bluuebunny/crossref_metadata_2025 huggingface dataset:
Endoglin Is Required for Hemangioblast Development.
Authors: Perlingeiro Rita R.
Published: October 2019

A critical role for endoglin (CD105) in early development has been demonstrated in mice deficient for this gene. Embryos homozygous for the endoglin mutation (eng−/−) fail to progress beyond 10.5 days postcoitum due primarily to vascular and cardiac abnormalities (Bordeau et al, 1999). Analysis of 9.5 dpc eng−/− embryos revealed abnormal vasculature and anemia of the yolk sac, suggesting that endoglin may be required for both blood and endothelial lineages. The hemangioblast, the bipotent precursor for hematopoietic and endothelial cells, can be assessed through the blast colony assay (BL-CFC) using a model system based on the in vitro differentiation of embryonic stem (ES) cells into embryoid bodies (EBs). To evaluate a role for endoglin in this early precursor, we differentiated eng−/−, eng+/−, and eng+/+ (wild-type) ES cells into EBs. At day 3 of EB differentiation, cells were disrupted and plated for blast colony formation in methylcellulose media containing vascular endothelial growth factor (VEGF), stem cell factor (SCF), and thrombopoietin (TPO). We found no difference in blast colony formation between heterozygous and wild-type ES cells. However, a significant reduction in the number of BL-CFCs was observed in eng−/− cells when compared to eng+/− or eng+/+ BL-CFCs (p < 0.001). Single eng−/−, eng+/−, and eng+/+ BL-CFCs gave rise to secondary hematopoietic colonies as well as endothelial cells, confirming their nature as hemangioblasts. These results suggest that although endoglin is required for hemangioblast development, its absence does not affect the bipotentiality of formed BL-CFCs. Since anemia was a feature of 9.5 dpc eng−/− yolk sac embryos, we also examined early erythropoiesis using the ES/EB system. For this purpose, eng−/−, eng+/−, and eng+/+ ES cells were differentiated into EBs for 4 days, at which time cells were disrupted and plated for primitive erythroid colonies (EryP) in methylcellulose media containing IL-3, IL-6, SCF, and Epo. We observed a reduction in the number of EryP colonies in eng−/− (p < 0.01) and eng+/− (p < 0.05) EBs when compared to controls (eng+/+). These results corroborate the anemia observed in vivo in the eng−/− embryos. We used RT-PCR and flow cytometry analysis to detect endoglin expression during a time course of EB differentiation. Endoglin is expressed in ES cells and disappears with differentiation. Expression re-appears at day 3 of differentiation, concomitantly with specification of the hemangioblast. Expression thereafter increases, correlating with mature endothelial cells at later time points. We did not find major differences in gene expression for Brachyury, Flk-1, Tie-2, embryonic and adult globins in a time course of EB differentiation for eng−/−, eng+/−, and eng+/+ ES cells. These data point out a role for endoglin, an ancillary receptor for several members of the transforming growth factor (TGF)-beta superfamily, in hemangioblast development.
Manteinance Treatment with Interferon (IFN) Is Not Necessary to Achieve Prolonged Progression Free Survival (PFS) in Patients with Indolent Non Hodgkin’s Lymphoma (NHL). Results from a Prospective Randomized Trial on 165 Patients.
Authors: Cannata Jimena , Nicolas Concepcion , Resident Raul Cordoba , Rayon Consuelo , Garcia-Laraña Jose , Lopez Javier , Perez Guillermo , Sanchez-Godoy Pedro , Garcia-Alfonso Pilar , Zamora Pilar , Fernandez-Rañada Jose-Maria , Arranz Reyes
Published: October 2019

INTRODUCTION. Treatment with IFN and chemotherapy induces a significant improvement of PFS in patients with low-grade NHL, specially in patients with responsive disease. However, the best IFN schedule is still pending as benefits are observed when IFN is administered associated to the induction therapy or, as maintenance treatment or, in both treatment phases.

OBJETIVE. To evaluate the additional benefit or not of one year maintenance treatment with IFN in patients who achieve response after an induction treatment with CVP and IFN for 3 months.

PATIENTS & METHODS. From February 1990- October 2002, 165 patients with low-grade NHL were recruited. Patients received treatment with CVP (median number 6 cycles) and IFN, 3MU/m2 three times a week during 3 months. Patients with complete or partial responses were then randomized to receive treatment with 12 months of IFN, 3MU/m2 three times a week vs observation. With a median of 4,7 years (range 0.3 to 11.7 years) for surviving patients, we analysed response, PFS and overall survival.

RESULTS. Patients median age was 63 years (range 28–89 years), with 65.5% having follicular (FL), 20% small lymphocyte and 14% marginal zone lymphoma. Most patients (83%) were in advance stage and 75% had extranodal involvement. Thirty-three percent of FL patients had a FLIPI index score > 3. Dose-intensity for cyclophosphamide and IFN in the induction treatment was 71% and 69%, respectively. Ten patients withdraw the study due to grade 3–4 toxicity events (6.5%).

Response : 86% of patients achieved a response, 80% complete. One hundred and twelve out of the 143 responding patients were randomised to receive IFN for 12 months (56 patients) vs observation (56 patients). Patients and disease characteristics for each group were similar. Median dose of maintenance IFN treatment was 67% (range 8%–113%). Fourteen patients (25%) withdraw the study, 7 due to IFN toxicity.

PFS : Median PFS of the whole series was 6.76 years, 6.6 years for patients in the maintenance IFN group vs 7.8 years for the observation group, p=0.7.

Overall survival at 12 years is 81% for both groups. Interestingly, 31 patients who achieved a good response but were not randomised showed a significantly shorter OS, p=0.001.

