bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteomics of Acute Myeloid Leukemia: Cytogenetic Risk Groups Differ Specifically in Their Proteome, Interactome and Posttranslational Protein Modifications.
Authors: Balkhi Mumtaz Y. , Geletu Mulu , Christopeit Maximilian , Behre Hermann M. , Behre Gerhard
Published: October 2019

Acute Myeloid Leukemia (AML) is characterized by specific cytogenetic aberrations which are strong determinants of prognostic outcome and therapeutic response. Because the clinical outcome in AML cytogenetic groups differs considerably, we hypothesized that cytogenetic risk groups of AML might differ specifically in their proteome, protein interaction pathways and posttranslational modifications (PTMs). Thus, we determined the proteome of 30 AML patients belonging to various cytogenetic groups based on two-dimensional gel electrophoresis and Nano LC coupled MALDI-TOF-TOF tandem mass spectrometry. We could identify substantial differences in the proteome, protein expression and peak pattern between cytogenetic risk groups of AML. The interactome analysis based on computational bioinformatics using Ingenuity analysis revealed major regulating networks: MAPK8 and MYC for complex aberrant karyotype AML, TP53 for t(8;21)-AML, TP53- MYC- PRKAC for 11q23-AML, JUN and MYC for inv(16)-AML. Most interestingly, peak explorer analysis revealed a modification of O-linked acetyl glucosamine of hnRNPH1 in AML patients with a 11q23 translocation, an acetylation of calreticulin in t(8;21) translocation AML, an increased intensity of dimethylated peptide of hnRNPA2/B1 in AML patients with translocations of t(8;21) and inv(16) in comparison to healthy bone marrow. We show for the first time that cytogenetic risk groups of AML differ specifically both in their proteome, interactome and PTMs. These findings lead to a new thinking about the pathogenesis of AML and has major therapeutic implications because PTMs are the primary drug targets.
A Phase II Study of Temozolomide Therapy for Poor Risk Patients Age ≥ 60 Years with Acute Myeloid Leukemia (AML) - Low Levels of MGMT May Predict for Response.
Authors: Brandwein Joseph M. , Schimmer Aaron D. , Schuh Andre C. , Gupta Vikas , Wells Richard A. , Yang Lin , Minden Mark D.
Published: October 2019

A previous Phase I study (Seiter K et al, 2002) had shown antileukemic activity of temozolomide (TMZ) in acute myeloid leukemia (AML). We conducted a Phase II study of TMZ in poor risk patients aged 60 years and over with AML. Patients were eligible if they had received no prior inductions and had one of the following: poor risk cytogenetics (complex abnormalities, deletions of chromsome 7 or 5, 11q23 abnormalities, inv[3]) or secondary leukemia (prior hematologic disorder or therapy-related leukemia). Patients who had not achieved remission or had relapsed after one prior induction course, regardless of other risk factors, were also eligible. Of 43 evaluable patients, 17 had received one prior induction and 26 had received no prior therapy - of the latter group, 12 had secondary leukemia and the remaining 14 had poor risk cytogenetics. The dose of TMZ was 200 mg/m2 orally daily for 7 days. Patients achieving partial response (PR) were eligible to receive a second identical course. Patients achieving complete response (CR or CRp) were eligible to receive TMZ 200 mg/m2 orally daily for 5/28 days for up to 6 postremission cycles. The treatment was well-tolerated, with mild to moderate myelosuppression and minimal nausea. Most patients received therapy on an outpatient basis. Four patients achieved complete response (3 CR and 1 CRp), 2 achieved PR and 2 had marrow aplasia at Day 30–35; the remaining 35 patients did not respond. Of the 4 patients achieving CR, one relapsed after 3 months, while the remaining patients remain in CR after 7, 12 and 29 months respectively. The 2 PR patients did not respond further to a second induction course. Because sensitivity to TMZ has been previously linked to expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), we assessed baseline levels of MGMT in leukemia blasts pre-treatment by Western blot. Of 24 evaluable samples, 10 had no detectable MGMT; this group included all 4 CR patients, one PR and one aplasia. Of the 14 with detectable MGMT there were no antileukemic responses. The remaining samples were either not available or not evaluable for MGMT. In conclusion, TMZ therapy was well tolerated and had antileukemic activity with approximately a 10% CR rate in older poor risk AML patients. MGMT appears to be predictive of response - all of the responders had undetectable MGMT activity, while no antileukemic activity was seen in MGMT positive cases. This assay may therefore be useful in selecting potential candidates for this treatment. Confirmation of this finding in a larger series is warranted. Furthermore, pharmacologic reduction in MGMT levels could potentially increase response rates to TMZ and warrants study.
Venous Thromboembolism Prophylaxis Practices in Acutely Ill Medical Patients with Heart Failure (NYHA Class III or IV): Findings from IMPROVE.
Authors: Pavanello Ricardo , Froehlich James B. , Tapson Victor , Bergmann Jean-Francois , Nakamura Mashio , Merli Geno , Pini Mario , Piovella Franco , Spencer Frederick A. , FitzGerald Gordon , Anderson Frederick A. ,   For the IMPROVE Investigators11
Published: October 2019

Background Acutely ill medical patients with heart failure have an increased risk for venous thromboembolism (VTE) and expert consensus guidelines recommend that they should receive VTE prophylaxis. However, little data is available on physician’s practices for providing prophylaxis to these patients. Our aim was to characterize VTE prophylaxis practices in acutely ill hospitalized medical patients with heart failure (NYHA class III or IV) enrolled in the International Medical Prevention Registry on Venous Thromboembolism (IMPROVE).

Methods Patient recruitment began in July 2002. Patients aged ≥ 18 years and hospitalized for ≥ 3 days with an acute medical illness are enrolled consecutively. Exclusion criteria are: therapeutic antithrombotic agents or thrombolytics at admission, major surgery or trauma during 3 months prior to admission, and VTE treatment within 24 hours of admission.

