continue to receive long term CPG 7909 at 0.12 mg/kg (58 total doses), 0.28 mg/kg (34 total doses) or 0.32 mg/kg (29 total doses) in a follow on protocol. Responses have been maintained up to 46 weeks.

Weekly doses up to 0.36 mg/kg have been well tolerated. Most reported adverse events have been of CTC grade 1 or 2. The most common are dose-related local injection site reactions (erythema, induration, edema, inflammation and pain) and mild or moderate flu-like symptoms (fatigue, rigors, fever, arthralgia). Four patients had CTC grade 3 drug related AEs: one decreased lymphocyte count (0.08 mg/kg), one increased gamma glutamyl transferase (0.16 mg/kg), one decreased absolute polys (0.36 mg/kg) and one fatigue (0.36 mg/kg).

Enrollment in the phase II portion of the study is ongoing and compares results of patients randomized to receive either 10 mg or 25 mg sc weekly for 24 weeks (equating to effective doses seen in dose escalation).

Clinical Response with CPG 7909 - 16 M, 12 F

Dose N Disease Stage CR PR SD PD 0.36 mg/kg 12 IB (7), IIB, III (3), IVA 0 2 6 4 0.32 mg/kg 4 IIA, IIB, IVA (2) 1 0 1 2 0.28 mg/kg 3 IB (2), III 0 1 2 0 0.24 mg/kg 3 IB, IIB (2) 0 1 1 1 0.16 mg/kg 3 IB (2), IIA 1 1 1 0 0.08 mg/kg 3 IB (2), IVA 0 0 0 3 28 7% 18% 39% 36%