bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Levels of Hepatocyte Growth Factor in Patients with DIC.
Authors: Kawasugi Kazuo , Noguchi Maho , Tashiro Haruko , Gotoh Moritaka , Shirafuji Naoki , Matsuda Juzo , Kinosita Tadatoshi
Published: October 2019

Injury of endothelial cells has been postulated as an initial trigger of the progression of DIC. Although hepatocyte growth factor (HGF) is a member of endothelium-specific growth factor, the relationship between HGF and DIC has not been described. To investigate the role of HGF, we measured plasma levels of HGF in patients with sepsis-associated (n=20) and acute promyelocytic leukemia (APL)-induced DIC (n=6). Plasma samples from those patients groups were assayed for HGF levels by enzyme-linked immunosorbent assay (ELIZA, TECHNE Corporation, USA). The VEGF levels were determined by one-step sandwich enzyme immunoassay (EIA, Chemicon International, USA).

The thrombin antithrombin complexes (TAT) levels were higher in both DIC patients as reported by others. In the septic patients with DIC, we found significant elevations in HGF levels compared to normal controls. Also, the HGF levels were elevated in the APL patients with DIC. However, we did not find any difference in plasma levels of VEGF in the APL and septic patients with DIC. There was a slight correlation between the TAT and HGF levels in both (septic and APL) patients groups with DIC. These results suggest that plasma levels of HGF may be candidates for a marker of DIC. It appears that HGF may contribute to the severity of DIC.

Sepsis associated DIC APL with DIC Controls (n=10) *(P<0.005) significantly different controls HGF 7949±71230* 11902±10726 846±216pg/ml VEGF 172±7.0 209±35 188±8pg/ml TAT 28.4±17.5* 22.5±15.3* <3 μg/ml
Prognostic Impact of MRD in the Bone Marrow of Children at First Isolated Extramedullary Relapse of ALL.
Authors: Hanzsch Nikola , Eckert Cornelia , Acquaviva Cecile , Fronkova Eva , von Stackelberg Arend , Stadt Udo zur , Schrauder Andre , Trka Jan , Seeger Karl , Henze Guenter , Cave Helene
Published: October 2019

Sensitive quantification of minimal residual disease (MRD) is an established method to assess treatment response in patients with bone marrow (BM) relapse of acute lymphoblastic leukemia (ALL). We have investigated, whether MRD can also determine the tumor load in the BM at cytologically isolated extramedullary (IEM) relapse and can consequently be associated with prognosis in this subgroup.

The extent of sub-cytological BM involvement at relapse diagnosis has been quantified in 53 patients with first relapse in the central nervous system (n=36), and in the testes (n=17) treated in Germany (n=33; 09′94 - 12′03) and Czech Republic (n=9; 10′01 - 04′04), or in France (n=11; 12′90 - 11′03) according to protocols ALL-REZ BFM, or to COOPRALL, respectively. MRD has been sensitively assessed by real-time-quantitative PCR using T-cell-receptor and immunoglobulin gene-rearrangements detected in the extramedullary or initial BM material. Stability of initial clonal markers was confirmed. A MRD-level of <10−4 was measured in 22 patients. Thirty-one patients showed a positive MRD-status: <10−3 - =/>10−4: n=9; <10−2 - =/>10−3: n=12; and >10−2: n=10. MRD-status was not associated with the relevant prognostic parameters sex, time-point of relapse, immunophenotype, and site, whereas it was associated with age at initial diagnosis (p=.057), and BCR/ABL translocation (p=.011). The probability of event-free survival (EFS) at 5 years was .55+/−.15 and the rate of subsequent relapses was 18% in patients without MRD detection at a level of 10−4 compared to .16+/−.10 and 52% in those with =/>10−4 (p(log-rank)=.16 and p=.028 respectively).

Our study reveals a large range of subcytological BM involvement in patients with IEM relapses of ALL. A relation to single prognostic factors and to the relapse rate can be found, but not to EFS.

The study was kindly supported by ‘Deutsche Kinderkrebsstiftung’, Germany, by ‘Kompetenznetz der Paediatrischen Onkologie’, BMBF, Germany; by ‘programme hospitalier de recherche clinique’ (PHRC 2002), France and by GAUK 62/2004, Czech Republic.
Co-Existence of Various Sub-Clones in TEL-AML1 Positive Acute Lymphoblastic Leukemia in Association with Sub-Microscopic Deletion of AML1.
Authors: Toren Amos , Rothman Rachel , Bielorai Bella , Reichart Malka , Amariglio Ninette , Rechavi Gideon , Traktenbrot Luba
Published: October 2019

The TEL/AML1 fusion gene is the most common gene rearrangement in pediatric acute lymphoblastic leukemia (ALL). Although considered to be a low risk leukemia it has a 20% risk of late relapse. The coexistence of different sub clones at diagnosis, based on polymerase chain reaction (PCR) studies of Ig/TCR gene rearrangement, was recently reported in this subtype of ALL. Their different response to chemotherapy may explain the emergence of certain sub clones at relapse, and may serve as a marker for minimal residual disease follow-up.

Several chromosomal rearrangements such as t(9;22), t(8;21), inv(16) and rearrangements of the MLL gene are frequently associated with submicroscopic deletions and some of them have prognostic significance. Such deletions were not reported in t(12;21) positive ALL.

Bone marrow cells from 76 pediatric patients with ALL at diagnosis were analyzed for the presence of the TEL/AML1 fusion gene by interphase fluorescence in situ hybridization (FISH). We used a new system of combined analysis enabling a very large-scale study of the cells of interest with regard to morphology, FISH and immunophenotyping. Fourteen patients were positive for the translocation. Four of them had several sub clones associated with various combinations of additional chromosomal abnormalities. The most striking was an atypical and unexpected hybridization pattern consistent with a submicroscopic deletion of the 5′ region of the AML1 breakpoint (intron2) not previously reported. We describe the use of a larger probe for AML1 (AML1/ETO) to exclude the possibility of insertion of TEL into the AML1 region without breakage and to reduce the false positivity due to optical fusion. This may enable a better monitoring of minimal residual disease in cases with submicroscopic deletion. All patients had some sub-clones with TEL deletion. Other abnormalities included trisomy and tetrasomy 21 as well as double TEL-AML1 fusion.