Results Of 6946 patients enrolled up to 31 March 2005 in 49 hospitals in 12 countries, 784 (11%) were heart failure patients. Compared with patients without heart failure, patients with heart failure were more likely to be in an ICU/CCU (13% vs 8%), immobile ≥ 4days (50% vs 30%), over 60 years old (85% vs 61%), perceived to be obese (20% vs 13%), or have respiratory failure (27% vs 17%; p<0.0001 for all). In total, only 51% of heart failure patients received pharmacologic prophylaxis and 61% received any type of prophylaxis. Pharmacologic prophylaxis type varied by region with low-molecular-weight heparin (LMWH) used less often, and unfractionated heparin (UFH) used more often in the USA compared with other participating countries (see Table). Aspirin and warfarin were used as VTE prophylaxis in 6% and 3% of heart failure patients, respectively. Intermittent pneumatic compression (IPC) was used more often in the USA than in other countries (24% vs 0.2%).

Conclusions Although acutely ill medical patients with heart failure are at risk of VTE and should receive prophylaxis, only 61% of these patients in IMPROVE actually received any type of prophylaxis. This reflects poor physician-awareness of the benefits of prophylaxis in this patient group and suggests that significant opportunity exists to improve physician practices.

Table. VTE prophylaxis in acutely ill medical patients with heart failure VTE prophylaxis (% patients) USA Other participating countries LMWH 15 46 UFH 27 13 Aspirin 8 4 Warfarin 5 1 Any pharmacologic prophylaxis 43 56 IPC 24 0.2 Elastic stockings 6 7
Inhibition of Factors Xa and Iia Results in Synergistic Inhibition of Thrombin Generation with Minimal Clot Time Elevation.
Authors: Welch Kathleen M. , Saiya-Cork Kamlai , Gould Weston R. , Leadley Robert J.
Published: October 2019

Inhibition of either coagulation factor Xa (FXa) or thrombin (FIIa) alters ex vivo biomarkers of coagulation and decreases thrombus size in animal models of experimental thrombosis. The objective of this study was to determine if combining FXa and FIIa inhibitors would synergistically reduce the magnitude of IIa generated without elevating markers of bleeding. A synergistic combination may translate into lower doses, more effective anticoagulation and better safety. To predict the FXa and FIIa inhibitor combinations with the maximum potential for synergy, PD 0313052, a potent, selective FXa inhibitor, and argatroban, a potent FIIa inhibitor, were each tested independently and in combination using an in vitro thrombin generation assay. Individually, PD 0313052 and argatroban reduced total thrombin generation (TG) in a concentration dependent manner with IC50’s of 497±148 and 882±193 nM, respectively. Subsequently, PD 0313052 and argatroban were combined in 96 well plates at concentrations ranging from 0.125x to 8x their respective IC50 concentrations. Analysis using the Bliss Independence Model identified statistically significant synergistic activity, with the greatest increase (33%) over simple additivity at 249 and 441 nM FXa to FIIa, respectively, both below their respective IC50 concentrations. Furthermore, combinations of PD 0313052 and argatroban were evaluated in an assay measuring activated clotting time (ACT). Although both PD 0313052 and argatroban dose-dependently elongated the ACT, the combination of 0.5x the TG IC50 concentrations, which demonstrated the greatest synergy in the TG assay, showed the smallest increase in the ACT, prolonging clotting time by only 15%. These data demonstrate that the combination of a specific factor Xa inhibitor and a specific IIa inhibitor can synergistically reduce thrombin generation without appreciable elevation of ACT, suggesting that dual inhibition of FXa and FIIa, using relatively low doses of each, may provide efficacious and safe treatment for thromboembolic diseases.
Mutations of the Tumor Suppressor Gene SOCS-1 in Classical Hodgkin Lymphoma Are Frequent and Associated with Nuclear Phospho-STAT5 Accumulation.
Authors: Weniger Marc A. , Melzner Ingo , Menz Christiane K. , Wegener Silke , Bucur Alexandra J. , Dorsch Karola , Mattfeldt Torsten , Barth Thomas F.E. , Möller Peter
Published: October 2019

The suppressors of cytokine signaling (SOCS) are critically involved in the regulation of cellular proliferation, survival, and apoptosis via cytokine-induced JAK/STAT signaling. SOCS-1 silencing by aberrant DNA methylation contributes to oncogenesis in various B-cell neoplasias and carcinomas. Recently, we showed an alternative loss of SOCS-1 function due to deleterious SOCS-1 mutations in a major subset of primary mediastinal B-cell lymphoma (PMBL) and in the PMBL line MedB-1, and a biallelic SOCS-1 deletion in PMBL line Karpas1106P (BLOOD, 105, 2535–42, 2005). For both cell lines our previous data demonstrated retarded JAK2 degradation and sustained phospho-JAK2 action leading to enhanced DNA binding of phospho-STAT5. Here we analysed SOCS-1 in laser-microdissected Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). We detected SOCS-1 mutations in HRS cells of eight of 19 cHL samples and in three of five Hodgkin lymphoma (HL)-derived cell lines by sequencing analysis. Moreover, we found a significant association between mutated SOCS-1 of isolated HRS cells and nuclear phospho-STAT5 accumulation in HRS cells of cHL tumor tissue (p<0.01). Collectively, these findings support the concept that PMBL and cHL share many overlapping features, and that defective tumor suppressor gene SOCS-1 triggers an oncogenic pathway operative in both lymphomas.
Optimum Storage Condition of Cord Blood before Cryopreservation.
Authors: Moldenhauer Anja , Wolf Juliane , Habermann Gudrun , Genter Gesche , Selders Lisa , Kiesewetter Holger , Salama Abdulgabar
Published: October 2019

Purpose. Different opinions regarding the storage conditions of cord blood before cryopreservation exist. Most experts recommend the storage at room temperature and the cyropreservation within the first 24 hours after delivery of the baby. However, the optimum storage temperature is unknown.