CONCLUSION. No additional increase on PFS has been observed when maintenance IFN is administered to patients responding to CVP and short term IFN induction therapy. With our data, very good response rates and long lasting remission are achieved with only 3 months IFN treatment. Treatment tolerance is therefore significantly better.
Can High Dose Therapy Change the Course of Low Grade Lymphoma? A Study Considering Patients as Their Own Control.
Authors: Vignot Stephane , Mounier Nicolas , Sergent Guillaume , Brice Pauline , Marolleau Jean-Pierre , Parquet Nathalie , Briere Josette , DeKerviller Eric , Madeleine Isabelle , Gisselbrecht Christian
Published: October 2019

Low grade lymphoma patients (pts) have an indolent evolution with median survival ranging between 8–10 years. During disease’s course, high dose therapy (HDT) and autologous stem-cell transplantation (ASCT) can be considered as an alternative to sequential chemotherapies. However, efficacy of this strategy remains controversial. The purpose of our study is to evaluate ASCT efficacy by comparing retrospectively for each pts disease free survival (DFS) after ASCT with DFS observed with pts’ last chemotherapy regimen (LCR) just before intensification. Between apr 1988 and feb 2002, 109 low grade lymphoma pts were treated with HDT and ASCT in our department, 61 were male, the median age was 49 yrs [range 28–65]. Histological subtypes were mostly follicular small cell (86 %). At time of diagnosis, LDH were normal for 85 pts; 60 pts had high tumor burden. IPI was 0 for 16 %, 1 for 70 % and 2 for 14 %. Prior to ASCT, pts had experienced a median of 2 progressions (range 1 to 5). At time of graft, 102 pts present complete or partial response and 7 pts present stable disease. Two principal intensification chemo regimens were used before ASCT: VP16/cyclophosphamide in 84 pts and BEAM in 12. TBI was associated for 86 pts. At June 2002, the median follow up was 6.4 yrs from diagnosis and 4.5 yrs from ASCT. 3 years after ASCT, survival rate was 72 % and DFS rate was 50 %. Median DFS decreased with nb of progression (p=0.02):

Median DFS according to nb of progression

Nb of progression 1 2 3 > 3 Nb pts (%) 17 (16) 57 (52) 28 (26) 7 (6) Median DFS in yrs 6.4 5.1 1.8 1.0

Considering pt with more than 1 progression (n=92) as his own control, DFS was longer after ASCT than after LCR for 61 % of pts. Median DFS was 2.5 yrs after ASCT and 2.0 yrs after LCR. At 3 yrs, DFS rate was 48 % after ASCT and 37 % after LCR (p<0,001):

Figure Figure

This study demonstrates that HDT and ASCT significantly increase DFS in comparison with the LCR for low grade lymphoma patients. Such methodology could be useful to evaluate new strategy incorporating monoclonal antibody.
Genetic Variants of Ataxia Telangiectasia Mutated (ATM) and Susceptibility for Childhood T-Lineage Acute Lymphoblastic Leukemia.
Authors: Meier Marrit , Den Boer Monique L. , Hall Andrew G. , Irving Julie , Passier Monique , Minto Lynne , Van Wering Elisabeth R. , Janka-Schaub Gritta E. , Pieters Rob
Published: October 2019

ATM was identified as the gene mutated in Ataxia Telangiectasia (AT), a degenerative neurological disorder that is characterized by, amongst others, an increased risk to develop a hematomalignancy at young age. Striking is the high incidence of T cell derived lymphoid malignancies amongst AT patients. As sporadic T-lineage Acute Lymphoblastic Leukemia (T-ALL) is particularly found in childhood, we have investigated the occurrence and frequency of mutations in 62 coding exons of the ATM gene in 41 children with T-ALL at initial diagnosis and their relation to susceptibility for T-ALL. Moreover, as ATM is implicated in cellular signaling events in response to DNA damaging agents, we have also investigated whether genetic variants of ATM are possibly related to cellular resistance to drugs and clinical outcome in childhood T-ALL. We have identified 18 different ATM alterations, of which 9 were not previously reported, in 68% of T-ALL patients. None of the alterations co-segregated with loss of heterozygosity of the ATM gene. The simultaneous presence of more than one genetic variant was observed in 32% of T-ALL patients compared to only 12% of healthy controls (P = 0.006). An increased frequency of ATM alterations was of no influence on the level of ATM mRNA and did not associate with cellular resistance to daunorubicin, prednisolone, vincristine and L-asparaginase nor with age at diagnosis, white blood cell count or long-term clinical outcome. However, the presence of specific genetic variants, i.e. S707P, F858L, T896A, P1054R and L1472W was related to an age younger than 10 years (P = 0.034) and an increased relapse-risk (P = 0.025). In addition, an increased frequency of carriers of these genetic variants was observed amongst T-ALL children (29%) compared to controls (11%) (P = 0.028). We conclude that ATM gene alterations might pre-dispose carriers to develop T-ALL in childhood, in particular involving specific genetic variants of ATM as their occurrence is significantly associated with a young age.
Genomewide Linkage Analysis of Chronic Lymphocytic Leukemia by Use of a High-Density Single Nucleotide Polymorphism Assay Containing 11,555 Markers.
Authors: Sellick Gabrielle S. , Allinson Ruth , Matutes Estella , Yuille Martin , Catovsky Daniel , Houlston Richard S.
Published: October 2019