The analysis of numerous sub-clones at presentation in these patients suggests clonal evolution at an early stage of the disease. These sub-clones may have different sensitivities to chemotherapy, and some of them may reappear at relapse. The frequency of AML1 deletion in t(12;21) in addition to other chromosomal abnormalities, is unknown. The involvement of these findings in the generation of leukemic sub clones, their prognostic significance and role in minimal residual disease follow-up deserves further studies in a large number of patients and a longer follow-up.
Discovery of Genes Involved in Experimental Pre-Leukemia to Leukemia Transition in Mice Using Expression Arrays.
Authors: Entin Igor A. , Kogan Scott C.
Published: October 2019

Acute myeloid leukemia (AML) is characterized by a block in differentiation of myeloid cells accompanied by suppression of normal production of blood cells and dissemination of immature cells from their normal hematopoietic environment. In order to gain insight into the mechanisms governing these features of leukemia, we have examined mRNA expression patterns in transgenic mice that express PML-RARA (associated with human acute promyelocytic leukemia, APL) and BCL2. Such mice represent a model system in which these genetic changes combine to initially arrest differentiation in a pre-leukemic state, with acute leukemia arising only upon the acquisition of additional genetic changes. In the present study we identified the set of genes whose transcription is altered in pre-leukemic and leukemic bone marrow in comparison to bone marrow from normal mice. We generated and analyzed three sets of gene expression data from cDNA microarrays: (1) PRE-LEUKEMIA vs. NORMAL (6600 substances), (2) LEUKEMIA vs. NORMAL (6677 substances) and (3) PRE-LEUKEMIA vs. LEUKEMIA (3762 substances). Greater than 90% of substances showed similar expression in the pre-leukemic and leukemic samples. Sixty unique genes were differentially expressed, in a statistically significant manner, with two-fold or more difference in expression, and presenting in all three datasets. Comparison of these 60 revealed genes to a published human AML expression dataset (Bullinger et al., N Engl J Med, 350:1605, 2004) identified eight genes with the same pattern of expression in our mouse model and in human APL, and four common genes with opposite expression. For mouse pre-leukemia vs. leukemia as compared to all human AMLs vs. APL, the eight genes that showed a similar change were: COL1A2, COL5A2, EIF5A, LAMR1, LGALS1, NOTCH2, SNN, and ACP5. Of particular interest to our laboratory are two genes: LGALS1 and NOTCH2. Among the identified genes the first is most over-expressed, and the second is most under-expressed in human APL. Next, we identified genes in our dataset co-expressed with LGALS1 or NOTCH2 (correlation coefficient >0.85). Among discovered genes were revealed transcription factors (NKX6-1, GTF3A, ZFP95, SOX11, HOXA4 and more), translation factors (EIF3S6, EIF5A, EIF4A1 and more), differentiation factors (RQCD1, NEUROD2, CREBBP/EP300 inhibitory protein 1 and more), oncogenes (Ski, COUP-TF1 and more) and others. We are currently seeking to identify which of these changes may contribute to AML pathogenesis. Gene profiling using such a comparative approach for analysis will allow better understanding of leukemogenesis, and can also be applied for studies of tumor progression in other malignancies.
Gene Expression Profiling Identifies Genes Associated with In Vitro Resistance to Daunorubicin and Cytarabine in Children with De Novo Acute Myelogenous Leukemia (AML) from the Pediatric Oncology Group (POG) Study # 9421.
Authors: Kuo Dennis J. , Lacayo Norman J. , Buck Steven , Yu Ron , Saraiya Chitra , Douglas Lorrie , Weinstein Howard , Chang Myron , Willman Cheryl L. , Tibshirani Robert , Dahl Gary V. , Sikic Branimir , Ravindranath Yaddanapudi
Published: October 2019

Daunorubicin and cytarabine are the most effective chemotherapeutic agents for AML. Using MTT (3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays as an in vitro test for sensitivity to these agents, investigators have found that leukemia cells from pediatric AML patients with Down Syndrome, which are more sensitive to cytarabine than blasts from non-Down Syndrome AML patients, have been associated with an increased ratio of deoxycytidine kinase to cytidine deaminase and an increased level of cystathionine-beta-synthetase [Ge, Jensen, Stout et al. Cancer Res2004; 64, 728–35]. Looking for related findings in non-Down syndrome AML, leukemic blasts from non-Down syndrome children with AML were examined utilizing gene expression profiling and MTT assays. Expression signatures from cDNA arrays were related to the drug sensitivity results to daunorubicin and cytarabine by supervised clustering. These results will then be analyzed to look for relationships to the patient’s clinical outcomes and identify genes that may be potential therapeutic targets for drug resistance reversal.

Bone marrow or blood specimens from 103 patients registered on the Pediatric Oncology Group (POG) protocol 9421 were studied. The diagnostic specimens were tested using MTT assays to measure the IC50 (drug concentration that causes 50% of the cells to die) for both daunorubicin and cytarabine. Samples from ninety-three de novo childhood AML patients were analyzed with a 43,760 element spotted array (containing 41,751 unique genes and expression sequence tags [ESTs]) from the Stanford University Microarray Core Facility. We selected the gene expression profile from 10 samples with the highest and lowest IC50’s for each drug. Then, we used Significance Analysis of Microarrays (SAM) to find significant differences in the gene expression between samples sensitive and resistant to each drug. A cluster of 118 overexpressed genes was associated with resistance to daunorubicin (e.g., BCL6, BAPX1, BCL2A1, MBD2, RAB31 and CDKN1A). A cluster of 24 overexpressed genes was associated with resistance to cytarabine (e.g., RIT1, SH3BP2, BCL2A1, NFKBIA, and PTPRE).