Methods. Cord blood units (n=46) were stored for 24, 48 and 72 hours at room temperature (RT) and 4°C. After storage, CD34(+) cells were isolated and analyzed for their cell count, flow cytometry profile, apoptosis rate, colony-forming capacity by methylcellulose assays, and transmigration capacity in response to stroma-derived factor 1. If possible, a direct-paired comparison was performed (n=15).

Results. The cell count of peripheral blood mononuclear cells was generally 10fold higher in cord blood units stored at RT than in cord blood units stored at 4°C, leading to a 10fold higher number of isolated CD34(+) cells. There was no difference in the frequency of CD34(+) cells after immunomagnetic isolation (>95%) or in the frequency of CD133(+) and CD45(+) cells. At 48 hours, the apoptosis rate was lower at RT than at 4°C (37.7±7.4 vs 49.9±4.2%). The plating efficiencies (PE) were highest after the units had been stored for 48 hours (RT 14.7±3.6%, 4°C 16.3±4%) independent of the storage temperature. The cells stored at RT for 48 hours had the highest transmigration capacity (54±11.9% vs 4°C 10.7±2%). Storage times of 24 and 72 hours resulted in PEs of on average below 10%. CD34(+)cells stored for 24 hours migrated less than the cells stored at 48 hours. Their transmigration capacity showed no temperature-dependent difference (RT 24.6±7.1 vs 4°C 36.1±10.2%).

Conclusion. Our data imply that CD34(+) cells from cord-blood units stored for 48 hours at room temperature have the highest plating efficiencies and transmigration capacities. A storage time for 48 hours before cryopreservation seems tolerable, since there was no increase of the apoptosis rate.
In Pediatric Mature B-Cell Non Hodgkin Lymphoma (NHL), Complex Karyotype or del(13q) Are Linked Prognostic Factors in Burkitt Lymphoma (BL) While 8q24/c-myc Rearrangement Is Associated with a Strong Adverse Effect in Diffuse Large B-Cell Lymphoma (DLBCL).
Authors: Poirel Hélène A. , Heerema Nyla A. , Swansbury John , Aupérin Anne , Launay Erika , Sanger Warren G. , Talley Polly , Raphael Martine , Perkins Sherrie , McCarthy Keith , Sposto Richard , Weston Claire , Gerrard Mary , Cairo Mitch S. , Bernheim Alain , Patte Catherine
Published: October 2019

Introduction: BL is the main childhood B-cell NHL, characterized by the 8q24 translocation [t(8q24)] leading to a c-myc deregulation. Additional chromosomal alterations have been described in BL as well as in DLBCL, but their prognostic value is not clearly evaluated, especially in children. We have already shown the prognostic value of complex karyotype in childhood B-cell NHL in general (Lugano 2005). We now study the role of c-myc rearrangement and of other chromosomal alterations in each sub entity, BL and DLBCL.

Methods: Karyotypes from 237 children enrolled in the FAB/LMB96 study were centrally reviewed by each participating cooperative group and obtained an international morphological consensus diagnosis: BL 76.8%, BL-like (BLL) 7.6%, DLBCL 12.2%, unclassifiable 3.4%.

Prognosis analyses (event free survival -EFS- and overall survival -OS-) were performed after adjustment on the national cooperative group, the therapeutic stratification group (C vs A & B), the morphological consensus classification (DLBCL vs BL & BLL), the LDH level (>2 vs <=2) & the CNS involvement.

Results: The BL/BLL were usually diploid with a t(8q24) in 92% cases and associated with other chromosomal aberrations in 69%, mainly +1q, del(13q), +7q. The DLBCL were more heterogeneous and more complex than BL/BLL. t(8q24) incidence (34%) was higher than in adult DLBCL and the pattern of chromosomal alterations varied according to the presence of t(8q24) [diploidy, +1q, del(6q), +7q] or not [hyperdiploidy, der(11q) and +12q].

Multivariate analysis identified two different associations of independent cytogenetic factors with worse prognosis on both OS and EFS. Model (A) for EFS: complex karyotype (more than 3 chromosomal alterations) [HR=3, p=0.0008] and t(8q24) [HR=5.1, p=0.04] or model (B) for EFS: del(13q) [HR=2.6, p=0.0008], +7q [HR=2.6, p=0.01] and t(8q24) [HR=5.7, p=0.03]. (+1q had no significant effect). In the BL entity (N=182), only the complexity [HR=2.5, p=0.02] persists in the model A and del(13q) [HR=4.2, p=0.002] in the model B. In the group of 29 DLBCL, the only significant variable (univariate analysis) is the t(8q24): the 3-yr EFS in DLBCL without t(8q24) is 100% while 50% in DLBCL with t(8q24) [p=0.0005].

Conclusion: In BL/BLL, the strong effect of complex karyotype is partly explained by the role of del(13q) and to a lesser extent by +7q. The strong prognosis effect of t(8q24) in B-cell mature childhood NHL is mainly due to its role in DLBCL. This study emphasizes the impact of cytogenetics in both diagnostic characterization and prognostic stratification of childhood mature B-cell NHL.
Constitutive Activation of AKT Contributes to the Pathogenesis and Survival of Blastoid Variants of Mantle Cell Lymphoma.
Authors: Rudelius Martina , Pittaluga Stefania , Pham Trinh H.-T. , Fountaine Thomas , Quintanilla-Martinez Leticia , Fend Falko , Jaffe Elaine S. , Raffeld Mark
Published: October 2019