A genetic basis for chronic lymphocytic leukemia (CLL) is indicated by studies demonstrating that the lifetime risk of developing the disease increases by a factor of seven when at least one first-degree relative is affected. Significant familial aggregation of CLL has been demonstrated, but the mode of inheritance is unknown. To date no genes which when mutated have unequivocally been shown to confer susceptibility to the disease. We have collected clinical data and biospecimens from 370 families comprising of at least two affected family members with CLL. Of these, samples from two or more individuals from 100 families were informative for linkage analysis. To identify a predisposition locus for CLL a high-density single nucleotide polymorphism (SNP)-based genomewide linkage search was conducted using the GeneChip Mapping 10k Xba array containing 11,555 SNP markers (Affymetrix Inc., Santa Clara, CA). Multipoint LOD scores were calculated, assuming both dominant and recessive inheritance and allowing for increased penetrance with age and genetic heterogeneity. Non-parametric linkage scores were also calculated. The results from these analyses will be presented.
BAFF Support Survival of Chronic Lymphocytic Leukemia B Cells by a Pathway Independent of Stromal Derived Factor-1α.
Authors: Nishio Mitsufumi , Tsukada Nobuhiro , Kitada Shinichi , Ohata Junko , Zvaifler Nathan J. , Reed John C. , Kipps Thomas J.
Published: October 2019

We examined the peripheral blood mononuclear cells (PBMC) of patients with chronic lymphocytic leukemia (CLL) for expression of B cell-activating factor of the TNF family (BAFF). Isolated CLL B cells had significantly lower levels of BAFF mRNA than did non-separated PBMC. Most of the BAFF mRNA in PBMC was due to contaminating CD14+ cells that previous studies found could differentiate into “nurselike” cells (NLC) when cultured with CLL B cells in vitro. We found NLC expressed high-levels of BAFF and stromal cell-derived factor-1 alpha (SDF-1α), in contrast to CLL B cells. CLL B cells cultured with exogenous recombinant human BAFF (rhBAFF), SDF-1α, or NLC sustained significantly greater viability than isolated CLL B cells cultured alone. The effect(s) of rhBAFF on leukemia cell survival appeared additive and distinct from that of SDF-1α, which in contrast to rhBAFF induced leukemia-cell phosphorylation of p44/42 mitogen-activated protein-kinase (ERK 1/2) and phosphorylation and activation of AKT at Ser473. However, rhBAFF, but not SDF-1α, could induce processing of p100 NF-κB2 to p52 and, like NLC, enhance and/or maintain CLL-cell expression of the anti-apoptotic protein Mcl-1. We conclude that BAFF can function in a paracrine manner to support leukemia cell survival via mechanisms that are distinct from those of SDF-1α and that NLC use multiple distinct pathways to support CLL-cell survival.
The Restricted VH-Gene Usage Is of Prognostic Value in B-Cell Chronic Lymphocytic Leukemia.
Authors: Philippé Jan , Van Bockstaele Femke , Smits Kaatje , Offner Fritz , Verhasselt Bruno , Janssens Ann
Published: October 2019

In B-cell chronic lymphocytic leukemia (B-CLL) the mutation status of the immunoglobulin variable heavy chain gene (VH) is known as a prognostic factor. We have investigated if specific VH-gene usage is of additional prognostic importance regarding survival. Peripheral blood samples from 147 B-CLL patients were analysed for VH usage. Recombinations occurring in at least 5% of cases were studied in depth. The most frequently used VH-gene segments were VH1-69 (10.9%), VH3-7 (7.5%), VH3-30 (6.8%), VH4-34 (6.8%), VH3-21 (6.1%), VH3-23 (6.1%), and VH3-33 (5.4%). The VH gene usage was compared with mutation status, cytogenetic abnormalities and survival. Comparison with age matched controls reveals the restricted VH gene usage in B-CLL. VH gene usage showed a distinct prognostic value (p=0.01) when the patients using VH genes with bad prognosis were grouped together (VH1-69, VH3-21, VH3-23 and VH3-33; 43% of these patients died) and were compared with the patients using VH genes with a relatively good prognosis (VH3-7, VH3-30 and VH4-34; mortality rate of only 13%). The prognostic significance holds true when all patients were included (p=0.03). Also the mutation status (p=0.04) and cytogenetics (p=0.03) showed a prognostic significance. The VH gene usage did not correlate with mutation status, nor with cytogenetics. On the contrary mutation status and cytogenetics were strongly correlated (p<0.00001). These findings are highly suggestive for VH gene usage to offer additional prognostic information to the other well established prognostic parameters. In conclusion, a bias to the specific VH gene segments involved in B-CLL supports the concept that B-CLL arises from B cells driven by specific antigens/superantigens which is different from a stochastic event in the elderly B-cell population. Moreover, the immunoglobulin specificity reveals specific subgroups with differing prognosis.
Predictive Value of Gene Expression with Respect to IgVH Mutational Status and Survival in Chronic Lymphocytic Leukemia.
Authors: Van’t Veer Mars B. , Brooymans Anne , Langerak Anton W. , Graveland Wilfried J. , Van Lom Kirsten , Valk Peter
Published: October 2019

Background: Chronic Lymphocytic Leukemia (CLL) has a variable clinical course, of which IgVH mutational status of the malignant B cells is a strong independent predictor for outcome.

Objective: We studied the expression of 5 different genes for their value to predict IgVH mutational status and overall survival (OS).

Methods: mRNA levels were quantified by real time PCR in lymphoprep separated but unsorted blood samples from 110 morphologically and fenotypically characterised CLL patients for the following genes: ZAP70, lipoprotein lipase (LPL), ADAM29, KIAA0610 and nRIP1. Expression levels were compared wih respect to IgVH mutational status (mutation = % germline < 98%), using logistic regression and with respect to OS (= time from day of laboratory tests to death due to any cause), using Cox regression analysis.