In summary, MTT assays and gene expression profiling have identified genes that correlate with daunorubicin and cytarabine resistance. Study of the relationship among drug resistance patterns, gene expression profiles and clinical outcome may allow us to better predict patients’ prognoses at diagnosis and also can be used to identify novel targets for drug resistance reversal.
Factors Affecting Thrombopoietin Production from Bone Marrow Stromal Cells.
Authors: McIntosh Bryan J. , Fox Norma E. , Kaushansky Kenneth
Published: October 2019

Thrombopoietin (TPO) is the primary regulator of megakaryopoiesis and therefore also the most important determinant of the number of platelets in circulation. The regulation of TPO blood concentration is complex, with at least a significant component mediated by removal and degradation of the hormone by the mature circulating platelet mass. In this way, if the production of TPO were fixed, when the number of platelets rises, an increased amount of TPO is removed from the plasma, resulting in an inverse relationship between platelet counts and TPO concentration. However, several studies hint at the existence of additional mechanisms in which TPO production is altered in response to physiological and pathologic conditions. We, and others, have previously reported that TPO mRNA is increased in the bone marrow of mice or humans after either immune- or radiation-mediated thrombocytopenia, although hepatic levels remain unchanged by this manipulation. To further explore the mechanism(s) of this effect, we utilized in vitro marrow stromal cell models to study the effects of blood proteins on TPO production. As an initial hypothesis we determined if platelet-derived proteins in serum might suppress TPO production from both a marrow stromal cell line, OP9, or primary murine marrow stromal cells derived from long-term hematopoietic cultures. As assessed by quantitative RT-PCR we found that TPO mRNA levels increase a mean of 2.8-fold±0.7 (n = 3) twelve hours following the removal of serum from the culture. Similar results were obtained from primary murine marrow stromal cells; TPO mRNA levels rose a mean of 2.9-fold±0.9 (n = 4) within sixteen hours of serum deprivation. In contrast, TPO transcript levels were unaffected by the same manipulation of serum in the hepatocyte cell line HEPA1c1c7. As the removal of serum might have induced a stress response in the cells, we tested whether other cell stressors might mimic this response; we found that neither UV irradiation nor treatment with toxic metals such as nickel, cobalt, or cadmium produced any rise in TPO mRNA in marrow stromal cells. Furthermore, to test whether a cell cycle arrest triggered by serum deprivation might mediate these effects, we treated stromal cells with cell cycle inhibitors, but failed to find any affect on TPO transcript levels in stromal cells. Further biochemical fractionation of serum suggested that one or more distinct proteins is responsible for this effect, demonstrated by the ability of both ammonium sulfate precipitation and ion exchange chromatography to partition the suppressive effects of serum. However, the active agent in serum is not TPO itself, as the addition of 150 ng/ml of the pure hormone did not suppress stromal cell TPO transcript levels. Knowledge of this novel regulatory mechanism should be useful in treating platelet disorders and perhaps also during stem cell transplantation, a setting in which TPO is known to play a vital role.
Evidence for Killing of Mesenchymal Stem Cells (MSC) by Autologous Natural Killer Lymphocytes.
Authors: Poggi Alessandro , Massaro Anna-Maria , Negrini Simone , Pierri Ivana , Balocco Manuela , Michelis Gianluca , Aquino Sara , Albarello Alessandra , Gobbi Marco , Zocchi Maria Raffaella
Published: October 2019

In this study, Mesenchymal Stem Cells (MSC) were obtained from bone marrow of 10 patients suffering from acute myeloid leukemia (AML), six M0/1 two M2, and two M5 (according to the FAB classification), 8 out of 10 in post-chemotherapy complete remission. These cells differentiated into adipocytes or osteoblasts under appropriate culture conditions. MSC were CD44+, CD73a+ CD73b+ CD105+, beta1 integrin+, ICAM1+, HLA-I+, HLA-II+ (variable proportions), CD45−, CD31−, CD34− and they constitutively expressed the stress-inducible MHC-related molecules MIC-A and the UL16 (induced at the surface of cells infected by cytomegalovirus) binding protein ULBP3. These molecules are reported ligands for the NKG2D receptor expressed by natural killer (NK) and CD8+ T lymphocytes, effector cells that are thought to play a role in host defence against tumors. NK cells have also been shown to regulate normal differentiation of hemopoietic precursor into the myeloid or lymphoid cell lineage. Moreover, it has been stated that NK cells are not able to damage autologous cells, as they receive negative signals through inhibitory receptors, including killer Ig-like receptors (KIR) or C-type lectin inhibitory receptors (CLIR), which bind to HLA-I discrete alleles. Surprisingly, we found that autologous IL2-activated, but not freshly isolated, NK cells lysed MSC, while T lymphocytes did not kill self or non-self MSC. Binding of ICAM-1 expressed by MSC to its receptor, the integrin LFA-1, expressed by NK cells plays a key role in MSC/NK interaction. More importantly, NKG2D/MICA and/or NKG2D/ULBP3 engagement is responsible for the delivery of lethal hit. Conversely, it appears that HLA-I molecules do not protect MSC from NK cell-mediated injury. Taken together, these data suggest that NK cells, when activated as it may occur during the first response to viral infections, are able to eliminate MSC, thus altering the normal interactions with hemopoietic precursors and possibly affecting their differentiation. This mechanism might also contribute to the development of aberrant precursors as observed in acute leukaemias.
Empirical Antifungal Therapy (Rx) with Caspofungin (CAS) vs Liposomal Amphotericin B (L-AmB) for Persistently Febrile Neutropenic Patients (Pts) with Acute Myeloid Leukemia (AML).
Authors: Maertens Johan A. , Teppler Hedy , Lupinacci Robert , Bourque Michael , DiNubile Mark , Sable Carole
Published: October 2019

Background: In a double-blind randomized trial of empirical antifungal Rx for persistently febrile neutropenic pts, CAS was as effective as and better tolerated than L-AmB. We now examine the results of this study in the subgroup of pts with AML.