The serine/threonine protein kinase AKT [protein kinase B (PKB)] plays a pivotal role in the tumorigenesis of many human malignancies, including hematopoietic neoplasms. AKT mediates cancer cell survival and cell growth through the phosphorylation of key regulatory proteins such as FRKHL-1, MDM2 and p27. We wished to determine whether the AKT signaling pathway might be involved in the pathogenesis of mantle cell lymphoma (MCL).The activated form of AKT (pAKT) and phosphorylation of several AKT targets, including FRKHL-1, MDM2 and p27 as well as the upstream modulators PTEN and TCL-1 were analyzed by immunoblotting in 31 MCL cases (16 classic MCL and 15 MCL of the blastoid variant) and in four MCL cell lines (Granta 519, NCEB, REC-1 and Z138). PI3KCA mutation analysis at two hotspot regions (Exons 9 and 20) were completed for all cases and cell lines. Subsequently, inhibition of the PIK3/AKT-pathway with LY294002, Wortmannin and AKT-inhibitor was performed in the four cell lines. pAKT was expressed in all 15 blastoid variants of MCL and in the 4 cell lines, with high expression in 13/15 cases and low expression in 2/15 cases. In contrast, only 2/16 typical MCL expressed pAKT, at low levels. Furthermore, pAKT expression in the blastoid variants and cell lines was accompanied by the phosphorylation of multiple downstream targets of activated AKT, including the cell cycle regulatory proteins p27, FRKHL-1, MDM-2 and the apoptosis associated protein bad. 42% of the blastoid MCL cases showed loss of PTEN, while only 2 (12%) of the typical cases showed diminished PTEN expression.TCL-1 expression levels were not correlated with AKT activation and no mutations of PI3KCA were detected. Functional studies of all four MCL lines demonstrated that the phosphorylation of the downstream effectors were regulated by activated AKT:Inhibition of the PI3K/AKT pathway by both 20 μM LY294002 and 8 μM Wortmannin or 40 μM AKT-inhibitor abrogated the phosphorylation of AKT, p27, FRKHL-1, bad and MDM2 after 24 to 48 hours of treatment, and resulted in G1 arrest after 24 hours of treatment and measurable apoptosis by 48 hours. We conclude that constitutive activation of AKT contributes to the pathogenesis of the blastoid variants of MCL. One possible mechanism of AKT activation is the loss of PTEN expression. The inhibition of the PIK3/AKT pathway in MCL cell lines leads to induction of cell cycle arrest and apoptosis, raising the possibility that PI3K/AKT inhibitors may be effective in the treatment of aggressive (blastoid) variants of MCLs.
Hormonal Regulation of the Anticoagulant Protein S.
Authors: Hughes Quintin , Staton Janelle , Watson Mark , Harvey Jennet , Baker Ross
Published: October 2019

The anticoagulant Protein S (PS) is coded for by the PROS1 gene and serves as a co-factor to APC in the inactivation of FVa and FVIIIa. It is produced predominately by the liver. Low PS levels are associated with an increased thrombotic risk. Clinically it is well documented that circulating PS levels are reduced in response to heightened estrogen (E2) levels (oral contraceptive use, pregnancy, etc.). However, an explanation as to how this is occurring at the molecular level has not yet been elucidated.

Two approaches have been utilized. Firstly, a liver carcinoma cell line, HepG2, was exposed to 5 and 10nM concentrations of testosterone (T) and E2 for 6, 12 and 24h at which point’s total RNA was extracted and quantitated using real-time PCR. Secondly, a reporter plasmid pQH5′[2]DsRed2 was constructed that has 948bp of the PROS1 promoter region (up to but not including the ATG start codon) fused to an enhanced green fluorescent protein (EGFP) reporter gene sequence. A second reporter gene, DsRed2, was incorporated into pQH5′[2]DsRed2, and is constitutively expressed by a cytomegalovirus (CMV) promoter. This provides a means of standardizing any change in expression of the EGFP reporter.

pQH5′[2]DsRed2 was transiently transfected into a breast cell carcinoma line MCF7 [chosen for its high levels of oestrogen receptor alpha (ERα)] and subsequently exposed to 5 and 10nM concentrations of E2 and T for 24h. Expression levels were assessed using a COULTER EPICS flow cytometer.

The HepG2 6, 12 and 24h PROS1 mRNA hormone response experiment showed an erratic response to E2. However there were consistently higher levels of PROS1 mRNA over time in response to T compared to the vehicle alone. Higher levels still were evident for the 10nM concentration.

The transient transfection of pQH5′[2]DsRed2 into MCF7s followed by a 24h exposure of either T or E2 at 5 and 10nM concentrations, showed a significant change in response to both the 5 and 10nM concentrations of E2, but little response to either of the T concentrations

These results suggest that E2 is exerting a direct effect on the promoter of PROS1, down regulating transcription, reflecting clinical observations. However, T may be affecting PROS1 mRNA stability via a yet undetermined mechanism. Therefore, hormonal regulation of PS may be occurring at several levels of the overall production process.
The Cytoplasmic Domain of Tissue Factor Inhibits Migration and Enhances Adhesion of Human Breast Cancer Cells.
Authors: Jiang Xiaofeng , Panetti Tracee S. , Bromberg Michael E.
Published: October 2019