Results: The predictive value of the expression of these 5 genes for IgVH mutational status is shown in table 1. Each of these genes showed to be an independent marker for the prediction of IgVH mutational status. LPL as a single marker was the best predictor for mutation. These factors taken together had a high predictive value towards mutational IgVH status (area under ROC curve = 0.93).

table 1

N Odds ratio P value ZAP70 no 85 1 (>5.2) yes 25 0.098 0.002 LPL no 73 1 (>0.04) yes 37 0.029 <0.001 ADAM29 no 73 1 (>30) yes 37 6.61 0.012 KIAA0610 no 81 1 (>0.25) yes 29 0.19 0.045 NRIP1 no 88 1 (>10) yes 22 48.73 0.008

The predictive value of IgVH mutation and the expression of these 5 genes for OS is shown in table 2. From the six parameters tested, IgVH mutational status and LPL were predictors for OS at a significant level of 5%, while ZAP70 was of borderline significance.

table 2

Conclusion: measured in unsorted CLL samples, all genes, and especially LPL, had predictive value towards the presence of IgVH mutation. Expression of LPL and absence of mutation were adverse prognostic factors for survival. Thus, the replacement of the assessment of both IgVH mutational status and ZAP70 by assessment of LPL levels for the prediction of prognosis in CLL patients may be considered.

N survival at 24months P value mutation no 36 70% yes 44 93% 0.041 ZAP70 no 66 87% yes 18 70% 0.078 LPL no 55 94% yes 29 66% 0.001 ADAM29 no 57 84% yes 27 84% 0.486 KIAA0610 no 63 88% yes 21 71% 0.133 NRIP no 71 81% yes 13 100% 0.167
Upregulation of Human Pim-2 in B-CLL, Involves Interactions between Oct-1, Oct-2, Bob-1 and NF-kB.
Authors: Dicken Yosef , Cohen Amos M. , Bessler Hanna , Levi-Hirsh Daphna , Arad Ariela , Merkel Drorit , Don Jeremy
Published: October 2019

hPim-2 is a proto-oncogene that encodes a serine/threonine kinase and inhibits apoptosis by phosphorylation of BAD. We have shown that hPim is upregulated in human non-Hodgkin’s lymphomas (NHL) and in chronic lymphocytic leukemia (B-CLL) and its cellular transcript levels in B-CLL correlates with lymphocyte doubling time. We found no mutations in the promoter region of hPim-2 in B-cells of 30 patients with CLL (~2000 bp upstream). The proximal promoter region of hPim-2 (600 bp) contains two adjacent NF-kB-binding elements, two adjacent Oct-binding elements and an SP1 element by bioinformatic analysis. Studies have recently shown that the transcription factor Oct-2 and the B-cell specific Oct cofactor Bob-1 are overexpressed in certain large B-cell lymphomas, whereas increased expression of Bob-1 has also been observed in T-cell neoplasms. Shift assays (EMSA) analysis, using nuclear extracts from B-CLL cells and various fragments of hPim-2 promoter region used as probes, revealed that complexes containing an Oct elements were consistently heavier in B-CLL extracts compared with control B-cells. Accordingly, Oct-1, Oct-2 and Bob-1 protein levels were significantly higher in B-CLL compared to healthy extracts. Moreover, chromatin immunoprecipitation (Chip) assays confirmed that in-vivo Oct-1+2 and Bob-1 are indeed physically attached to the hPim-2 promoter, and that this interaction is significantly more intensive in B-CLL cells than in control B-cells. Furthermore, we have found in addition that the p52 isoform subunit of NF-kB predominates the interaction with the kB element in the hPim-2 promoter in B-CLL cells, as compared to the p50 isoform observed in control B-cells. To determine whether these interactions are transcriptionaly significant, we fused the luciferase reporter gene to various promoter fragments, and monitored luciferase expression in-vitro after incubation with either B-CLL or normal B-cell extracts. Luciferase expression was consistently higher when Oct element-containing fragment was incubated with B-CLL cell extracts. Together, these results suggest that the upregulation of hPim-2 in B-CLL is due to enhanced expression and transcriptional activity of the Oct-1+2 and Bob-1 complex and that it might synergistically act with the p52 containing NF-kB transcription factor.
Human Platelets Contain Forms of Factor V in Disulfide-Linkage with Multimerin.
Authors: Hayward Catherine P.M. , Fuller Nola , Zheng Shilun , Adam Frederic , Jeimy Samira , Horsewood Ian , Quinn-Allen Mary Ann , Kane William H.
Published: October 2019

Factor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factor V is stored complexed with the polymeric α-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains, were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Because unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that all unusually large factor V in platelets was associated with multimerin and it remained associated in 0.5 M salt. Multimerin immunodepletion of the normal pooled platelet lysate removed 100 ± 0% of multimerin and 47.0 ± 2.4% of total factor V antigen, whereas sham immunodepletion removed 12.0 ± 3.0 % of multimerin and 4.0 ± 4.0% of factor V antigen (means ± 1 S.D. for 3 experiments). Analyses of serial factor V immunopurified samples indicated that platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage. The suggestion that only a subpopulation of multimerin was covalently linked to factor V was consistent with the estimated 17 fold molar excess of multimerin subunits to factor V molecules in platelets. The disulfide-linked complexes of multimerin and factor V in platelets, that are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Multimerin could function to hold about half of the platelet pool of factor V in covalent and noncovalent linkages, until granule release occurs and thrombin cleavages liberate factor Va for prothrombinase assembly on the platelet surface, akin to the way supporting scaffolds hold pieces of plastic models in a unit until their removal for model assembly is desired.
Demystifying Tissue Factor.
Authors: Butenas Saulius , Gissel Matthew T. , Bouchard Beth A. , Brummel Kathleen E. , Parhami-Seren Behnaz , Mann Kenneth G.
Published: October 2019