Methods: Randomization to CAS (70 mg x 1, then 50 mg/d) or L-AmB (3 mg/kg/d) was stratified by risk category [high risk = allogeneic hematopoietic stem cell transplant or relapsed acute leukemia] and use of antifungal prophylaxis. The primary efficacy endpoint was % of treated pts with documented fever and neutropenia who had a successful outcome defined by all the following: successful Rx of baseline (BL) invasive fungal infection (IFI) (if any), no breakthrough (BT) IFI to 7 d post-Rx, survival @7d post-Rx, no premature discontinuation (DC) due to lack of efficacy or study drug toxicity, and fever resolution x 48 hr during neutropenia.

Results: 703/1095 (64%) of the treated pts had AML, including 364/556 (65%) and 339/539 (63%) in the CAS and L-AmB groups, respectively. Demographic characteristics were similar in AML pts in both Rx groups. 27% of CAS and 22% of L-AmB pts with AML were high risk. Median days of Rx were: CAS, 12; L-AmB, 11. The table shows % AML pts with a successful outcome by Rx group.

% AML pts with a successful outcome by Rx group

CAS (N=364) L-AmB (N=339) Difference (CAS - L-AmB) 95% CI for difference between treatment groups † 7/14 CAS and 5/16 L-AmB pts had successful Rx of BL IFI. Success Rx of BL IFI † 50 31 19 (−16, 53) No BT IFI 93 95 −2 (−5, 2) Suvival @ 7d post-Rx 92 88 4 (0, 8) No premature DC 87 86 2 (−3, 7) Fever resolution 48 46 2 (−5, 9) All of the above 40 38 2 (−5, 10)

The composite success rates for the high risk AML pts were 46/99 (46%) for CAS and 31/76 (41%) for L-AmB.

Conclusions: In this post hoc subgroup analysis, CAS provided an effective and generally well-tolerated option for the empirical Rx of persistently febrile neutropenic pts with AML.
Oligoclonal/Multiclonal Pattern of Kaposi Sarcoma-Associated Herpesvirus (KSHV/HHV-8) Episomes in Primary Effusion Lymphoma Cells.
Authors: Boulander Emmanuelle , Duprez Renan , Delabesse Eric , Gabarre Jean , Macintyre Elizabeth , Gessain Antoine
Published: October 2019

Primary effusion lymphoma (PEL) is a rare non-Hodgkin lymphoma of B-cell lineage origin, developed in the serous body cavities and mostly observed in the course of HIV infection. PEL tumor cells are latently infected with Kaposi sarcoma-associated herpesvirus (KSHV) and in most cases, co-infected with Epstein-Barr virus (EBV). As a virological marker of clonality in PEL, we analyzed the fused terminal repeat regions (TR) of KSHV episomes using pulsed-field gel electrophoresis and Southern blot hybridization, in 15 primary PEL tumors including 10 EBV+ cases, and in 6 PEL cell lines. The cellular clonality was assessed on the same genomic DNA samples, by Southern blot and PCR detection of monoclonal immunoglobulin heavy chain (IgH) VDJ gene rearrangements, associated in the co-infected PEL with Southern blot analysis of the fused termini of EBV episomes. Monoclonal IgH gene rearrangements were detected in 87% (13/15) of primary PEL tumors using Southern blot, in 73% (11/15) using PCR analysis, and in all tumors considering both methods. All the co-infected PEL also displayed a monoclonal EBV infection. However, only 5 primary PEL tumors were found to be monoclonally infected with KSHV. In the 10 remaining cases, as well as in the KSHV+/EBV-negative BC-3 cell line, we found a biclonal (2 bands)(n=3) or an oligoclonal/multiclonal pattern (3–6 bands)(n=7) of KSHV episomes. KSHV infection of non-tumoral contaminating cells, superinfection mechanisms from lytically infected tumor cells, or viral integration events might explain such discordant findings between the viral and cellular clonality of PEL.
Thrombopoietin as an Independent Prognostic Marker in Chronic Lymphocytic Leukemia.
Authors: Koller Charles Asa , Bekele B. Nebiyou , Zhou Xian , Park Charles , Estrove Zeef , O’Brien Susan , Keating Michael , Jilani Iman , Giles Francis J. , Kantarjian Hagop M. , Albitar Maher
Published: October 2019

Several cytokines and growth factors are involved in the regulation of megakaryocytopoieses and platelet formation. Interleukin-11 (IL-11), IL-6, IL-3, IL-1b, and thrombopoietin (TPO) act synergistically to promote proliferation and maturation of megakaryocytes. Recombinant IL-11 and TPO are under clinical investigation as supplements to stimulate thrombopoiesis in patients with cancer. We investigated the plasma levels of TPO in 127 patients with chronic lymphocytic leukemia (CLL) and correlated these levels with platelet counts and various laboratory and clinical characteristics. TPO levels were significantly higher in patients with CLL (median, 232 pg/mL; range, 26.4–2714.5 pg/mL) than in healthy control subjects (P <0.01). More importantly, higher levels of TPO were associated with advanced Rai stage (P <0.0001), higher b-2-microglublin (b2-M) levels (P<0.001), and the presence of mutation in the IgVH gene (P <0.001). TPO levels were inversely correlated with platelet count (P = 0.002). Univariate Cox proportional hazard models demonstrated that high TPO levels in CLL patients were associated with shorter survival (P <0.0001); multivariate analysis demonstrated that this association was independent of the IgVH mutation status, b2-M levels, and Rai stage. Recursive partitioning showed that a cut-point of 639.4 pg/mL separated CLL patients into two major groups with a significant difference in survival (P <0.0001). When b2-M level was added to this model, its effect was masked by the effects of TPO in patients with TPO >639.4 pg/mL. However, among patients with TPO levels =639.4 pg/mL, those with b2-M levels >4.95 mg/L had significantly shorter survival than those with lower b2-M levels. These data not only emphasize the importance of TPO in reflecting the aggressiveness of CLL, but also suggest that TPO and b2-M can be used to predict clinical behavior in this disease and may replace the need for determining IgVH mutation status. Further studies are needed to determine if the prognostic role of TPO is due to its direct effects on the growth of the leukemic clone or simply reflects a general failure in the homeostatic regulation of various cytokines.