Tissue factor (TF) is a 47 kDa transmembrane glycoprotein that when complexed with its cofactor, factor VIIa (FVIIa), initiates blood coagulation. Apart from hemostasis, TF has been shown to have roles in cellular signaling, development, inflammation, metastasis and angiogenesis. We showed previously that both the cytoplasmic and extracellular domains of TF are required for the full metastatic effect of TF. Recently, we showed that TF-FVIIa-FXa complex induces cellular signaling in human breast cancer cells and is associated with enhanced cell migration and prevention of apoptosis. However, the role of the cytoplasmic domain of TF in tumor cell function is not fully known. In the present study, the role of the cytoplasmic domain of TF in cell migration and adhesion was investigated using the Adr-MCF-7 cell line, a multidrug resistant subline of the human breast cancer cell line, MCF-7. The Adr-MCF-7 cell line has high endogenous expression of TF and expression of PAR1 and PAR2. Adr-MCF-7 cells were retrovirally transfected with either a cDNA construct encoding a FLAG epitope tag fused to the transmembrane and cytoplasmic domains of TF (known as FLAG-TFCD) or vector (LXSN) alone as a control, and stable, polyclonal cell lines selected using G418. Expression of the FLAG-TFCD construct was verified by RT-PCR, Western blot analysis and flow cytometry. To test the effect of overexpression of the FLAG-TFCD construct on cell motility a modified Boyden chamber chemotaxis assay was used. The control LXSN cell line had a nearly 9 fold increase in cell migration [33.5± 3.2 cells/hpf (mean± SEM)]using the combination of rFVIIa (10 nM) and FX (150 nM) as the chemoattractant compared with 0.1% bovine serum albumin (BSA) [3.9± 1.2 cells/hpf]. In contrast, the FLAG-TFCD cell line had no increase in migration of using the combination of rFVIIa and FX [6.6± 0.57 cells/hpf] compared with BSA [5.4± 0.67 cells/hpf]. We then examined the ability of the transfected cell lines to adhere to type IV collagen. The number of adherent cells for the transfected cell line, FLAG-TFCD, was nearly 3 fold higher than that for the LXSN line using a colorimetric MTS assay (0.295± 0.041 vs 0.119± 0.011). Moreover, treatment of the FLAG-TFCD cells with the combination of rFVIIa and FX increased the adhesion by nearly 2 fold compared with untreated FLAG-TFCD cells (0.561±0.055 vs 0.296±0.044). In summary, overexpression of TF cytoplasmic domain leads to inhibition of tumor cell migration and enhancement of cell adhesion and potentially acts as a dominant negative in these cellular processes. These data suggest that a function of the cytoplasmic domain of TF in metastasis is to regulate tumor cell migration and adhesion.
Enhancement of Tissue Factor Dependent Procoagulant Activity and Factor Xa Generation by Alpha2-Macroglobulin.
Authors: Bhattacharjee Gourab , Arandjelovic Sanja , Mackman Nigel , Gonias Steven L. , Edgington Thomas S.
Published: October 2019

Alpha2-macroglobulin (α2M) is a broad-spectrum proteinase inhibitor (M~718,000) which is unique in its ability to inhibit proteinases from all four mechanistic classes. α2M inhibits coagulation proteinases, such as thrombin and factor Xa, and fibrinolytic proteinases, such as plasmin. α2M is also a major carrier of growth factors and may regulate growth factor activity. The primary receptors for α2M are the low density lipoprotein receptor related protein (LRP, KD~5 nM) and a higher affinity receptor recently identified as the 78 kDa glucose regulated protein (Grp78, KD~50 pM). In its native conformation α2M is present in plasma at very high concentrations (up to 5 μM). Upon binding to serum proteinases or small primary amines α2M is converted to the receptor-recognized form (α2M*) and then binds to LRP. LRP-associated α2M-proteinase complexes are rapidly internalized and degraded by phagocytic cells such as macrophages. Previous work in our laboratory has demonstrated that Grp78 binds to tissue factor (TF) and inhibits its functional activity. We now demonstrate that α2M* enhances TF-dependent procoagulant activity and factor Xa generation and this function is dependent on α2M* binding to LRP, but not Grp78, since receptor-associated protein (RAP) blocks the response. The effects of α2M on TF-dependent procoagulant activity are most pronounced in RAW 264.7 macrophage-like cells, but also observed in THP-1 monocytic cells. We are performing additional studies, using both α2M mutants and LRP-deficient cells, to further elucidate this novel mechanism by which α2M* may regulate hemostasis.
Gene Therapy for Severe Combined Immunodeficiency X1.
Authors: Hacein-Bey-Abina S. , Schmidt M. , Le Deist F. , Garrigue A. , Borkhardt A. , Wulffraat N. , Alexander I.E. , Wintergerst U. , Leiva L.E. , Delabesse E. , Macintyre E. , Gougeon M.L. , Gyapay G. , Weinssenbach J. , Fraser C. , de Saint-Basile G. , Casanova J.L. , Von Kalle C. , Fischer A. , Cavazzana-Calvo M.
Published: October 2019

We have previously reported that ex vivo retroviraly-mediated gc gene transfer into CD34 (+) bone marrow precursor cells led to the correction of the immunodeficiency in 9 out of 10 patients with X-linked severe combined immunodeficiency. Follow-up now reaches more that 6 years for the first 2 treated patients. Patients’immune function has been restored. The distribution of both TCR Vb family usage and TCR Vb CDR3 length still reveals a broadly diversified T cell repertoire. Moreover 6 years after treatment the thymus is still seeded by transduced progenitor cells as attested by the presence of TRECS in peripheral blood RTE. Among these patients, three (P4, P5 and P10) developed at 30 to 34 months after gene therapy a monoclonal T cell proliferation requiring a chemotherapy. P4 received also an allogenic HSCT from a MUD but died 26 months after the occurence of the lymphoproliferation. For P5 and P10, chemotherapy has led to an overall control of the clonal proliferation. These two patients are doing well and P5 is off treatment with a good immunological recovery. Genetic analysis of the blastic cells showed that in the two first cases the vector had integrated within or upstream of the LMO2 locus causing an insertional activation of LMO2 transcription. The last case revealed the involvement of several targeted sites, but their exact contribution to the lymphoproliferation is still under investigation. The repeated involvment of LMO2 as a site of vector integration in the proliferating T-cells points to an insertional activation of this gene as at least one of the causes of the oncogenic process. However, the long latency observed in all cases (> 30 months) suggests that additional “hits” have been required for overt desease. Synergy with gc expression and thereby induced proliferative signals (explaining occurrence in SCID-X1 patients only) is the most obvious hypothesis which we are trying to analyse in a mouse model. A deep analysis of retroviral integration patterns has been performed on patients’PBMCS by LAM-PCR to estimate the frequency of potentially harmful integration events and to assess the risk factors associated with the LTR’s strong enhancer effect of the MLV-based retroviral vector. 708 unique integration sites (IS) have been obtained from all analysed patients post-gene therapy and among them, 577 could be mapped unequivocally to the human genome. *Most of these insertions (63%) are located in the vicinity of 10kb or within the coding sequence of a known gene*. A significant peak of insertion frequency is related closely to the transcription strart site *among the 577 IS, 43 are common integration sites. Among the latter, we found out a high selection of genes involved in human oncogenic process.
A Novel Thrombin Fluorogenic Substrate of High Affinity, Catalytic Efficiency and Selectivity.
Authors: Baird T. Regan , Jacobs Margaret , Tinklepaugh Adam , Gross Peter , Furie Barbara C. , Furie Bruce
Published: October 2019