Tissue factor (TF) is an integral membrane protein, which is the key initiator of blood coagulation in vivo. Due to the limited availability of natural TF, recombinant proteins of various lengths and origins have been extensively used in research and clinical laboratories worldwide. Experimental results acquired with recombinant TF proteins are frequently used for the understanding of the coagulation processes occurring in vivo, although there is a lack of data confirming the structural and functional identity of natural TF proteins from various sources and recombinant ones. In the current study, human TF from cultured monocytes and purified from placenta were compared with three different species of recombinant TF: 1–218 (extracellular domain only), 1–242 (lacking cytoplasmic domain) and 1–263 (full-length). Anti-TF mAbs gave 93–98% inhibition of TF activity for all TF species tested, in both natural and relipidated preparations. It was established that purified placental TF has a higher affinity for factor VIIa (Kd 0.13 nM) than recombinant counterparts 1–242 and 1–263 (Kd 0.50–0.80 nM). Similarly, placental TF is more efficient in factor X activation by the extrinsic Xase than recombinant TF 1–242 (the second order rate constants are 3.0x107 and 0.7x107 M−1s−1, respectively). We explored the use of these TF species as well as monocyte TF (purified/relipidated and present on LPS-stimulated monocytes) for the initiation of thrombin generation in two in vitro models of blood coagulation. At equimolar concentrations (5 pM; determined by immunoassay), when evaluated in synthetic plasma reconstituted with 2x108/ml platelets, recombinant TF 1-263 provided an initiation phase of ~4 min. Placental TF and relipidated monocyte TF had similar profiles of thrombin generation with an initiation phase of ~3 min. In contrast, 0.5 pM TF on LPS-stimulated monocytes gave an initiation phase of ~1 min. Even at 0.05 pM concentration, monocyte TF was as active as any relipidated protein at 5.0 pM. A similar pattern of relative TF activity was observed in whole blood and plasma PT clotting assays. TF on stimulated monocytes gave the highest activity, exceeding that of any relipidated protein by 100–200-fold. Recombinant TF 1–242 was more active than recombinant TF 1–263 and placental TF in the PT assay but less active in synthetic plasma and whole blood. The lowest overall activity was observed for relipidated monocyte TF. Our data suggest that TF proteins from different sources are different with respect to their functional properties.
Enhanced Capacity of Monocytes from Patients with Essential Thrombocytemia to Generate Tissue Factor. Possible Mechanism of Thrombosis.
Authors: Kornberg Abraham , Koren-Michowitz Maya , Rahimi-Levene Naomi , Voltchek Yulia , Rapoport Micha
Published: October 2019

The main cause of thrombosis, a major complication of essential thrombocythemia (ET), is the high platelet count. In contrast, thrombosis is rare in reactive thormbocytosis (RT), indicating that thrombocytosis can not be the sole cause of thrombosis. Moreover, thrombosis occurs in ET even after reduction of the platelet count. Tissue factor (TF) is the most potent activator of coagulation. Monocytes are a main source of TF and monocyte TF is increased in various disorders including solid tumors and polycythemia vera that are associated with increased incidence of thrombosis. The decreased incidence of thrombosis that was reported recently in ET after treatment with hydroxyurea compared to anagralide was attributed to a decrease in leukocytes that occurs only after hydroxyurea. Based on this information, we studied the capacity of monocytes from patients with ET to generate TF. Peripheral blood mono-nuclear cells (PBMC) were isolated on Ficoll-hypague from 14 patients with ET and 10 with RT due to iron deficiency. Monocytes were counted by anti CD14 and were the same in both groups (18%). Monocytes were purified from lymphocytes by adherence to plastic surface. Cells were incubated for 16 hours with and without endotoxin.TF activity was measured in the cells by modified PT and TF antigen by ELISA. TF activity and antigen were 1.4–1.7x 10−5 U/monocyte and 21–31x10−5 pg/monocyte in unstimulated PBMC from normal controls (NC), RT and ET. Stimulated PBMC from NC and RT showed a similar increase of TF activity and antigen (2.5 and 3.8 fold).However, PBMC from ET generated 12.9 and 14.4 times more TF activity and antigen (p<.005). Similar increase was found in ET after reduction of platelet counts. Endotoxin-stimulated purified lymphocytes from four ET patients generated weak TF while purified monocytes generated TF levels similar to PBMC. Three ET patients compared to none of RT patients developed thrombosis. The increased capacity of monocytes to generate TF may be an additional mechanism of thrombus formation in ET and should be included in the list of hypercoagulability syndromes.
Down Regulation of Thrombin Procoagulant Activity by Prothrombin Activation Fragment 1.2.
Authors: Dasgupta Swapan K. , Thiagarajan Perumal
Published: October 2019

Prothrombin is the precursor of thrombin, the central enzyme in coagulation. Under physiological condition, prothrombin activation catalyzed by the fully assembled prothrombinase complex via an ordered, sequential reaction with primary cleavage at Arg320-lle321 followed by the second cleavage at Arg271-Thr272, forming fragment 1.2 and thrombin. The fragment 1.2 contains the amino-terminal gla domain and the two kringle domains. It has been shown that the kringle 2 domain in fragment 2 can bind to the exosite II of thrombin and inhibit allosterically its activity by inducing conformational changes at the active site. Nascent thrombin that is generated on platelet surface will remains non-covalenty bound to fragment 1.2 by kringle 2-exosite II interaction. To determine whether this interaction can modulate thrombin activity, we tested the effect of anionic phospholipid-bound fragment 1.2 (0–10 μM) on thrombin clotting activity. Phospholipid-bound fragment 1.2 inhibited fibrinogen clotting in a concentration-dependent manner but had no significant effect on esterase activity towards S2238 (D-Phe-Pip-Arg-pNA.2HCI) suggesting a competitive inhibition of fibrinogen binding. We also labeled thrombin at the active site with fluorescein-FPRCK (5-fluorescein-D-Phe-Pro-Arg chloromethyl-ketone) and monitored fluorescence changes following fragment 1.2 binding in a spectrofluorometer. Anionic phospholipid-bound fragment 1.2 induced changes in the active site similar to fragment 2 with half maximal effect at ~8 μM. We tested the effect of fragment 1.2 on fluorescein-FPR thrombin binding to platelets. Fragment 1.2 inhibited thrombin binding to platelet. Consistent with these findings fragment 1.2 inhibited thrombin-induced aggregation of gel filtered platelets in a concentration-dependant manner. These results suggest that membrane-bound prothrombin fragment 1.2 may play an important role in hemostasis by down regulating thrombin procoagulant activity because of its interaction with exosite 2.
The Oncogenic Potential of the Homeobox Gene Cdx2 Is Depending on the MAPK Pathway and Can Be Antagonized by the MEK1/2 Inhibitor PD98059 in a Mouse Model of t(12;13)(p13;q12) Positive AML.
Authors: Rawat Vijay P.S. , Naidu Vegi M. , Schessl Christina , Cusan Monica , Humphries R.Keith , Lynch J. P. , Hiddemann W. , Feuring-Buske Michaela , Buske Christian
Published: October 2019