Figure Figure
Regulation of Tissue Factor Signaling by Tissue Factor Pathway Inhibitor.
Authors: Ahamed Jasimuddin , Belting Mattias , Ruf Wolfram
Published: October 2019

Tissue factor initiates the coagulation cascade as well as cellular signaling through the activation of cell surface protease activated receptors (PARs), relevant to inflammation, tumor metastasis and angiogenesis. Tissue factor pathway inhibitor-1 (TFPI-1) is the major regulator of TF initiated coagulation, but recombinant TFPI-1 (rTFPI-1) has shown limited efficacy on sepsis-associated inflammatory responses and mortality. In TNF-stimulated human umbilical vein endothelial cells (HUVECs), we demonstrate with specific blocking antibodies that endogenous TFPI effectively controls TF procoagulant activity as well as TF-dependent signaling through PAR1 and PAR2. The effect of rTFPI-1 on coagulation and signaling was tested in TF-transfected CHO-cells or in HUVECs transduced with TF to overcome the inhibitory threshold of endogenous TFPI-1. Under conditions that specifically measure signaling of the ternary TF-VIIa-Xa complex, 2.5–5 nM rTFPI-1 inhibited TF-dependent Xa generation by >80% but the signaling of the ternary complex through PAR1 at these concentrations of rTFPI-1 was only marginally inhibited. Moreover, studies with proteoglycan-deficient CHO cells provided genetic evidence that PG binding facilitates the inhibitory activities of rTFPI-1 on coagulation and signaling. In HUVECs that co-expressed PAR2 with TF, three different signaling responses of the ternary TF-VIIa-Xa complex were inhibited by ~ 50% at 1.2–2.5 nM rTFPI-1, but the inhibitory potency of rTFPI-1 to block Xa generation was consistently higher relative to its effect on signaling. These data demonstrate that coagulation initiation phase signaling can occur in the absence of the activation of coagulation. The results also establish that endogenously expressed TFPI-1, known to be entirely GPI-anchored in endothelial cells, is a potent, negative regulator of ternary complex-mediated PAR signaling. The efficacy of rTPFI-1 appears to be influenced by the presence of PAR2, further emphasizing the close reciprocal signaling linkage of PAR2 and TF demonstrated in angiogenesis and cellular signaling targeting the TF cytoplasmic domain by phosphorylation.
Enhanced Primary CML Stem Cell Elimination by Bryostatin Priming with Imatinib Mesylate In Vitro.
Authors: Jorgensen Heather G. , Allan Elaine K. , Mountford Joanne C. , Richmond Linda , Elliott Moira A. , Holyoake Tessa L.
Published: October 2019

In chronic myeloid leukemia (CML), CD34+ quiescent stem cells are sensitive to the anti-proliferative but not the pro-apoptotic effects of imatinib mesylate (IM; Gleevec®, Glivec®). Bryostatin-1 (bryo), a Protein Kinase C modulator, was investigated to determine whether it would increase IM-mediated apoptosis either through initiation of cycling of G0/G1 Ph + cells or reversal of the IM-induced anti-proliferative response. The Ph+ K562 cell line and primary CD34+ CML cells were studied. Cell cycle status was assayed by propidium iodide uptake. Following exposure to IM, 86 ± 1 % (mean ± SEM) of surviving K562 cells was in G0/G1 as compared to 52 ± 2 % in the control (p < 0.001). After accounting for IM-induced cell kill, the absolute number of viable G0/G1 cells was significantly increased, confirming the anti-proliferative, anti-apoptotic effect. However, priming for 24 hours with bryo both increased K562 total cell kill and normalised the percentage of cells recovered in G0/G1 thus reducing their absolute number. To assess the effect of priming with bryo on primary cells, endpoints on two levels were investigated: firstly, at the total cell, and secondly, at the stem cell level. For total primary CML CD34+ cells, as compared to untreated control (= 100 %), 56 ± 2 % survived 96 hours treatment with 5 μM IM, whereas 37 ± 2 and 24 ± 2 % survived 1 and 10 nM bryo alone, respectively (n = 7). Priming with bryo prior to IM (5 μM) significantly enhanced bryo-induced total cell death (19 ± 2 and 12 ± 1 % surviving 1 and 10 nM bryo then IM, respectively; p < 0.03). Critically, within the G0/G1 CD34+ cells, 59 ± 3 % of input cells survived IM (5 μM) alone (n = 7). Bryo alone (1 or 10 nM) significantly reduced the proportion of cells recovered in G0/G1 (35 ± 3 and 23 ± 2 %) relative to both the untreated control and IM treatment alone (p < 0.02). Bryo in scheduled combination with IM, further significantly reduced the number of cells recovered in G0/G1 (16 ± 3 and 9 ± 1 %) with respect to IM alone (p < 0.001) and bryo alone (p < 0.05). Thus, it was the combination that was more potent than each individual agent. These results suggest that carefully scheduled drug combinations may prove more efficacious within the Ph+ stem cell compartment than IM monotherapy. Specifically, inclusion of an agent to antagonise the anti-proliferative effect of IM is warranted.
Incidence and Prognosis of RTKs and RAS Mutations in CBF AML. A Retrospective Study of French Adult ALFA and Pediatric LAME Trials.
Authors: Boissel Nicolas , Leroy Hugues , De Botton Stephane , Leblanc Thierry , Philippe Nathalie , Baruchel Andre , Hermine Olivier , Dombret Herve , Preudhomme Claude
Published: October 2019

Introduction: In core binding factor (CBF) acute myeloid leukemia (AML), the disruption of CBFa (AML1) or CBFb genes through t(8;21) or inv(16) impairs normal hematopoietic differentiation, but is considered to be insufficient to induce leukemogenesis. The need of an additionnal oncogenic event that promotes cellular proliferation has been suggested. This proliferative signal may be delivered by mutated receptor tyrosine kinases (RTKs) or RAS proto-oncogenes. In a retrospective study, we evaluate the incidence and the prognosis of RTKs (FLT3/D835, KIT/D816 and KIT/Ex8 insertion/deletion) and N/K-RAS mutations in patients with CBF AML.