Thrombin is a serine protease with multiple functions, including the conversion of fibrinogen to fibrin, platelet activation, activation of Factor VIII and Factor V. Although many low molecular weight substrates have been developed for the study of thrombin catalytic activity, our interest in analyzing thrombin activity in the blood of a living mouse required development of a new class of thrombin substrates of high affinity and high selectivity whose product upon hydrolysis could be visualized by intravital fluorescence microscopy. We have developed a novel substrate for thrombin using the fluorochrome Alexa 488 and the quencher QSY35. Alexa 488 is conjugated to the N-terminus of a 12 amino acid peptide based upon the thrombin cleavage site in the α-chain of fibrinogen and the quencher is coupled to the C-terminus of the peptide, yielding Alexa 488-KGGVR-GPRVVEA-QSY35. We term this substrate FBG-12. Through fluorescence energy resonance transfer, the emission from Alexa 488 is absorbed by the quencher QSY 35, thus minimizing fluorescence, since the two moieties have overlapping spectral properties and are separated by a Förster radius less than 44 Å. Thrombin hydrolyzes the peptide yielding the N-terminal fragment, Alexa 488-KGGVR-COOH, which is highly fluorescent, and the C-terminal fragment NH2-GPRVVEA-QSY35 peptide which, being physically separated from the Alexa 488, no longer quenches the fluorochrome. The peptide was synthesized by solid phase peptide synthesis, its N-terminus and C-terminus were modified with Alexa 488 and QSY35 respectively, and its identity confirmed by mass spectroscopy and protein sequencing. The kinetic properties of FBG-12 were determined in vitro. Hydrolysis of the substrate by thrombin resulted in a linear increase in fluorescence at 525 nm over time and was dependent on enzyme concentration. The fluorescence of the product of thrombin hydrolysis of FBG-12 was 120-fold greater than that of the substrate. Michaelis-Menten kinetic analysis of thrombin hydrolysis of the fluorogenic substrate FBG-12 revealed a Km of 2 μM, a kcat of 759 s−1, and a kcat/Km of 3795 x 106 M−1 s−1. To determine the selectivity of this substrate, other plasma serine proteases were analyzed for their ability to hydrolyze FBG-12. Factor Xa, Factor VIIa, and activated protein C did not hydrolyze FBG-12. Factor XIa (Km 11 μM, kcat 20s−1, kcat/Km = 20 x 106 M−1 s−1) did hydrolyze FBG-12, although the reaction was inefficient compared to thrombin. Our characterization of FBG-12 suggests that this substrate is hydrolyzed efficiently and selectively by thrombin. Its spectral properties and solubility in a physiologic environment make FBG-12 a suitable substrate for the detection of thrombin activity via intravital imaging during thrombus formation in vivo.
Mobilization of Stem Cells with DT-PACE Plus Pegfilgrastim vs. DT-PACE Plus Filgrastim in Previously Treated MM Patients.
Authors: Tricot Guido , Reiner Maureen , Burns Michael , Zangari Maurizio , van Rhee Frits , Barlogie Bart , Cottler-Fox Michele
Published: October 2019

Mobilization with chemotherapy (CT) plus hematopoietic growth factors (HGFs) is superior to mobilization with HGFs only. For stem cell mobilization after chemotherapy HGFs are typically given once or twice a day for approximately 14 days. The aim of this study was to evaluate whether mobilization of peripheral blood stem cells (PBSC) with DT-PACE plus pegfilgrastim was equivalent to mobilization with DT-PACE + filgrastim. Patients analyzed were enrolled in two consecutive studies 2001–12 (N=97) and 2003–41 (N=72) and included only patients who had received ≥ 2 cycles of prior therapy, but no prior transplant. Both protocols employed a single cycle of induction with DT-PACE (Lee et al. JCO2003;21:2732) followed by stem cell collection with either filgrastim 5μg/kg bid until completion of stem cell collection (2001–12) or pegfilgrastim 6mg on days 6 and 13 (2003–41). Patients then proceeded with tandem transplants, one consolidation cycle with DT-PACE and two years of maintenance with thalidomide (100mg daily) and dexamethasone (4 mg/day, days 1–4 q 3 weeks). Group characteristics were compared using the Kruskall-Wallis test. Time to hematopoietic recovery after transplantation was analyzed with Kaplan-Meier plots, and comparison was performed using the logrank statistic. The two studies had comparable characteristics, except that more patients ≥ 65 years were enrolled in 2003–41 (35% vs 13%; p=.001). The median number of collection days was 2 in both studies (p=.8). The median number of CD34 cells/kg (x106) collected per day was 9.7 (2001–12) vs 12.7 (2003–41) (p=.2). The total number of CD34 cell/kg (x106) collected was 20.4 (2001–12) versus 25.4 (2003–41) (p=.2). The median number of CD34 cells/kg (x106) infused after the first transplant was 4.1 (2001–12) and 4.5 (2003–41) (p=.5). The time to recover ANC > 500/μl and platelets > 20,000/μl without transfusion was shorter for 2003–41 patients (13 versus 15 days; p< .0001) (Figure 1). Mobilization of PBSC with pegfilgrastim is easier for patients because they only receive 2 injections and is as effective as filgrastim when combined with DT-PACE. Moreover, mobilization with pegfilgrastim may result in a more rapid hematologic recovery after transplantation.