In AML the translocation t(12;13)(p13;q12) results in the ectopic expression of the homeobox gene Cdx2 and the expression of the ETV6-CDX2 fusion. We have recently shown that myeloid leukemogenesis is induced by the ectopic expression of the proto-oncogene Cdx2 and not by the ETV6-CDX2 fusion gene in a murine model of t(12;13) AML. To characterize the contribution of different Cdx2 motifs to the transforming capacity of the gene we generated different mutants, inactivating the DNA binding homeodomain (N51S-Cdx2), or the PBX1-interacting motif (W167A-Cdx2), or deleting the N-terminal portion of Cdx2 (N-Cdx2). Expression of Cdx2 and the different mutants were induced in primary murine bone marrow cells by retroviral gene transfer, using an MSCV based retroviral construct with an IRES-YFP cassette. Expression of Cdx2 and the W167A-Cdx2 mutant significantly increased primary colony formation (3-fold) (n=3;p<0.001) with a higher number of CFU-G/GM colonies (p<0.015). Furthermore, both constructs enhanced the replating capacity of clonogenic progenitors with an 80-100fold increase in secondary colonies (p<0.005). In addition, both constructs induced the outgrowth of blast colonies (2700fold; p<0.02). In contrast, cells transduced with N51S-Cdx2 and N-Cdx2 lost their clonogenic potential after replating. In vivo all mice transplanted with cells expressing Cdx2 or the W167A-Cdx2 mutant developed transplantable AML. However, in Cdx2 leukemic mice > 90% of the cells co-expressed Gr-1+ and Mac1+, whereas in W167A mice 40% of the leukemic population were Gr-1+ only. The N51S mutant induced a distinct leukemia phenotype with 90 % Gr-1+/c-Kit+. We extended structure-function analyses, inactivating the phosphorylation site (S60) in the Cdx2 transactivation domain, previously shown to be regulated by the MAPK family. We confirmed that oncogenic Cdx2 is phophorylated at the N-terminal in primary BM cells by Western blotting using a P-Cdx2-S60 specific antibody. S60 position mutation slightly reduced the hematopoietic activity of wild-type Cdx2. Incubation with the MEK1 inhibitor PD98059 inhibited phosphorylation, decreased the frequency of CFU-S 8fold (n=7; p<0.001) and blocked growth of leukemic Cdx2 transfected blasts in vitro. In contrast, the p38 inhibitor SB2059 did not prevent phosphorylation and was unable to antagonize Cdx2 induced transformation. These data demonstrate that the transforming activity of Cdx2 and the phenotype of Cdx2 induced leukemias is depending on the functional integrity of distinct Cdx2 domains. Furthermore, our data link the oncogenic potential of Cdx2 directly to the MAPK signaling, opening the possibility to counteract Cdx2 associated leukemogenesis by kinase inhibitors.
Epigenetic Suppression of the CTNNA1 Gene, Encoding the α-Catenin Protein, which Is Located in the 5q31 Critical Deleted Region in Malignant Myeloid Disorders with del(5q).
Authors: Liu Ting Xi , Becker Michael , Hsu Karl , Jelinek Jaroslav , Deng Min , Bloomfield Clara , Issa Jean-Pierre , Clarke Michael F. , Look A. Thomas
Published: October 2019