Patients: Genomic DNA were obtained from the bone marrow or blood of 81 patients under 60 years with CBF AML (t(8;21)/inv(16) : 48/33) : 21 treated in the pediatric LAME trial (27/6) and 60 in the adult ALFA trial (33/15) between 1990 and 2004. The median follow-up is 5 years.

Results: RTKs mutations are found in 15/74 (20%) patients. KIT mutations are present in 11/74 (15%) patients, at a lower rate than previously described. KIT/D816 mutations are observed in 2/49 (4%) t(8;21) patients and 1/32 (3%) inv(16) patients. KIT/Ex8 mutations are slightly more present in inv(16) than in t(8;21) patients (5/32 vs 3/42, 16% vs 7%). One of the 5/81 patients (6%) with FLT3/D835 mutations also presents a KIT/Ex8 mutation. RAS mutations rates are significantly higher in inv(16) than in t(8;21) patients (11/33 vs 4/38, 33% vs 11%), with mostly N-RAS mutations (75%). No correlation between RTKs or RAS mutations and age or WBC is observed. Four patients did not achieve complete remission (CR rate : 95%), 2 with FLT3/D835 and 3 with RAS mutations. In univariate analysis, RTKs mutations are significantly associated with a shorter OS and EFS (p=.001 and p=.02, resp.) KIT mutations are associated with a shorter EFS and RFS (p=.002 and p=.003) in t(8;21) patients but do not affect the outcome of inv(16) patients in our study. Patients with FLT3/D835 mutations have significantly shorter OS, EFS and RFS (p<.0001, p<.0001 and p=.0007, resp.). The outcome of each subgroup of CBF AML is affected by FLT3/D835 status. RAS mutations have no impact on outcome. Multivariate analysis show a persistent significant prognostic impact of RTKs mutations on OS, RFS and EFS.

Conclusion: Inv(16) and t(8;21) cytogenetics are classically associated with a low risk of relapse. Patients with CBF AML are no more considered by many teams for allogeneic stem cell transplantation, even with a related donor. However, screening for RTKs mutations may identify patients with a more adverse outcome and thus susceptible to benefit from intensified protocols or tyrosine kinase inhibitors.
Novel Stratification Scheme for AML Based on a Large-Scale DNA Microarray Analysis of CD133-Positive Blasts.
Authors: Mano Hiroyuki , Yamashita Yoshihiro
Published: October 2019

AML is a clonal disorder of immature hematopoietic blasts and has a variable clinical outcome. Current classification of AML is based predominantly on the cytogenetic abnormalities and morphology of the malignant blasts and is not always helpful for optimization of treatment strategy. It is, for instance, very difficult to predict the prognosis of AML patients with a normal karyotype, who constitute ~50% of the AML population. DNA microarray analysis has the potential to provide a novel stratification scheme for AML patients, which is based on gene expression profile, and might help to predict the prognosis of, and optimize the treatment strategy for, each affected individual. However, leukemic blasts derived from bone marrow (BM) of AML-related disorders, are not homogeneous. The blasts may constitute from 20% to almost 100% of mononuclear cells (MNCs) in the marrow. Furthermore, given that many leukemic blasts possess the ability to differentiate to a certain extent, the marrow of AML patients contains not only the immature blasts (leukemic stem clone) but also differentiated blasts. A simple comparison of BM MNCs among heterogeneous AML patients is thus likely to reveal a large number of changes in gene expression that only reflect differences either in the percentage of blasts or in the differentiation ability of the blasts. To minimize such population-shift effects in microarray analyses, we established a large-scale cell depository “Blast Bank” for the storage of CD133 (AC133)-positive hematopoietic stem cell-like fractions from individuals with a wide range of hematopoietic disorders. In the present study, we have used Affymetrix HGU133 A&B microarrays to measure the expression profiles of ~33,000 genes in the Blast Bank specimens of 99 adults with AML-related disorders: 83 individuals with AML and 16 patients in the RAEB stage of MDS. In contrast to the previous microarray analyses of BM MNCs of AML, unsupervised hierarchical clustering of the subjects based on the expression profile did not separate the patients into FAB subtype-matched subgroups. Comparison of gene expression profile between the long-time and short-time survivors has identified a small number of outcome-related genes. Supervised class prediction, based on these genes, with k-nearest neighbor method or Cox proportional hazard model both succeeded to clearly separate individuals into subgroups with statistically distinct prognoses. Our analysis may pave a way toward the expression profile-based novel stratification scheme for AML.
Over-Expression of Flt3 Induces the Expression of NFkB Target Genes, cIAP-1 and IL-6.
Authors: Takahashi Shinichiro , Harigae Hideo , Inomata Mitsue , Ishii Keiko Kumura , Licht Jonathan D. , Sasaki Takeshi , Kaku Mitsuo
Published: October 2019