Cumulative incidence analysis Recovery of ANC to 500 and Platelets > 20K 2003-41 vs. 2001-12, Transplant 1 Cumulative incidence analysis Recovery of ANC to 500 and Platelets > 20K 2003-41 vs. 2001-12, Transplant 1
Varying Response to Imatinib in CML Patients with BCR-ABLM244V Mutation.
Authors: Khorashad Jamshid S. , Anand Mona , Marin David , Andreasson Catharina , Ghorashian Sara , Apperley Jane F. , Goldman John M. , Kaeda Jaspal S.
Published: October 2019

BCR-ABL kinase domain mutation in a CML patient with clinical resistance to imatinib is generally accepted as ‘causal’. Here we report the kinetics of the mutant BCR-ABL transcripts in 6 CML patients with the M244V mutation that are at variance with the accepted view. Prior to starting imatinib, one patient in chronic phase (CP) was previously untreated, 4 patients in CP had previously received alfa-interferon, and the sixth had received an autograft but had subsequently progressed to accelerated phase (AP). Patients were started on 400 mg/day dosage which was escalated to 600, 800 or 1000 mg/day following little or no response. Philadelphia chromosome was the only cytogenetic abnormality detected at the time of diagnosis and at start of imatinib treatment. The median time from diagnosis to starting imatinib was 19 months (range: 3 – 120 months). The M224V mutations were detected initially as a result of routine screening by Sanger’s direct sequencing of cDNA from blood of patients on imatinib, at which time the mutant clone in each patient already constituted >80% of BCR-ABL transcripts, measured by QR-PCR. We then retrospectively measured levels of mutated BCR-ABL transcripts in all the stored samples, median 14.5 samples (5 – 28), by pyrosequencing. Of the 4 patients for whom there were pre-imatinib samples the BCR-ABLM244V allele was identified in one case (previously treated with IFN-alfa) at 3%, but was not detected in the other three. In the 2 patients for whom there were no pre-imatinib samples, the BCR-ABLM244V allele was present at 3.0% and 93% respectively in the earliest available sample. In the 5 patients whose earliest samples showed low or absent mutant transcripts the non-mutated BCR-ABL population was progressively replaced by BCR-ABLM244V-positive cells, eventually becoming the dominant clone with a median time from first sample to maximal level of 35 months (11 – 54 months). In three patients total BCR-ABL transcripts decreased and they achieved complete cytogenetic remission (CCyR). In the other three patients (including the newly diagnosed patient) there was little or no reduction in BCR-ABL transcript numbers and all failed to achieve CCyR. One patient in this latter group acquired trisomy 8, reported 40 months prior to blastic phase; she died following allogeneic SCT. The response to increased imatinib dose in three patients is consistent with the reported IC50 for the M244V mutation, i.e. moderate resistance, but the response in the other three patients is not. In summary, the response to imatinib in 6 patients with the same BCR-ABL mutation is variable. Furthermore, these observations imply the mutation is unlikely to be the sole cause of the resistance in at least in 3 of the patients. The observed heterogeneous phenotype of these 6 CML patients is likely to be due to a myriad of undefined contributory factors.
Imatinib and Leptomycin B Are Effective in Overcoming Imatinib-Resistance Due to Bcr-Abl Amplification or Clonal Evolution but Not Due to Bcr-Abl Kinase Domain Mutation.
Authors: Kancha Rama K. , Miething Cornelius , Bubnoff Nikolas V. , Gotze Katharina , Peschel Christian , Duyster Justus
Published: October 2019

Constitutive activity of the Bcr-Abl kinase has been shown to be the causative event in the molecular pathogenesis of CML and Ph+ ALL. Even though Imatinib, a small molecule inhibitor of the Abl kinase, is very effective in treating the disease, development of resistance to this drug is a common phenomenon in advanced stage disease. Using Leptomycin B (LMB), a nuclear export blocker, Vigneri et al. have shown that Bcr-Abl kinase activity trapped in the nucleus induces cell death, thus being a new potential treatment option for Bcr-Abl expressing diseases. To test whether this approach might be effective in the case of imatinib resistant leukaemia several Bcr-Abl positive, imatinib-resistant Ba/F3 clones were established and characterized: 1. Ba/F3 cells resistant due to a point mutation in the Abl kinase domain at position T315I. 2. Ba/F3 cells resistant due to amplification of the Bcr-Abl kinase. 3. Ba/F3 cell clones resistant to imatinib with clear features of clonal evolution. All four cell lines were tested for the efficacy of a combined treatment approach with imatinib and leptomycin B. This combination was very effective in inducing apoptosis in imatinib resistant Ba/F3 cells displaying Bcr-Abl amplification. One out of two cell lines with signs of clonal evolution also responded to this combination treatment. However, imatinib resistant cells expressing the Bcr-AblT315I mutant kinase were completely resistant to this approach. Thus, these results are in agreement with the proposed hypothesis of Vigneri and wang that the combined effect of leptomycin B and imatinib requires Bcr-Abl kinase inhibition (which is not achieved in cells expressing Bcr-Abl T315I) by imatinib and nuclear entrapment by LMB To check the feasibility of this combined treatment for purging purposes, in vitro colony forming assays were performed with mixed cultures of Bcr-Abl positive BaF3 cells and primary murine bone marrow. All colonies formed after combined treatment with imatinib and LMB were derived from BM cells, indicating selective toxicity towards Bcr-Abl positive cells while sparing primary murine bone marrow cells. We are currently evaluating the effectivity of the combined LMB/imatinib treatment as purging strategy in a murine bone marrow transplantation model.
Psychological Outcome after Hematopoietic Cell Transplantation for Sickle Cell Disease.
Authors: Pradere Jerome , Drain Elise , Taieb Olivier , Bernaudin Francoise , Champion Marie , Bonnet Doris , Dutray Benoit , Abbal Tahar , Chaudre Geraldine , Kuentz Mathieu , Coic Lena , Gluckman Eliane , de Montalembert Mariane , Dhedin Nathalie , Vernant Jean-Paul , Benkerrou Malika , Bertrand Yves , Vannier Jean-Pierre , Steschenko Dominique , Bordigoni Pierre , Bachir Dora , Galacteros Frederic , Casanova Jean-Laurent , Fischer Alain , Rose Marie
Published: October 2019