Recurring interstitial loss of all or part of the long arm of chromosome 5, del(5q), is associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Although the affected genes have not been identified, two critical deleted regions (CDR) on 5q have been established, a proximal CDR located in 5q31, and another distal CDR in 5q32–33. We investigated the expression of 28 genes located in the 5q31 CDR, in enriched populations of normal hematopoietic stem cells (HSCs) and leukemic initiating cells (L-ICs) by the reverse transcription polymerase chain reaction (RT-PCR). Of the 28 5q CDR genes investigated, 12 (42%) were expressed in HSCs (CD34+Thy-1+CD38-Lin-), including HNRPA0 (Hs.77492), LOC51306 (Hs.82035), SMAP (Hs.5464), CDC23 (Hs.153546), LOC51307 (Hs.102469), LOC51780 (Hs.24125), EGRI (Hs.326035), ETF1 (Hs.77324), HSBPA9B (Hs.3069), CTNNA1 (Hs.178452), MATR3 (Hs.223745) and UBE2D2 (Hs.108332). We also isolated L-ICs (CD34+CD123+CD38-Lin-) from 11 cases of MDS/AML with 5q-, and investigated their expression levels of these 12 HSC genes. Expression of CTNNA1 was not detected in the L-ICs of 8 of the 11 cases of del(5q) MDS/AML using standard RT-PCR, while the other 11 genes were expressed in each patient at levels comparable to those of HSCs. Quantitative analysis of CTNNA1 expression in L-ICs by real-time RT-PCR confirmed expression levels <30% of HSC levels in 4 of the 6 cases tested with del(5q) MDS/AML. Analysis of protein expression by immunofluorescence also verified a lack of detectable CTNNA1 in L-ICs for the two del(5q) cases tested. CTNNA1 expression levels were normal in each of the 11 non-del(5q) MDS/AML cases tested. Methylation of the CTNNA1 promoter region correlated with the downregulation of CTNNA1 transcript in the del(5q) HL-60 cell line, in which CTNNA1 expression was partially restored following treatment with 5-aza-2-deoxycytidine. Our results suggest that epigenetic suppression of the expression of the intact CTNNA1 allele may contribute to the pathogenesis of MDS/AML with del(5q). We are currently using mouse and zebrafish models to study the role of CTNNA1 loss-of-function in del(5q) MDS/AML leukemogenesis.
cDNA Microarray Analysis of Genes Up-Regulated by Treatment with 5-AZA-2′-Deoxycytidine in Combination with Trichostatin A Identifies Aberrant Metallothionein 1H Promoter Methylation at a High Frequency Iin Human AML.
Authors: Scott Stuart A. , Pearson Derek S. , Bainbridge Matthew N. , Dong Wei-Feng , Takahashi Naoto , Sheridan David P. , Ichinohasama Ryo , Geyer C. R. , DeCoteau John F.
Published: October 2019

Gene promoter methylation is a major mechanism of transcriptional silencing in cancer cells that is mediated by CpG methylation and histone deacetylase activity, with methylation being dominant. Importantly, pharmacological treatment of cancer cells with inhibitors of DNA methylation and histone deacetylation have been shown to synergistically reactivate transcription of previously silenced genes. In an effort to identify novel genes silenced by DNA methylation in human acute myeloid leukemia (AML), we used cDNA microarray technology to screen for genes upregulated in the human cell line AML193 following treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, Trichostatin A (TSA). Analysis of 1,700 gene microarrays in triplicate revealed seven genes consistently upregulated by combined 5-Aza-dC and TSA treatment, all of which were confirmed by semi-quantitative RT-PCR. Interestingly, four of these genes (MT1H, MT1E, MT2A, MT1G) are members of the metallothionein family of cysteine-rich small molecular weight proteins. These proteins are considered to be important mediators of cellular detoxification and are induced by toxic heavy metals, UV irradiation and reactive oxygen species. As well, MT1G promoter methylation has been implicated in the pathogenesis of sporadic papillary thyroid carcinoma. The methylation status of candidate metallothionein genes reactivated by 5-Aza-dC and TSA treatment in AML193 cells was assessed by methylation specific PCR (MSP) in seven human AML cell lines. Methylated promoter alleles were detected in MT1H, MT1E and MT1G in 71%, 29% and 29% of cell lines, respectively, and there was no methylation of MT2A in any of the cell lines tested. To investigate for the occurrence of MT1H, MT1E and MT1G methylation in human AML in vivo, we analyzed AML patient blasts (n=39) and non-leukemic controls (n=13) by MSP. MT1H, MT1E and MT1G methylation was detected in 51%, 0% and 3% of AML patient samples, respectively, and there was no methylation of any of these genes in control samples. Our findings implicate MT1H promoter methylation in the pathogenesis of human AML and suggest that the use of cDNA microarray technology following pharmacological manipulation is a useful approach for identifying novel epigenetically silenced genes in this disease.
Differential Effects on Cell Cycle Regulators by AML1-ETO and a C-Terminal Truncated AML1-ETO (From Tumor Suppressor to Oncogene).
Authors: Peterson Luke F. , Yan Ming , Boyapati Anita , Zhang Dong-Er
Published: October 2019

The fusion protein AML1-ETO created by the 8;21 translocation involved in de novo acute myeloid leukemia requires secondary mutational events to promote leukemia. Here, we report that the loss of the molecular events associated with AML1-ETO C-terminus including an NCoR/SMRT interacting domain transforms AML1-ETO into a potent leukemogenic protein in mice. Furthermore, we present evidence of aberrant protein expression of cell cycle regulators in a hematopoietic cell line expressing AML1-ETO compared to the leukemogenic truncated form of AML1-ETO (AML1-ETOtr). Our studies show that AML1-ETO and AML1-ETOtr are biochemically isolated from the same cellular compartments, that they can oligomerize, and that they can both efficiently immunoprecipitate the transcription factor Gfi-1. However, contrary to AML1-ETO, AML1-ETOtr does not promote growth arrest. Western analyses show that cell cycle promoting factors cyclin D3 and cyclin A are decreased by AML1-ETO, while their RNA levels remain the same. In addition, an increase in the cdk-inhibitor p21WAF1 is observed in the presence of both proteins, while only AML1-ETO induces high expression of the cdk-inhibitor p27KIP1. These changes are associated with a deregulation of the SCF ubiquitin E3 ligase component Skp2 that is involved in controlling the levels of both cyclins and cdk-inhibitors. These observations suggest that AML1-ETO has tumor suppressor activity. Additional mutations to bypass this effect can change it into an oncogenic protein. Therefore, our results lead to a new model of AML1-ETO in leukemogenesis, i.e., the gain/loss of function of cell cycle regulators to promote cell cycle progression or disruption of molecular events associated with AML1-ETO C-terminal NCoR/SMRT interacting domain are required for AML1-ETO involved leukemogenesis. The disrupted molecular events may include 1) the loss of factor(s) physically interacting with the C-terminal domain of AML1-ETO, 2) the alteration by mutagenesis of signaling pathways downstream of AML1-ETO C-terminal domain, and 3) the dysfunction within AML1-ETO C-terminal domain by truncations or mutations.
A Prospective Randomized Trial of Oral Melphalan, Prednisone, Thalidomide (MPT) vs Oral Melphalan, Prednisone (MP): An Interim Analysis.
Authors: Palumbo Antonio , Bertola Alessandra , Musto Pellegrino , Caravita Tommaso , Callea Vincenzo , Cangialosi Clotilde , Liberati Anna Marina , Niscola Pasquale , Catalano Lucio , Grasso Mariella , Lauta Vito Michele , Petti Maria Concetta , Morandi Sergio , Galli Monica , Bringhen Sara , Cavallo Federica , Falco Patrizia , Boccadoro Mario
Published: October 2019