Nuclear factor kappa B (NFkB) regulates transcription of many genes involved in the immune response, including cytokines (e.g. IL-6) and growth factors. NFkB suppresses apoptosis as well, through various mechanisms including activation of cellular inhibitor of apoptosis-1 (cIAP-1). NFkB can be activated by the activation of Ras/phosphatidylinositol 3′-kinase (PI3K)/protein kinase B pathway in acute myeloblastic leukemia (AML). It was reported that in large proportion of primary AML cells, NFkB is constitutively activated, and the target genes are upregulated. Flt3 is a member of receptor tyrosine kinases. Flt3 receptor phosphorylation/activation results in the activation of downstream kinase pathways, like Ras, PI3K, leading to abnormal cell growth and aberrant gene regulation. Increased levels of Flt3 transcript are observed in a large number of AML specimens, and the over-expression of Flt3 contributes to the phosphorylation of Flt3 and activation of this pathway. In this study, we addressed the role of Flt3 over-expression in the activation of NFkB, which is a target molecule of various kinase pathways. We first tested the effect of Flt3 expression to NFkB responsive reporter by transient transfection and revealed the significant induction of the reporter. Next, we generated Flt3 transgenic BaF3 (BaF3-Flt3) cells that stably over-expressing Flt3 to confirm this effect. The introduction of NFkB-responsive reporter, as well as serum response element (SRE)-luciferase fused reporter into these cells resulted in the activation of the reporters. These results suggest that Flt3 over-expression mediated signal transduction activates NFkB pathways and affects growth factor pathways as well. Real time quantitative PCR demonstrated that in BaF3-Flt3 cells, mRNA expression of IL-6 and cIAP-1, both known target genes of NFkB, were increased. AG1296 or PDTC, potent inhibitors of Flt3 and NFkB respectively, completely abrogated the induction of these genes, suggesting the importance of Flt3-NFkB pathway to these effects. Moreover, we measured the expression level of Flt3 and NFkB target genes in 24 primary AML samples. As a result, there was a tendency of moderate to weak positive correlation between Flt3 and NFkB target genes. (Flt3 vs IL-6 [Spearman’s rank correlation coefficient: r= 0.35], Flt3 vs cIAP-1 [r=0.16]). These facts may also indicate the positive influence of Flt3 over-expression to NFkB target genes. Overall, these suggest a role of Flt3 over-expression in the activation of NFkB pathway in AML.
R399H Mutated ETS Domain Leads to a Functional ETV6 Double-Knock-Out in Acute Leukemia and Completely Alters the Physiological Function of ETV6.
Authors: Hennig Heike , Hillmer Frauke , Becker Annegret , Podleschny Martina , Pukrop Tobias , Truemper Lorenz H. , Griesinger Frank
Published: October 2019

ETV6 is the only known transcriptional repressor which plays an important role in the malignant transformation of haematopoietic cells in different leukemias.

In most cases of ETV6-AML1 + leukemias, the second ETV6 allele is knocked out by deletions. Other mechanisms of knocking out the second ETV6 allele in acute leukemias carrying ETV6 fusion genes, have not been described as of yet. Recently, we have published that the MT-ALL cell line with multilineage differentiation potential carries an ETV6-ARG fusion gene. The second ETV6 allele carries a point mutation in the highly conserved ETS-domain (G to A, bp 1220), leading to the replacement of arginine, normally directly binding to DNA, by histidine (R399H). Therefore, we investigated whether the R399H point mutation leads to a functional double knock-out and will reduce the DNA binding affinity of ETV6.

For the ETV6 binding studies the single point mutated and wild type ETS-domains were amplified and the proteins were expressed. Electro mobility shift assays (EMSA) were performed with the EBS (ETS binding site) carrying the specific GGAA core motif. The wild type ETS domain (ETSwt) incubated with the EBS showed a clear shift of the protein DNA complex loaded on a gel, but not the point mutated ETS domain (ETSmt). In order to quantify the binding of the ETSwt and mt-domains to EBS we established a transient transfection reporter gene assay. For this assay we constructed vectors which contain the EBS fused to the TK promoter, firefly luciferase serves as reporter gene. After transient cotransfections of HeLa cells with plasmids which expressed the ETSwt- or ETSmt-domain together with the vector containing the EBS(wt or mt), the activities of luciferase were measured in a tube luminometer. As expected, cotransfection of ETSwt and EBSwt reduced luciferase activity up to 75% compared with control vector and EBSwt cotransfection. Cotransfection of ETSmt with EBSwt did not lead to a reduction of luciferase activity. The specificity of the reporter gene analysis was controlled by using EBSmt.

These results show that the transcriptional repressor function of ETV6 is dependent on binding of ETV6-ETS domain to EBS and that the point mutation completely alters the physiological function of ETV6. Further studies would explain whether the multilineage differentiation potential of MT-ALL is dependent on ETV6 double-knock-out.
Randomized Clinical Trial Comparing Melphalan-Prednisone (MP), MP-Thalidomide (MP-THAL) and High-Dose Therapy Using Melphalan 100 MG/M2 (MEL100) for Newly Diagnosed Myeloma Patients Aged 65–75 Years. Interim Analysis of the IFM 99-06 Trial on 350 Patients.
Authors: Facon Thierry , Mary Jean Yves , Hulin Cyril , Benboubker Lofti , Attal Michel , Harousseau Jean Luc , Pegourie Brigitte , Renaud Marc , Guillerm Gaelle , Voillat Laurent , Chaleteix Carine , Troncy Jacques , Monconduit Mathieu , Casassus Philippe , Maisonneuve Herve , Dorvaux Veronique , Jaubert Jerome , Dib Mamoun , Doyen Chantal
Published: October 2019