Hematopoietic cell transplantation (HCT) is the only potentially curative treatment for sickle cell disease (SCD). The probability of event-free survival is currently between 85 and 90% after matched sibling-donor HCT. This intensive treatment conducts to a major change for patients and families. The purpose of the present study was to determine the psychological impact on individuals and their families of this recovery process after HCT. The hypothesis was that the recovery is a process with different aspects (medical, psychological, individual, familial and cultural) which could be discordant. This study was supported by the Etablissement Français des Greffes (EFG) and conducted by child and adolescent psychiatrists, psychologists and a medical anthropologist. The assessment consisted in two semi-structured interviews adressed to the patients and their families for studying their illness narratives and questionnaires for quality of life, post-traumatic stress disorder (PTSD), depression and anxiety. 28 patients (17 males, 11 females) with SCD treated in France by HCT (22 by bone marrow and 6 by cord blood transplantation) and their families were evaluated at least one year (median 5 years, range, 1–14 years) after HCT at the median age of 14 years (range, 4–24 years). Among these 28 patients, 7 had prior history of severe vasculopathy and 6 of stroke before the HCT. At evaluation, 27 patients were free from SCD symptoms (but 3 had moderate chronic GVHD), 1 had graft rejection. 9 patients (4 males, 5 females) (median age 16 years, range 7–23 years) presented psychiatric symptoms as inhibition, depression, hypomaniac symptoms, anxiety, stutter. A psychiatric care was indicated for these 9 subjects. No diagnosis of current PTSD was found. Psychological distress was reported by parents. 21 patients considered themselves as cured, 4 of them in an incomplete way (none with stroke before the HCT, 3 with moderate chronic GVHD), and 3 not cured at all (none with stroke before the HCT, 1 with graft rejection). The study of the narratives illustrated the major individual, familial and cultural changes after the HCT. The recovery needed complex adjustments. Patients had to cope with new potentialities and requirements: school and relationships with peers, identity issues (disease transmission, infertility) and family changes. This study underlies the importance of psychological interventions for improving the outcome of patients with SCD treated by HCT and their families.
5′-ABL Gene Deletion on Derivative Chromosome 9 Detected by FISH: Prognostic Significance in CML Patients after Allogeneic Hematopoietic Stem Cell Transplantation (HSCT).
Authors: Chaudhri Naeem A. , Aljurf Mahmoud D. , Sahovic Entezam A. , Almhareb Fahad , Al-Sharif Fahad , Al-Zahrani Hazzaa , Al-Omar Hamad , Al-Shanqeeti Ali , Zaidi Syed , Morshed Manzur , Al-Anazi Khalid , El-Hassan Ibrahim , Al-Lahham Mohammad , Iqbal Mohammad A.
Published: October 2019

BACKGROUND AND OBJECTIVE

Large cryptic deletions of the 5′-ABL regions on derivative chromosome 9 can be identified by FISH in 10%–15% of Ph+ CML patients. This abnormality may be associated with unfavorable prognosis with conventional therapies. There is limited data regarding the prognosis of 5′-ABL gene deletion abnormality in recipients of allogeneic HSCT. We performed an analysis of our prospectively collected database to evaluate the prognostic importance of this karyotypic abnormality in HSCT patients.

METHODS:

Between1989–2004, 24 allogeneic HSCT patients with 5′-ABL gene deletion detected by FISH using ES-BCR/ABL probe were identified. The diagnosis was CML CP1, 16(66.6%), CML CP2, 5(20.8%), CML AP 2(8.3%), CML blastic phase 1(4.1%). Sex ratio: M/F 8/17; median age: 25.06 (14.7–51.9) years. All donors were HLA matched identical sibling. Stem cell source was BM in 15(62.5%), PB in 9(37.5%). Mean CD34 cells x106/kg dose was, BM 5.0 (2.05–7.67), PB 7.18 (1.64–10.71). Conditioning regimen was BU/CY2 in 19, CY/TBI in 4 and FLU/TBI in 1 patient.

A second group of 206 Ph+ CML pts was evaluated for comparison. These were patients with CML without 5′-ABL gene deletions that underwent allogeneic SCT from 1989–2004. This group was matched for age, sex, conditioning regimen, type of SCT, stem cell source and HLA matched sibling donors. The diagnosis was CML CP 148(71.8%), CML CP2 37(18.0%) AP 21(10.2%).

RESULTS:

After a median follow up of 17.5 mths in group 1 (del 9) and 17.05 mths in group 2 (without del 9), relapse occurred in 11/24(45.8%) in group 1 and 48/206(23.3%) in group 2, indicating RFS of 54.2% vs 76.7% respectively (P= .03). A sub-group analysis was made between CML CP1 patients only, between both groups. With 16 patients in group 1 and 148 pts in group 2 with a median FU of 21.5 vs 26.5 mths, the RFS was 7/16(43.7%) vs 118/148 (79.7%) respectively (P=. 001).

CONCLUSION

Patients with 5′-ABL gene deletions on derivative chromosome 9 have a significantly higher relapse rate even after high dose chemotherapy and allogeneic HSCT as compared to patients without this abnormality. We recommend close monitoring of these patients by RT-PCR to detect early molecular relapse and treatment with DLI and/or Imatinib Mesylate.