In newly diagnosed multiple myeloma (MM) patients, the combination melphalan, prednisone and thalidomide induces a fast tumor response with a high complete remission rate. In a prospective randomized trial, we compare the efficacy and toxicity of oral MPT and MP.

An interim analysis was conducted after the first 200 newly diagnosed myeloma patients, median age 72, range 56–85, entered the study, between January 2002 and June 2004. At present, 116 patients were evaluated for toxicity and 102 patients for response on an intent to treat basis. The MPT regimen included 6 monthly courses of oral melphalan 4 mg/sqm and prednisone 40 mg/sqm for 7 days every month plus thalidomide 100 mg/day continuously until any sign of progressive disease or relapse. The dose of thalidomide was reduced to 50% when grade II toxicity occurred, and suspended for any grade III. On December 2003, the protocol was amended and enoxaparin prophilaxys was added. The MP regimen was as MPT without thalidomide. The end points of the study were: response, EFS, OS and toxicity.

The response rate among patients who received MPT was: 25.9% immunofixation negative CR (CR), 5.5% immunofixation positive near CR (nCR) 48.2% partial remission (PR) (M-protein reduction 50–99%), 9.3% stable disease (SD) (M-protein reduction 0–49%) and 11.1% progressive disease (PD). The response rate after MP was 4.2% CR, 0% nCR, 43.6% PR, 23% SD and 29.2% PD. Response was followed by significant improvement of performance status, skeletal pain, anemia and transfusion requirement.

After a median follow up of 15 months, 38 patients relapsed: 11 (29%) after MPT and 27 (71%) after MP. The EFS @ 26 months was 67.8% for MPT and 32.4% for MP (P <0.001). The median OS has not been reached.

Treatment-related mortality was 5% after MPT (1 septicemia, 1 pulmonary thrombo-embolism and 1 heart failure), and 1.9% after MP (1 myocardial infarction). The major adverse events of MPT vs MP were: deep-vein thrombosis (19.3% vs 1.9%), grade III-IV infections (12.9% vs 1.9%), grade I-II neurotoxicity (35.5% vs 5.5%), grade III-IV hematologic toxicity (22.6% vs 27.6%). Thalidomide discontinuation was recorded in 33.8% of patients (8 thrombo-embolic events, 4 neurotoxicities, 4 constipations, 2 infections, 3 miscellaneous); dose-reduction in 24.2% of patients (8 neurotoxicities, 3 constipations, 4 miscellaneous).

MPT significantly improves response rate and EFS in elderly myeloma patients with a median age of 72 years. These results are similar to those observed after transplant.

An update of these data will be presented.
Mutation of Cellular Retinoic Acid-Binding Protein-II (Crabp-II) Does Not Account for Resistance to All-Trans Retinoic Acid (ATRA) in Relapse Acute Promyelocytic Leukemia (APL).
Authors: Sankoorikal Binu-John , Zhou Da-Cheng , Wiernik Peter H. , Gallagher Robert E.
Published: October 2019

An increase in CRABP-II has frequently been invoked as a cause of resistance to ATRA in APL due to cytoplasmic sequestration and catabolism of ATRA. However, recent evidence indicates that CRABP-II has a positive rather than a negative role in ATRA activity by facilitating delivery of ATRA to retinoic acid receptor-alpha (RARα) associated with ATRA target genes in the cell nucleus or/and by serving as a co-activator of RARα-regulated transcription. This implies that if CRABP-II has a role in the development of ATRA resistance in APL, this would more likely occur through a deficiency rather than from an increase in CRABP-II. We previously reported that CRABP-II is constitutively expressed at similar levels in pretreatment and relapse APL cells (Zhou, et al, Cancer Res58, 5770, 1998), suggesting that putative CRABP-II deficiency is not due to the loss of CRABP-II expression. To investigate the alternative possibility that CRABP-II deficiency might arise through inactivating mutations, we sequenced the entire coding region of CRABP-II from 18 cases of APL who had relapsed from prior ATRA-containing treatment regimens. In 8 cases RNA was transcribed by reverse transcriptase to cDNA and amplified by polymerase chain reaction (PCR), using primers anchored in the 5′ and 3′ untranslated region of mRNA; in 10 cases, genomic DNA was PCR amplified for sequence analysis, using primers anchored in the introns between the 4 exons of the gene. No CRABP-II mutations were identified. The samples tested included 11 first-relapse cases, 2 of whom were refractory to reinduction therapy with intravenous liposomal-ATRA, and 7 multiple relapse cases, all of whom were clinically refractory to ATRA and had, additionally, relapsed from arsenic trioxide therapy. Also, no mutations were found in 3 APL patients who had relapsed from chemotherapy-induced remissions or in 3 APL cell lines (NB4, UF-1 and AP-1060). Two heterozygous base substitutions were incidentally identified in CRABP-II intron 2 in a chemotherapy-only treated patient. These results indicate that CRABP-II mutations rarely, if ever, contribute to ATRA-resistance or disease relapse in APL.