The results of the IFM 95-01 trial comparing MP to Dexamethasone-based regimens were consistent with the fact that the standard MP remained the reference treatment for patients (pts) older than 65 years with multiple myeloma (MM) (Blood2003;102,147a). In May 2000, the IFM designed a new trial, IFM 99-06, for pts aged 65–75 years, comparing standard MP (12 courses at 6 weeks intervals) to MP-THAL (same MP, THAL at the maximum tolerated dose but ≤400 mg/day, stopped at the end of MP) and a MEL100-based treatment (intermediate-dose MEL procedure developped by the Italian MM study group); VADx2, cyclophosphamide 3g/m2 for stem cells collection, 2 courses of MEL100. IFM99-06 is planned to randomize 500 pts, according to a 3 (MP) - 2 (MP-THAL) - 2 (MEL100) allocation, and schedules two main comparisons ; MP vs MP-THAL and MP vs MEL100. The primary end point is overall survival. In the absence of clear toxicity data for MP-THAL and MEL100 in elderly MM pts, in the setting of a large randomized study, two interim analyses were planned, after the enrollment of 200 (40%) and 350 (70%) pts (followed at least 4 months), to be reviewed by an independent expert review committee (R.Schots / B.Van Camp, Brussels, Belgium ; M. Boccadoro, Torino, Italy ; S.Chevret, Paris-St-Louis, France). The first interim analysis was performed during the first months of 2003 (reference date for analysis January 2, 2003 ; 200 pts enrolled between May, 2000 and June, 2002). The experts considered that (i) the adverse events were as expected with the treatments used (ii) no clear-cut advantage or disadvantage of either MP-THAL or MEL100 over MP was observed according to the statistical rule of the first interim analysis (iii) the trial should pursue as planned. Following a request of the french regulatory agency AFSSAPS, we were asked to look rapidly at some thalidomide toxicities and at the feasability of MEL100. The incidence of deep veinous thrombosis (DVT) was 6%, 9% and 3.5% for MP, MP-THAL and MEL100, respectively (no toxic death due to DVT). Peripheral neuropathy was observed in 25% of MP-THAL pts (14/57 pts, at a median time of 12 months). MEL100 was feasible ; approximately 80% of the pts received the cyclophosphamide and 65% received the 2 MEL100 treatments. No toxic death was noted among 74 courses of MEL100 (39 pts). The second interim analysis, performed on 350 pts (382 pts enrolled on August 1, 2004) is in progress during July/August 2004 (date of point June 1, 2004). Results of the second interim analysis will be presented at the meeting.
Maternal Anti-HPA1a Antibody Level as Predictive Value in Neonatal Alloimmune Trombocytopenic Purpura (NAITP).
Authors: Killie Mette K. , Kjeldsen-Kragh Jens , Skogen Bjørn , Husebekk Anne
Published: October 2019

NAITP is suspected if a neonate unexpectedly presents with petecchiae and is severely trombocytopenic. The thrombocytopenia is caused by maternal alloantibodies to platelet-specific antigens, most often anti-HPA 1a. A mother who has previously given birth to an affected child is usually considered at high risk for giving birth to a trombocytopenic child also in the subsequent pregnancy. The aim of the present study was to compare to what extent quantification of anti-HPA 1a versus obstetric history can predict the severity of thrombocytopenia in new-borns. Anti-HPA 1a was repeatedly quantified in 152 immunized pregnant women who were included in a large Norwegian prospective study. Sixty-eight of the women gave birth to thrombocytopenic children of which 42 suffered severe thrombocytopenia (plt. count <50x109/l). Ten women had previously given birth to children with symptomatic NAITP. Five of these did not give birth to children with severe thrombocytopenia in their subsequent pregnancy. Positive predictive values for thrombocytopenia (<150x109/l) were 0.75 using obstetric history and 0.80 for anti-HPA 1a antibody level above 100 Arbitrary Units (AU). Positive predictive values for severe NAITP (<50x109/l) were hence 0.50 and 0.51 for obstetric history and antibody level. However, the diagnostic sensitivity for obstetric history was only 0.12, whereas anti-HPA 1a level above 100 AU had a diagnostic sensitivity of 0.88. In addition, we found that the level of anti-HPA1a tend to increase in first pregnancy and decrease in subsequent non-compatible pregnancies. This observation is in accordance with the fact that half of the women, who gave birth to NAITP children in their first pregnancy, did not get an affected child in their next pregnancy. In conclusion, obstetric history only identifies a few of the affected cases since all first time cases of sever NAITP are missed. Both obstetric history and antibody level has comparable positive predictive values for severe NAITP. However, the level of anti-HPA 1a antibody would be the best marker to identify severe NAITP cases because of independence of parity and obstetric history.
Can IVIg Preparations with Specificity for Soluble Antigens Mimic the Therapeutic Effects of IVIg in the Treatment of Immune Thrombocytopenia?.
Authors: Siragam Vinayakumar , Brinc Davor , Crow Andrew R. , Song Seng , Freedman John , Lazarus Alan H.
Published: October 2019

Intravenous immunoglobulin (IVIg) mediates protection from the effects of immune thrombocytopenic purpura (ITP). In addition, an IVIg specific for a cell-associated antigen (anti-D) is also able to increase platelet counts in D positive subjects with ITP. Whether an IVIg directed to a soluble antigen can likewise be beneficial in ITP is, however, unknown. A murine model of ITP was used to determine the effectiveness of IgG specific to soluble antigens in treating immune thrombocytopenia. Ovalbumin (OVA) was selected as the primary target antigen because it can be used in its soluble form or can be coupled to syngeneic red blood cells (OVA-RBC), and the same anti-OVA antibody could be used with both OVA and OVA-RBC. Mice were injected with either preformed soluble OVA + anti-OVA, OVA-RBC’s presensitised with anti-OVA, or appropriate control preparations, followed by an anti-platelet antibody to induce thrombocytopenia. Both experimental regimes, but none of the control preparations, protected mice from ITP. Similar to IVIg, soluble OVA + anti-OVA did not have any effect on thrombocytopenia in mice lacking the inhibitory receptor FcγRIIB (FcγRIIB−/−). In contrast, injection of anti-OVA sensitised OVA-RBC’s did ameliorate thrombocytopenia in FcγRIIB−/− mice. Finally, mice injected with IgG specific for the endogenous soluble antigens, albumin and transferrin, also inhibited ITP in an FcγRIIB-dependent manner. We conclude that IgG antibodies directed to soluble antigens can inhibit or reverse immune thrombocytopenia in an FcγRIIB-dependent manner.